Yves Vandenbrouck
email: yves.vandenbrouck@cea.fr

Lydie Lane
email: lydie.lane@sib.swiss

Christine Carapito
email: ccarapito@unistra.fr

Paula Duek
email: paula.duek@sib.swiss

Karine Rondel
email: karine.rondel@univ-rennes1.fr

Christophe Bruley
email: christophe.bruley@cea.fr

Charlotte Macron
email: c.macron@unistra.fr

Anne Gonzalez De Peredo
email: anne.gonzalez-de-peredo@ipbs.fr

Yohann Coute
email: yohann.coute@cea.fr

Karima Chaoui
email: karima.chaoui@ipbs.fr

Yohann Couté

Emmanuelle Com
email: emmanuelle.com@univ-rennes1.fr

Alain Gateau
email: alain.gateau@sib.swiss

Anne-Marie Hesse
email: anne-marie.hesse@cea.fr

Marlene Marcellin
email: marlene.marcellin@ipbs.fr

Loren Méar
email: loren.mear@univ-rennes1.fr

Emmanuelle Mouton-Barbosa
email: emmanuelle.mouton@ipbs.fr

Thibault Robin
email: thibault.robin@etu.unige.ch

Odile Burlet- Schiltz

Sarah Cianferani
email: sarah.cianferani@unistra.frobs

Myriam Ferro
email: myriam.ferro@cea.fr

Thomas Fréour
email: thomas.freour@chu-nantes.fr

Cecilia Lindskog
email: cecilia.lindskog@igp.uu.se

Jérôme Garin
email: jerome.garin@cea.fr

Charles Pineau
email: charles.pineau@inserm.fr

Looking for Missing Proteins in the Proteome of Human Spermatozoa: An Update

Keywords: human proteome project, spermatozoon, missing proteins, mass spectrometry proteomics, immunohistochemistry, bioinformatics, data mining, cilia Journal of Proteome Research

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The Chromosome-Centric Human Proteome Project (C-HPP) aims to catalogue the protein gene products encoded by the human genome, in a gene-centric manner [START_REF] Paik | The Chromosome-Centric Human Proteome Project for cataloging proteins encoded in the genome[END_REF] .

As part of this project, neXProt 2 has been confirmed as the reference knowledgebase for human protein annotation [START_REF] Omenn | Metrics for the Human Proteome Project 2015: Progress on the Human Proteome and Guidelines for High-Confidence Protein Identification[END_REF] . Numerous initiatives were launched worldwide to search for so-called missing proteins -proteins predicted by genomic or transcriptomic analysis, but not yet validated experimentally by mass-spectrometry or antibody-based techniques. These proteins are annotated with a "Protein Existence" (PE) score of 2 when they are predicted by transcriptomics analysis, 3 when they are predicted by genomic analysis and have homologs in distant species, and 4 when they are only predicted by genomic analysis in human or other mammals. The most recent neXtProt release (2016-01-11) contains 2949 such missing proteins. It was suggested by Lane and collaborators 4 that proteins that have been systematically missed might be expressed only in a few organs or cell types. The very high number of testis-specific genes that have been described [START_REF] Chalmel | Global human tissue profiling and protein network analysis reveals distinct levels of transcriptional germline-specificity and identifies target genes for male infertility[END_REF] supports the hypothesis that the testis is a promising organ in which to search for elements of the missing proteome 6 7 . The testis' main function is well known: to produce male gametes, known as spermatozoa (commonly called sperm). Human spermatozoa are produced at a rate of about 1,000 cells/sec 8 by a complex, intricate, tightly controlled and specialized process known as spermatogenesis 9 10 . Spermiogenesis is the final stage of Journal of Proteome Research spermatogenesis, which sees the maturation of spermatids into mature, motile spermatozoa. The fact that the numbers of couples consulting for difficulties related to conceiving has increased in recent years, and that sperm quality has been shown to be altered in one in seven men, for example with abnormal motility or morphology [START_REF] Krausz | Male infertility: pathogenesis and clinical diagnosis. Best practice & research[END_REF] , makes further study of these cells even more topically relevant.

Large numbers of spermatozoa can be recovered in highly pure preparation through non-invasive procedures, making it possible to access the final proteome of the germ cell lineage, and providing access to a large number of germ cell-specific proteins. Thus, MS-based proteomics studies of spermatozoa have generated highly relevant data [START_REF] Rolland | Computational and Mass-Spectrometry-Based Workflow for the Discovery and Validation of Missing Human Proteins: Application to Chromosomes 2 and 14[END_REF] . Knowledge of the mature sperm proteome will significantly contribute to sperm biology and help us to better understand fertility issues.

In a recent study 13 , the Proteomics French Infrastructure (ProFI; www.profiproteomics.fr) described a step-by-step strategy combining bioinformatics and MS-based experiments to identify and validate missing proteins based on database search results from a compendium of MS/MS datasets. The datasets used were generated using 40 human cell line/tissue type/body fluid samples. In addition to the peptide-and protein-level false discovery rate (FDR), supplementary MSbased criteria were used for validation, such as peptide spectrum match (PSM) quality as assessed by an expert eye, spectral dot-product -calculated based on the fragment intensities of the native spectrum (endogenous peptide) and a reference spectrum (synthetic peptide) -and LC-SRM assays that were specifically developed to target proteotypic peptides. Some of these criteria were also used in a concomitant study [START_REF] Jumeau | Human Spermatozoa as a Model for Detecting Missing Proteins in the Context of the Chromosome-Centric Human Proteome Project[END_REF] involving transchromosome-based data analysis on a high quality mass spectrometry data set to catalogue missing proteins in total protein extracts from isolated human spermatozoa. This analysis validated 89 missing proteins based on version 1.0 of the HPP guidelines (http://www.thehpp.org/guidelines/). The distribution of two interesting candidates (C2orf57 and TEX37) was further studied by immunohistochemistry in the adult testis, and their expression was confirmed in postmeiotic germ cells. Finally, based on analyses of transcript abundance during human spermatogenesis, we concluded that it would be possible to characterize additional missing proteins in ejaculated spermatozoa.

The study presented in this paper originated with the Franco-Swiss contribution to the C-HPP initiative to map chromosomes 14 (France) and 2 (Switzerland) by identifying additional missing proteins. Here, we combine the search for proteins that are currently classed as "missing" with an extensive examination of the sperm proteome. A single pool of human spermatozoa was treated by a range of approaches, and the most recent version of the guidelines for the identification of missing proteins was followed (Deutsch et al., submitted;

http://www.thehpp.org/guidelines/). We thus performed an in-depth analysis of human sperm using different fractionation/separation protocols along with different protein extraction procedures. Through MS/MS analysis, 4727 distinct protein groups were identified that passed the 1% PSM-, peptide-and protein-level FDR thresholds.

Mapping of unique peptides against the most recent neXtProt release (2016-01-11) revealed 235 proteins (201 PE2, 22 PE3, 12 PE4) that are still considered missing by the C-H , and nine proteins annotated with a PE5 (uncertain) status in neXtProt. 8

Materials and methods

Ethics and donor consent

The study protocol "Study of Normal and Pathological Human Spermatogenesis" was approved by the local ethics committee. The protocol was then registered as No.

PFS09-015 at the French Biomedicine Agency. Informed consent was obtained from donors where appropriate.

Sample collection and preparation

Human semen samples were collected from five healthy donors of unproven fertility at Nantes University Hospital (France). The donors gave informed consent for the use of their semen for research purposes, and samples were anonymized. Semen samples were all obtained on-site by masturbation following 2 to 7 days of sexual abstinence. After 30 min liquefaction at room temperature under gentle agitation, 1 ml of each sample was taken. Aliquots were pooled and a protease inhibitor mix (protease inhibitor cocktail tablets, complete mini EDTA-free, Roche, Meylan, France) was added according to the manufacturer's instructions. To separate sperm cells from seminal plasma and round cells, the pooled sperm sample was loaded onto 1 mL of a 50% suspension of silica particles (SupraSperm, Origio, Malov, Denmark) diluted in Sperm Washing medium (Origio, Malov, Denmark). The sample was centrifuged at 400 x g for 15 min at room temperature. The sperm pellet was then washed once by resuspension in 3 mL of Phosphate-buffered saline (PBS) and centrifuged again at 400 x g for 5 min at room temperature. The supernatant was removed and the cell pellet was flash frozen in liquid nitrogen. analyses performed in this study can be found in Supplementary Material.

Protein extraction, digestion and liquid chromatography

MS/MS data analysis

Peak lists were generated from the original LC-MS/MS raw data using the Mascot Distiller tool (version 2.5. database searches were first assembled to allow competition between target and decoy peptides for each MS/MS query. Finally, the total number of validated hits was computed as Ntarget+Ndecoy, the number of false positive hits was estimated as 2xNdecoy, and the FDR was then computed as 2xNdecoy/(Ntarget+Ndecoy). Proline software automatically determined a threshold Mascot e-value to filter peptides, and computed the FDR as described so as to automatically adjust it to 1%. At protein level, a composite score was computed for each protein group, based on the MudPIT scoring method implemented in Mascot: for each non-duplicate peptide identifying a protein group, the difference between its Mascot score and its homology threshold was computed and these "score offsets" were then summed before adding them to the average homology (or identity) thresholds for the peptide. Therefore, less significant peptide matches contributed less to the total protein score. Protein groups were filtered by applying a threshold to this MudPIT protein score to obtain a final

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protein-level FDR of 1%. To optimize discrimination between true positive and true negative protein hits, the software applies a selection scheme approach, by adjusting the FDR separately for the subset of proteins identified by more than one validated peptide, and then for the single-peptide hits. In accordance with version 2.0. identified and validated at 1% were merged to create a unique combination of amino acid sequences and a list of PTMs located on that sequence which were aggregated in a single "representative" PSM. The newly created PSM were then grouped into proteins and protein families [START_REF] Nesvizhskii | Interpretation of shotgun proteomic data: the protein inference problem[END_REF] . The resulting dataset therefore provides a nonredundant view of the identified proteins present in the original sample.

Detection of missing proteins

The sequence of each peptide identified was searched in all the splicing isoform sequences present in neXtProt release 2016-01-11, using the pepx program developed in-house (https://github.com/calipho-sib/pepx). The method is based on a 6-mer amino acid index that is regenerated at each release; the 6 aa length was chosen as it significantly speeds up the mapping process. Leucine and isoleucine were considered equivalent. A peptide is considered to match an isoform sequence when all the 6-mers covering the peptide return the same sequence. Peptides were subsequently checked against the retrieved isoform sequence(s) to ensure an exact

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string match. All matches to splicing isoforms derived from a single entry were considered relevant for the identification of the entry.

To further validate the identification of missing proteins, a second round of peptideto-protein mapping was performed taking into account the 2.5 million variants described in neXtProt (SNPs and disease mutations). Currently, pepx only considers a single amino acid substitution or deletion in the 6-mer; substitutions and deletions more than 1 aa in length, as well as insertions, are not taken into account.

Consequently, pepx returns a match if single amino acid variations in the isoform sequence are spaced at least 5 amino acids apart. Peptides matching more than one entry when variants were taken into account were excluded as they are potentially not proteotypic.

predicted proteotypic peptides per protein, when possible. Thus, a total of 100 peptides were synthesized (Supplementary

Results and discussion

Overall workflow

The overall workflow for the detection and validation of missing proteins is illustrated in Figure 1 fragmentation spectra and LC-PRM assays). In parallel, the full list of missing or uncertain protein entries (PE2-5) was mined by gathering additional information from public resources, bioinformatics analysis and the literature. This information was used to select a subset of high priority proteins for further immunohistochemistry analysis on human testes sections.

Analysis of the human sperm proteome

Because the workflow involved a range of enrichment strategies and separation protocols, including peptide pre-fractionation protocols based on high pH reverse phase (HpH-RP) chromatography that have been shown to be orthogonal to subsequent online reverse phase nano-LC separation of peptides [START_REF] Gilar | Orthogonality of separation in two-dimensional liquid chromatography[END_REF] , sensitivity was high and coverage extensive. This type of "cover all bases" approach has been shown to be particularly efficient for improving the detection of missing proteins [START_REF] Kitata | Mining Missing Membrane Proteins by High-pH Reverse-Phase StageTip Fractionation and Multiple Reaction Monitoring Mass Spectrometry[END_REF] .

Validation was subsequently performed for each results file (.dat) through the targetdecoy approach [START_REF] Elias | Target-decoy search strategy for mass spectrometrybased proteomics[END_REF] , using the in-house developed Proline software (http://proline.profiproteomics.fr/), by adjusting the FDR to 1%, at PSM-and proteinlevel. In a second step, individual results files were combined for each dataset and a 1% protein-level FDR was applied to comply with the HPP data interpretation 2. Protein identification and their distribution across the five MS datasets were then compared to assess their contribution to total human sperm proteome data sets (Figure 1). The Venn diagram shows that 1526 proteins detected were present in all five datasets. In addition, each fractionation/separation method used in this study provided a significant added-value in terms of proteome coverage. Thus, Triton X-100 insoluble and soluble fractions, whole cell lysate analyzed by 1D SDS-PAGE, high-pH reverse phase peptide fractionation and long gradient runs allowed gains of 8.3, 6.7, 6.7, 3.8 and 0.8%, respectively. These results clearly emphasize the complementarity of the different enrichment techniques when seeking to obtain exhaustive (or as exhaustive as possible) proteome coverage.

Iin 2014, Amaral et al. 20 published a sperm proteome comprising 6198 proteins identified by combined MS-based analysis based on 30 LC-MS/MS proteomics studies. Crossing identifier lists between their data and the sperm proteome produced here revealed that our analysis yielded 1140 additional proteins. However, further investigation will be necessary to ensure a fair assessment as Amaral et al.

applied different validation criteria to ours (e.g., identification of at least two peptides with a protein-level FDR < 5%).

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Focusing on missing proteins identified from the sperm proteome

Missing proteins were detected as described in Materials and Methods using the most recent neXtProt release (2016-01-11). The first step in this detection took all possible splice isoforms and I/L ambiguities into account, but no single amino-acid variants. This produced a list of 235 missing proteins (PE2-4) and nine uncertain proteins (PE5). Among the PE2-4 protein entries, 188 were identified and validated (<1% FDR) by at least two or more distinct uniquely-mapping peptide sequences of length ≥ 9 amino acids, while the remaining 47 were associated with only one unique peptide ≥ 9 amino acids (Table 1). We named this subset of missing proteins "one-hit

wonders" and considered it separately for further MS-based analysis (see next section). In fact, 5 of these protein entries were identified with 2 (A2RUU4, Q5GH77, Q8NG35, Q8WTQ4) or 3 (Q6PI97) unique peptides, but since only one of these had a length equal to or greater than 9 amino-acids (Table 2), these entries were nevertheless considered to be "one-hit wonders". Among the full set of 235 proteins considered to be missing by neXtProt (PE2-4), 180 have no annotated function, while 56 are predicted to have at least one transmembrane helix (TMH). Full details (description, number of unique peptides, chromosome location, etc.) on the missing proteins are reported in Table 2 and Supplementary Table 3. The Venn diagram illustrated in Figure 3A shows how each fractionation protocol contributed to the identification of the whole set of missing proteins (PE5 included). Thirty-three proteins were detected in all five datasets, whereas 63 were specifically detected in a given dataset (30 in "insoluble fraction-1D gel", 9 in "soluble fraction-1D gel" gel, 17

in "whole lysate-1D gel", 6 in "peptide HpH-RP" and 1 in "whole lysate-long runs").

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We noticed that among the 30 proteins only detected in the insoluble fraction, around one-third (9 proteins) were annotated with at least one TMH (see Supplementary

Table 3) illustrating the benefit of preliminary subcellular fractionation for the identification of hydrophobic proteins. Unsurprisingly, only a small number of proteins (8) with at least one TMH were detected in all five sperm datasets, and an even smaller number of them were specifically detected when protocols starting with the soluble fraction or a whole cell lysate were applied (5 in "soluble fraction-1D gel", 3 in "whole lysate-1D gel", 2 in "peptide HpH-RP" and none in "whole lysate-long runs").

The missing proteins identified in sperm were found to be distributed across all chromosomes, except chromosome Y and chromosome 21, with the highest number (21 proteins) coded by genes present on chromosome 1 (Figure 3B). In terms of coverage of missing proteins (PE5 included), around 80% were supported by two or more distinct uniquely-mapping peptide sequences of length ≥ 9 amino acids, with some proteins very well covered (up to 78 peptides). The other 20% of missing proteins (52 proteins) were identified by only one unique peptide sequence of length ≥ 9 amino acids (Figure 3C).

To comply with recent C-HPP guidelines (version 2.0.1; http://www.thehpp.org/guidelines/), we also considered alternative mappings of all peptides of length >= 9 amino acids mapping to PE2-5 proteins by taking the 2.5 million single amino acid variants available in neXtProt into account. This analysis indicated that 13 peptides mapping to 12 missing (PE2-4) proteins could correspond to an alternative peptide sequence. Peptide 'TKMGLYYSYFK' maps uniquely to DPY19L2P1 (Q6NXN4), but if reported SNPs are considered, it could also map to the PE1 proteins DPY19L2 (Q6NUT2) and DPY19L1 (Q2PZI1). Peptide 'TPPYQGDVPLGIR' maps uniquely to the PE2 protein SPAG11A (Q6PDA7), but if reported SNPs are considered, it could also map to the paralog SPAG11B (Q08648), which was also identified in this study with two other unique peptides. Likewise, the PE2 protein LRRC37A (A6NMS7) was identified by two peptides 'NAFEENDFMENTNMPEGTISENTNYNHPPEADSAGTAFNLGPTVK' and 'SKDLTHAISILESAK'. If reported SNPs are considered these peptides could also map to the PE2 protein LRRC37A2 (A6NM11) and the PE1 protein LRRC37A3 (O60309), respectively. Therefore, DPY19L2P1, SPAG11A and LRRC37A will need further investigation to validate their existence at protein level. Peptides 'QNVQQNEDASQYEESILTK' and 'QNVQQNEDATQYEESILTK' were both validated by PRM (see below) but only differ by one residue (S or T) and map to two close paralogs, RSPH10B2 (B2RC85) and RSPH10B (P0C881), respectively. An S71T variant has been reported, thus, the two paralogs cannot be distinguished based on these peptides. Since RSPH10B2 and RSPH10B only differ by three amino acids in total, it is very difficult to find other suitable unique peptides for validation. However, the identification of RSPH10B2 (B2RC85) was confirmed by PRM using the additional peptide 'EEEFNTWVNNTYVFFVNTLFHAYK'. The PE2 protein ZDHHC11 (Q9H8X9) was identified by 2 unique peptides, but one of them ('GVLQQGAGALGSSAQGVK') could also map to its paralog ZDHHC11B if SNP were considered. The six other peptides that lost their unicity when SNP were taken into account map to proteins for which there were more than three peptides of length ≥9 amino acids.

In the small group of nine proteins with a PE5 status (uncertain), three were identified and validated (<1% FDR) by at least two or more distinct uniquely-mapping peptide

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sequences of length ≥ 9 amino acids. ATXN3L (Q9H3M9) was identified by six peptides -of which one would lose its unicity if SNPs were considered. This protein has now been characterized as a deubiquitinylase 22 23 24 25 , and its entry in UniProtKB/Swiss-Prot is currently under revision by the curators. HSP90AB4P

(Q58FF6) and GK3P (Q14409), identified, respectively, by two and three peptides ≥ 9

amino acids in length are annotated as pseudogenes in most protein databases.

Their status should be revised based on the results presented here.

The six others (PRSS41, LINC00521, FTH1P19, FAM205BP, SPATA31D3 and SPATA31D4) were detected with only one distinct uniquely-mapping peptide of length ≥ 9 amino acids. PRSS41 (Q7RTY9) is the ortholog of the recentlycharacterized testis-specific serine protease Prss41/Tessp-1 [START_REF] Yoneda | A testis-specific serine protease, Prss41/Tessp-1, is necessary for the progression of meiosis during murine in vitro spermatogenesis[END_REF] , and should no longer be considered as a pseudogene -the PE5 status of the entry is under revision by UniProtKB/Swiss-Prot curators. The putative uncharacterized protein encoded by LINC00521 (Q8NCU1) was detected by a 26-amino acid peptide for which no other match in the human proteome was found, even when possible variants were considered. The identification of the putative pseudogene FTH1P19 (P0C7X4) is also plausible since the peptide identified could only match the validated FTH1 protein (P02794) if both the rare D172N variant and an unknown S164A variant were considered. Nevertheless, according to the current HPP guidelines, the identifications of PRSS41, LINC00521 and FTH1P19 still need to be confirmed using other peptides.

The three remaining identifications are more dubious. Indeed, the peptide identifying FAM205BP (Q63HN1) could also match FAM205A (Q6ZU69), a PE1 protein, if its

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very common (30%) M499V variant form was considered. Likewise, as discussed in Jumeau et al. [START_REF] Jumeau | Human Spermatozoa as a Model for Detecting Missing Proteins in the Context of the Chromosome-Centric Human Proteome Project[END_REF] , the peptides mapping to SPATA31D4 (Q6ZUB0) and SPATA31D3 (P0C874) do not confidently identify one protein or the other. This issue cannot be resolved without access to data relative to the genomic sequences of the donors, as both variants SPATA31D3 R882G (dbSNP:rs815819) and SPATA31D4 G882R

(dbSNP:rs138456481) may be present in the pooled sample studied.

Investigating one-hit wonder missing proteins using MS-based criteria

As one-hit wonder proteins could potentially correspond to incorrect PSM assignment or false positives that passed the 1% FDR threshold, the recent HPP guidelines recommend that additional MS-based analyses be performed to provide further proteomics evidence (Deutsch et al., submitted; http://www.thehpp.org/guidelines/).

We therefore investigated our subset of one-hit wonder missing or uncertain proteins using additional MS-based criteria, as described in 13 . The first criterion relied on a blinded inspection of each MS/MS spectrum for the 52 missing or uncertain proteins (47 PE2-PE4; 5 PE5), all of which were identified by a unique peptide ≥ 9 amino acids in length.

The quality control of the PSMs corresponding to these unique peptides was carried between the experimental and theoretical spectra. A subset of 18 peptides were assigned a "high" quality tag by three out of the four sites and were therefore preferentially selected for further MS validation (data not shown). In addition to these 18 candidates, other one hit wonders were selected that were awarded a majority rather than a consensual "high" quality attribute. A supplementary filter was applied by retaining only peptides that could be synthesized (i.e., peptides shorter than 25 amino acids). This filter was necessary as further validation steps required the availability of a synthetic peptide for each candidate to be validated. Thus, of the initial 52 "one-hit wonders" we ended up with a final list of 36 peptides mapping to 34 missing proteins (PE2-4) and 2 uncertain proteins (PE5) for further assessment (among the 18 that had a unanimous high-quality vote, 17 peptides were selected, see Supplementary Table 4). Synthetic labelled peptide versions of all 36 peptides were ordered to allow systematic comparison of identifications with a synthetic version of the peptide (i.e., same charge, same instrument fragmentation conditions).

The goal of this step was to increase confidence in the identification of each peptide.

To allow readers to assess spectrum quality and peak intensity patterns between the endogenous peptide and its synthetic counterpart peptide for themselves, an example spectrum for synthetic peptides is shown alongside the naturally-derived peptides for protein entry Q9H693 in Figure 4A; likewise all other comparative spectra are presented in Supplementary Figure 1. To objectively assess peptide 'VEAALPYWVPLSLRPR' (protein entry Q9H693; shown in Figure 4A), we calculated the spectral dot-product score (SDPscore) [START_REF] Ye | Open MS/MS spectral library search to identify unanticipated post-translational modifications and increase spectral identification rate[END_REF] which corresponds to the spectral correlation score calculated for the intensities of all common singly-charged b-and y-

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ions of the reference spectrum and the native spectrum, as in our previous studies 13 .

A SDPscore of 0.954 was obtained for this peptide, indicating that its MS/MS fragmentation pattern is very similar to the pattern obtained for the reference synthetic peptide.

Additional experimental validation using targeted LC-PRM assays

In a final MS-based validation attempt, targeted MS assays were developed for the 36 candidate "one-hit wonder" proteins to try to re-detect their proteotypic peptide coeluting with its synthetic labeled counterpart. Samples for these assays were prepared independently. In addition to the original unique peptide identified, two additional predicted proteotypic peptides were selected, when possible, and synthesized for all 36 proteins (Supplementary Table 4). A total of 100 labeled peptides were synthesized. These peptides were mixed with protein digests and the heavy and light forms were targeted for analysis using parallel reaction monitoring scanning on a high resolution Q-Orbitrap mass spectrometer. In a first attempt, all proteotypic peptides were targeted in an unfractionated total protein extract prepared in stacking gel bands. This allowed us to unambiguously detect perfectly coeluting specific light/heavy transition groups for 24 proteotypic peptides corresponding to 21 of the 36 proteins. Examples of light/heavy transition group coelution are presented for protein Q9H693 for its initial peptide (Figure 4B) and for one additional predicted peptide that was detected thanks to the increased sensitivity of targeted assays (Figure 4C). Subsequently, to attempt to validate more candidates, the remaining undetected peptides were targeted in the insoluble proteins fraction after preliminary

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separation on 1D SDS-PAGE. Samples were prepared as previously described for the total proteome analysis (protocol ii). The insoluble fraction was chosen for these targeted assays as a majority of the one hit wonders (18 out of the 36, Supplementary Table 4) were identified in samples prepared by this protocol. Thus, our chances of validating peptides were greater with samples prepared by this protocol. These final MS experiments unambiguously validated 27 additional peptides belonging to 20 out of the 36 proteins, not all the same as the previously validated 21 proteins. Thus, in total, 24 out of the 36 proteins were validated with additional predicted and specifically targeted peptides. This successful validation with additional peptides confirms the utility of highly sensitive targeted assays compared to non-targeted data-dependent LC-MS/MS acquisitions, and shows that this approach is suitable for unambiguous validation of missing proteins. All the results obtained with targeted LC-PRM assays can be found in Supplementary Table 4 and Supplementary Figure 1.

Bioanalysis of the missing proteins

For all the missing proteins identified by MS-based analysis, the chromosomal location, PE status, predicted number of TMH, and functional annotation were retrieved from neXtProt (see Table 2 and Supplementary Table 3 for details). Using a previously described methodology [START_REF] Jumeau | Human Spermatozoa as a Model for Detecting Missing Proteins in the Context of the Chromosome-Centric Human Proteome Project[END_REF] , we also extensively mined publicly available transcriptome data, i.e., the "Human testis gene expression program" described by Chalmel and collaborators 5 to check whether the missing and uncertain proteins identified in the present study corresponded to genes carrying the testis signature,

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related to the onset of human spermatogenesis, or whether they corresponded to genes expressed only at the very end of spermiogenesis.

Only 132 of the 235 missing proteins and three of the nine uncertain proteins identified in this study corresponded to genes referenced in the "Human testis gene expression program" [START_REF] Chalmel | Global human tissue profiling and protein network analysis reveals distinct levels of transcriptional germline-specificity and identifies target genes for male infertility[END_REF] , with an increasing expression in seminiferous tubules containing post-meiotic germ cells (Johnsen score >=7). Up to 76 (+ 2 uncertain) of these proteins corresponded to genes specifically expressed in the testis (SET), 31

(+ 1 uncertain) corresponded to genes preferentially expressed in the testis (PET), and 25 corresponded to genes with intermediate (IE) or ubiquitous (UE) expression in the testis (Supplemental Table 3). This information was not used to select candidates for validation, but it is important to help understand the results of the immunohistochemistry experiments. Of note is that 26 and 2 proteins corresponding respectively to SET and PET genes are present in the current list of 1057 testisenriched proteins in Human Protein Atlas, an update of the initial list from from Djureinovic et al. [START_REF] Djureinovic | The human testis-specific proteome defined by transcriptomics and antibody-based profiling[END_REF] . That suggests the spermatozoon has great potential for the identification of additional missing proteins. It also shows that proteins that are not considered as highly enriched in the testis might concentrate in germ cells during late spermiogenesis and become accessible in the spermatozoa. However, it is also important to note that a significant subset of missing proteins identified in the present study corresponded to genes that are testis-specific but do not belong to the "Human testis gene expression program" as their expression is not enriched in seminiferous tubules containing post-meiotic germ cells (data not shown).

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To date, up to 111 of the 244 PE2-5 proteins identified have been subjected to extensive data and literature mining (Supplementary Table 5). The information gleaned from this data mining was used to establish priorities for further antibodybased studies. The first selection criterion was based on transcriptomics analysis.

RNA sequencing results from the Human Protein Atlas database indicated that 88 of these 111 proteins were either specifically expressed in testis, or were enriched in a small group of tissues in which testis has one of the two highest expression levels (column C). These proteins were assigned a very high score (dark green). The eight proteins which were enriched in a small group of tissues including testis, but for which expression levels in testis were not among the two highest ones were assigned with a lower score (light green). In contrast, a low (red) priority score was assigned to six proteins which were shown either to be specifically expressed in organs other than testis, or to be undetectable by RNA sequencing 5 43 . The remaining proteins are either ubiquitously expressed or expressed at low levels in testis and were assigned a neutral score (white).

The second selection criterion was based on phylogenetic profiling. We sought and report (column D) on the presence of homologs in S. cerevisiae and a number of ciliated organisms from distant groups. The 18 proteins for which homologs were present in at least three of the ciliated groups were assigned with a very high priority score (dark green) because we hypothesized that they could be involved in ciliogenesis. The 17 having homologs in one or two of the ciliated groups, but not in yeast were assigned a high priority score (green). In contrast, a low priority score (red) was assigned to the three proteins that were found to be conserved in yeast as they are not expected to play a specific role in ciliogenesis or human reproduction.

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The third selection criterion was based on the phenotypes observed in knock-out mice (column E). The four proteins for which a deletion led to a reproduction phenotype, were assigned a very high priority score (dark green). The ten proteins whose deletion had no effect on fertility were assigned a low priority score (red).

The fourth selection criterion was based on information retrieved from literature mining (gene expression regulation, protein protein interactions, function). This information is summarized in column F. Proteins for which published information suggested a putative role in spermatogenesis, ciliogenesis, or cilia function (85 proteins) were assigned a very high (dark green, 28 proteins) or high (green, 56 proteins) priority score. In the course of literature mining, we noticed that up to 76 of these 111 proteins had already been detected by mass spectrometry in human testis [START_REF] Liu | Scanning of novel cancer/testis proteins by human testis proteomic analysis[END_REF] or sperm 28 29 30 . However, these identifications have not yet been curated by PeptideAtlas or neXtProt; thus, the existence of these proteins was not considered validated at the time of writing.

The fifth selection criterion was based on immunohistochemistry data retrieved from the Human Protein Atlas (column G). The nine proteins that were specifically observed in germ cells, or associated with ciliary or cytoskeletal structures were assigned a very high priority score (dark green). Twelve other proteins observed in testis or ciliated cells were assigned a high priority score (green). Conversely, four proteins which were not seen in testis but were clearly seen in other tissues were assigned a low priority score (red).

Based on the combination of all these criteria, each of the 111 PE2-5 proteins was assigned a score grading the potential relevance of the protein in sperm and testis biology as high/medium/low. Seven entries which were initially classed as of

Journal of Proteome Research

high/medium interest were down-graded to low interest proteins because their localization in human sperm cells had already been published (column H). This left a total of 33 "high interest" proteins. Among them, we selected the 26 for which a Human Protein Atlas antibody was available which passed the Protein Arrays (PA)

test with a single peak, corresponding to interaction only with its own antigen. We selected 12 additional proteins from among the 42 scored as "medium interest", and one from among the 36 scored as "low interest" (columns I and J).

Orthogonal immunohistochemistry evidence

Immunohistochemical studies were undertaken to provide non-MS-based evidence for the expression of the 39 missing proteins selected based on this data mining process. Specific immunohistochemistry staining in human testes was obtained for 16 missing proteins using antibodies from the Human Protein Atlas without need for further technical improvement (see Supplemental Table 6). Results from these experiments show that all 16 selected proteins displayed immunoreactive signals at various intensities in germ cells at all stages of their development (Figures 5A,5B).

No staining above background levels was visible in interstitial cells or somatic cells in the seminiferous tubules for any of the antibodies (Figures 5A,5B). The staining intensity for missing proteins increased significantly from pre-meiotic and meiotic germ cells onwards (for C19orf81, C20orf85, SSMEM1, CCT8L2, WDR88, SAMD15, WDR93 and NOXRED1) or post-meiotic germ cells onwards (for CXorf58, C17orf105, C10orf53, C10orf67, FAM187B, AXDND1, GOT1L1 and CFAP46). This profile was to be expected as all proteins, except C10orf67, C19orf81 and WDR93, corresponded to genes in the TGEP, and their expression was expected to gradually

Journal of Proteome Research

increase in later stages of sexual maturation 5 (Supplemental Tables 3 and6). The expression levels for genes coding for C10orf67, C19orf81 and WDR93 may be below the threshold required to be part of the TGEP, even though immunoreactivity was observed in the germ cell lineage. Finally, CFAP46 is the ortholog of Chlamydomonas FAP46, which is known to be part of the central apparatus of the cilium axoneme and to play a role in cilium movement [START_REF] Brown | A FAP46 mutant provides new insights into the function and assembly of the C1d complex of the ciliary central apparatus[END_REF] . In the adult testis, immunostaining for CFAP46 was annular in late spermatids, a pattern that is typical of migration that will continue further down the midpiece or flagellum. Interestingly, CFAP46 expression has been shown to be downregulated in patients with primary ciliary dyskinesia [START_REF] Geremek | Ciliary genes are down-regulated in bronchial tissue of primary ciliary dyskinesia patients[END_REF] . That, together with our observations, is in favor of a central role in sperm movement.

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FAM187B is an orphan protein with no curated comments in UniProtKB/Swiss-Prot, except an indication that it is a transmembrane protein [START_REF] Wang | Mapping of the N-linked glycoproteome of human spermatozoa[END_REF] . The very peculiar localization of the immunohistochemistry staining for this protein, in the cytoplasmic region of elongating spermatids, suggests that FAM187B may play a role in cytoplasm displacement and elimination that take place during spermiogenesis.

Interestingly, FMA187B mRNA expression in sperm has been proposed as a valuable diagnostic indicator of sperm survival, fertility and capacity to promote early embryogenesis [START_REF] Georgiadis | High quality RNA in semen and sperm: isolation, analysis and potential application in clinical testing[END_REF] .

AXDND1 is an intracellular protein which is selectively expressed in the nasopharynx, bronchus, testis and fallopian tubes, according to HPA immunochemistry data. It is highly conserved in Vertebrates and in the Choanoflagellate Salpingoeca, and contains an axonemal dynein light chain domain (IPR019347). Interestingly, the outer arm dynein complex is the main propulsive force generator for ciliary/flagellar beating. The staining pattern for the protein, positive in the cytoplasmic region of elongating spermatids undergoing extensive remodelling, matches with a possible role for the protein in mobility of the sperm flagellum.

Conclusion

In this study, MS-based analysis of sperm samples detected 235 missing (PE2-4) and 9 uncertain (PE5) proteins. Among these, 206 missing and 4 uncertain proteins were validated with at least two or more distinct peptide sequences with ≥ 9 amino

Journal of Proteome Research

acids that mapped only to a single protein entry, even when possible variants were considered. In line with version 2.0 of the HPP Data Interpretation Guidelines, these 210 proteins can therefore be considered as validated. Twenty-four of these proteins were confirmed by LC-PRM assays, and 16 by IHC on human testis sections. IHC studies not only allowed us to confirm the existence of the proteins in sperm, but also to hypothesize a biological role for some of them (i.e., CXorf58, C20orf85, CFAP46, FAM187B and AXDND1). The combination of LC-PRM and IHC was clearly instrumental in validating two "one hit wonders": CXorf58 and C19orf81.

The importance of considering possible variants was illustrated by the cases of eight proteins, including three PE5, identified with peptides that lost their proteotypicity when possible variants were considered. These eight proteins therefore cannot be considered validated with our data.

The remaining 26 proteins were detected with only one unique peptide ≥ 9 amino acids. Six of these peptides were confirmed by LC-PRM and for three others, manual inspection unanimously indicated high quality LC-MS spectra. Thus, these nine identifications are reported here with confidence. However, the current HPP guidelines require MS-based validation of additional peptides for these proteins, or antibody detection to definitively validate their existence. The 17 other peptides passed the FDR criterion, but visual examination of their spectra indicated insufficient quality to warrant further study. Hence, the identification of these 17 proteins can only be considered dubious.

The Swiss-French collaborative project investigating the human sperm proteome in the context of the C-HPP started three years ago. In our previous article were identified in the present study. Except for TMEM239 (Q8WW34), detected with a single 24-amino acid peptide, all proteins were detected with at least 2 peptides, and often many more (Supplementary Table 7). The coverage of each protein was considerably improved by the use of cutting edge instruments (i.e., Q-Exactive;

Thermo Scientific) and sample fractionation.

We are confident that the present data can be used to validate the existence of 210 missing or uncertain proteins and are looking forward to integration of these validations in neXtProt once they have been reanalyzed by PeptideAtlas and combined with data from the other C-HPP teams interested in the testis or sperm proteome. In the meantime, the investigation of the human sperm proteome continues in our laboratories together with extensive data mining on the remaining set of missing proteins presented in this study. The information gleaned will help to extend our knowledge on the potential roles of these proteins in sperm function and/or maturation. Indeed, some of the proteins identified here may present a high clinical potential, and could also benefit the Biology and Disease driven HPP (B/D-HPP) that aims to explore the impact of proteomic technologies applied to a focused area of life science and health [START_REF] Aebersold | The biology/disease-driven human proteome project (B/D-HPP): Journal of Proteome Research enabling protein research for the life sciences community[END_REF] .

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Notes:

The authors declare no competing financial interest.

Tables Table 1: Description of missing proteins (PE2-PE4) detected in this study. see Table 2 and Supplementary Table 3 for details).

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Data mining and bioinformatics analysis

  -tandem mass spectrometry (LC-MS/MS) analysesMS/MS analysis of pooled sperm was performed using four different protocols based on a range of protein extraction procedures: i.) total cell lysate followed by a 1D SDS-PAGE separation (23 gel slices); ii.) separation of Triton X-100 soluble and insoluble fractions followed by a 1D SDS-PAGE separation (20 gel slices per fraction); iii.) total cell lysate, in-gel digestion, peptides analyzed by nano-LC with long gradient runs; iv.) total cell lysate, in-gel digestion, peptides fractionated by high-pH reversed-phase (Hp-RP) chromatography. For all protocols, tryptic peptides were analyzed by highresolution MS instruments (Q-Exactive). These experiments were performed by the three proteomics platforms making up ProFI (Grenoble, Strasbourg and Toulouse). A detailed description of the protein fractionation using Triton X-100, protein extraction and digestion, and liquid chromatography-tandem mass spectrometry (LC-MS/MS)

  out by visual validation by at least two mass spectrometry experts from each of the three sites of the ProFI infrastructure (Grenoble, Strasbourg, Toulouse) and from the Protim core facility (Rennes). PSM quality was classed in two categories: low and high. A high classification was based on the following spectral features: (i) the Journal of Proteome Research presence of y-ion and b-ion series; (ii) peak intensities; (iii) quality of the match

All 16 missing

 16 proteins whose expression was demonstrated in situ deserve further study to determine their role in sperm biology. However, due to their very specific expression patterns, 5 of them call for an immediate focus. Indeed, based on our immunohistochemistry data, staining for CXorf58, C20orf85 and CFAP46 was concentrated in late spermatids at the level of the acrosome under formation, a sperm-specific organelle essential for fertilization, with CFAP46 and CXorf58 displaying spectacular annular staining. Expression of FAM187B and AXDND1 displayed a slightly different profile in the adult testis, with immunoreactivity concentrated in the cytoplasmic region of elongating and elongated spermatids undergoing intense remodeling. This staining profile has previously been shown to be associated with the expression of proteins playing a role in sperm maturation.CXorf58 is an orphan protein with no curated functional comments in UniProtKB/Swiss-Prot; TargetP and MitoProt prediction programs predict a mitochondrial localization. Its expression in sperm cells was confirmed by PRM based on endogenous peptide 'SFFDEAPAFSGGR', detected only in the insoluble fraction, and the additional peptide 'DISAQIIQR'. The staining pattern observed in our immunohistochemistry experiment suggests that this protein is mainly located at the lower part of the head and midpiece of spermatids. This position is in favor of a link to mitochondria. Interestingly, mitochondria are grouped in the midpiece of mature Journal of Proteome Research spermatozoa, and numerous studies support the proposal that these organelles are important for sperm function and fertilization (for a review, see[START_REF] Rajender | Mitochondria, spermatogenesis and male infertility[END_REF] ).C20orf85 is also an orphan protein. It is expressed in the epithelium of the airways 32 , with levels increasing sharply during mucociliary differentiation. Its expression clusters with that of genes involved in regulation of cytoskeletal organization and intracellular transport[START_REF] Ross | Transcriptional profiling of mucociliary differentiation in human airway epithelial cells[END_REF] . In the adult testis, C20orf85 immunoreactivity was very strongly concentrated in the acrosome as it formed in elongating spermatids. The protein might migrate further down to the midpiece or flagellum in mature sperm. In yeast two-hybrid experiments, the murine C20orf85 ortholog (1700021F07Rik) was shown to interact with CCNB1IP1, a putative ubiquitin E3 ligase that is essential for chiasmata formation, and hence fertility 34 . Together with these observations, the altered expression of C20orf85 in asthenozoospermic patients 20 suggests a possible role in sperm movement.

Figure 1 :

 1 Figure 1: Flowchart illustrating the strategies used to identify and validate missing

Figure 2 :

 2 Figure 2: Contribution of the different fractionation protocols to identification of

Figure 3 :

 3 Figure 3: Missing proteins detected in the sperm proteome. A. Venn diagram

Figure 4 :

 4 Figure 4: MS validation of one-hit wonder missing protein, example of protein

Figure 5A :Figure 5B :

 5A5B Figure 5A: Antibody staining for orphan proteins CXorf58 (Q96LI9), C19orf81

Figure 1 .

 1 Figure 1. Flowchart illustrating the strategies used to identify and validate missing proteins detected in the human sperm proteome.

Figure 2 .Figure 3 .

 23 Figure 2. Contribution of the different fractionation protocols to identification of spermatozoa proteins.

Figure 4 .

 4 Figure 4. MS validation of one-hit wonder missing protein, example of protein Q9H693 (C16orf95)

Table 4

 4

	). The labeled peptides

Data mining to select missing proteins for further characterization.

	endogenous peroxidase was blocked with Inhibitor-D, 3% H2O2, (Ventana) for 8 min
	at 37 °C. After rinsing in TBS, slides were incubated at 37 °C for 60 min with
	polyclonal rabbit antibodies specific for the selected missing proteins (Atlas
	Antibodies) diluted in TBS containing 0.2% Tween -20 (v/v) and 3% BSA (TBST-
	-BSA). The antibody dilutions used are listed in Supplementary Table 6. Non-immune
	rabbit serum (1:1000) was used as a negative control. After several washes in TBS,
	sections were incubated for 16 min with a biotinylated goat anti-rabbit antibody
	(Roche) at a final dilution of 1:500 in TBST--BSA. Signal was enhanced using the
	Ventana DABMap Kit or Ventana OMNIMap kit. Sections were then counterstained
	for 16 min with hematoxylin (commercial solution, Microm), and for 4 min with bluing
	reagent (commercial solution, Microm) before rinsing with milliQ water. After removal
	from the instrument, slides were manually dehydrated and mounted in Eukitt
	(Labnord, Villeneuve d'Ascq, France). Finally, immunohistology images were
	obtained using NDP.Scan acquisition software (v2.5, Hamamatsu) and visualized
	For each protein identified by MS, the tissue expression profile based on RNA with NDP.View2 software (Hamamatsu). Representative images are shown.
	sequencing analysis was retrieved from the Human Protein Atlas portal (version 14)
	(www.proteinatlas.org/). The evolutionary conservation profile was determined by a
	BLAST analysis using UniProtKB "Reference Proteomes" as target. In addition,
	homologs were systematically searched for in a number of ciliated organisms from
	distant groups including Choanoflagellida (Salpingoeca, Monosiga), Chlorophyta
	(Micromonas, Volvox, Chlamydomonas), Ciliophora (Paramecium, Oxytricha,
	Stylonychia, Tetrahymena, Ichthyophthirius), Trypanosomatidae (Trypanosoma,
	Phytomonas, Leishmania, Angomonas, Leptomonas), Cryptophyta (Guillardia),
	Naegleria gruberi and Flagellated protozoan (Bodo saltans). For each protein and all

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	guidelines, version 2.0.1 (http://www.thehpp.org/guidelines/, guideline #9) (see
	Materials and methods) . Supplementary Table 1 lists PSM-, peptide-, and protein-
	level FDR values along with the total number of true positives and false positives at
	each level for the five sperm sample preparation methods (individual results files for
	each fraction and after combination). After MS/MS data processing and filtering, a
	total of 4727 distinct protein groups passed the 1% PSM-, peptide-and protein-level
	criteria. Detailed information on the proteins identified from the five MS datasets are
	reported in Supplementary Table

Table 2 :

 2 List of missing proteins (PE2-4) identified in the five MS/MS datasets. Accession numbers and number of transmembrane helices (No. TMH) were retrieved from UniprotKB; Gene names and chromosome location are as referenced in neXtProt.

			Journal of Proteome Research
	Entry	Gene names	no. of unique peptide	chromosome	neXtProt PE level	no. of TMH
	A0AVI2	FER1L5	1	2q11.2	Evidence at transcript level	1
	A1A4V9	CCDC189 C16orf93	10	16p11.2	Evidence at transcript level	0
	A1L453	PRSS38 MPN2	2	1q42.13	Evidence at transcript level	0
	A2RUU4	CLPSL1 C6orf127	2	6p21.31	Evidence at transcript level	0
	A4D1F6	LRRD1	13	7q21.2	Evidence at transcript level	0
	A4D256	CDC14C CDC14B2	1	7p12.3	Evidence at transcript level	1
	A4QMS7	C5orf49	5	5p15.31	Evidence at transcript level	0
	A5D8W1	CFAP69 C7orf63	25	7q21.13	Evidence at transcript level	0
	A5PLK6	RGSL1 RGSL RGSL2	5	1q25.3	Evidence at transcript level	1
	A6H8Z2	FAM221B C9orf128	7	9p13.3	Evidence at transcript level	0
	A6NCJ1	C19orf71	10	19p13.3	Predicted	0
	A6NCL2	LRCOL1	2	12q24.33	Evidence at transcript level	0
	A6NCM1	IQCA1L IQCA1P1	2	7q36.1	Inferred from homology	0
	A6NCN8		3	12p13.33	Predicted	0
	A6NE01	FAM186A	6	12q13.12	Evidence at transcript level	0
	A6NE52	WDR97 KIAA1875	11	8q24.3	Evidence at transcript level	0
	A6NEN9	CXorf65	4	Xq13.1	Predicted	0
	A6NF34	ANTXRL	8	10q11.22	Inferred from homology	1
	A6NFU0	FAM187A	4	17q21.31	Inferred from homology	1
	A6NFZ4	FAM24A	4	10q26.13	Inferred from homology	0
	A6NGB0	TMEM191C	1	22q11.21	Inferred from homology	1
	A6NGY3	C5orf52	2	5q33.3	Evidence at transcript level	0

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Acknowledgements:

This work was partially funded through the French National Agency for Research (ANR) (grant ANR-10-INBS-08; ProFI project, "Infrastructures Nationales en Biologie et Santé"; "Investissements d'Avenir" call). This work was also supported by grants from Biogenouest and Conseil Régional de Bretagne awarded to CP. We are grateful to Monique Zahn and Maighread Gallagher-Gambarelli for suggestions on language usage. We thank Marine Seffals for technical support with immunohistochemistry experiments on the H2P2 core facility (Université de Rennes 1, US18, UMS3480 Biosit, Biogenouest, Rennes, France). We particularly thank Blandine Guével, Véronique Dupierris, Mélanie Lagarrigue, Jean-Philippe Menetrey, Mathieu Schaeffer, Régis Lavigne and Christine Kervarrec for their technical assistance.

The mass spectrometry data used for proteomics have been deposited with the ProteomeXchange Consortium via the PRIDE partner repository under dataset identifier PXD003947

Data availability

All MS proteomics data, including reference files (readme, search database, .dat files) form a complete submission with the ProteomeXchange Consortium [START_REF] Vizcaino | Journal of Proteome Research ProteomeXchange provides globally coordinated proteomics data submission and dissemination[END_REF] . Data were submitted via the PRIDE partner repository under dataset identifiers PXD003947 and 10.6019/PXD003947.

Additional MS-based validation (MS/MS analysis of synthetic peptides, comparison of reference/endogenous fragmentation spectra and LC-PRM analysis).

Synthetic heavy labeled peptides were purchased (crude PEPotec™, Thermo Fisher Scientific) for 36 "one-hit wonder" candidates selected based on visual inspection of PSMs. The 36 peptides initially identified were synthesized along with two additional

Supporting Information:

This material is available free of charge via http://pubs.acs.org/. Supplementary Material: 1. Shotgun LC-MS/MS analyses. 2. LC-MS/MS analysis of labeled synthetic peptides and comparison of fragmentation spectra. 3. How targeted LC-PRM assays were developed (details).

Supplementary Figure 1: MS/MS spectra for the 36 endogenous peptides and their synthetic reference counterparts combined with LC-PRM results for the 36 peptides, additional predicted proteotypic peptides and their labeled synthetic counterparts. MS validation results are presented for all 36 selected one-hit wonder proteins in the same way as data for protein Q9H693 are presented in Figure 4 of the manuscript. A.

Missing protein sequence with the unique identified peptide highlighted in blue; MS/MS spectrum of the endogenous peptide correlated with the MS/MS spectrum of its labeled synthetic counterpart. B. MS2 traces extracted from LC-PRM data with dotp calculated for the endogenous and labeled peptides and rdotp calculated for light/heavy correlation. C. MS2 traces extracted from LC-PRM data targeting additional predicted peptides. The dotp for the endogenous and labeled peptide(s) and the rdotp for light/heavy correlation were calculated.

Supplementary Table 1: PSM-, peptide-, and protein-level FDR values along with the total number of expected true-and false-positives at each level for each sperm proteome dataset and the combined dataset (tab 1: total cell lysate followed by a 1D SDS-PAGE separation of proteins (23 gel slices), tab 2: total cell lysate, in-gel digestion of proteins, and total peptide analysis by nanoLC with long gradient runs, Journal of Proteome Research tab 3: total cell lysate, in-gel digestion of proteins, and peptide fractionation by high-pH reversed-phase (HpH-RP) chromatography; tabs 4 and 5: fractionation of proteins into Triton X-100-soluble and -insoluble fractions, followed by 1D SDS-PAGE separation of proteins (20 gel slices per fraction); tab 6: combined dataset corresponding to the combination of results for the five proteome datasets).

Supplementary Table 2:

List of proteins identified and validated with a protein-level 1% FDR for each fraction (detailed information): tab 1: total cell lysate followed by a 1D SDS-PAGE separation of proteins (23 gel slices), tab 2: total cell lysate, in-gel digestion of proteins, and total peptide analysis by nanoLC with long gradient runs, tab 3: total cell lysate, in-gel digestion of proteins, and peptide fractionation by high-pH reversed-phase (HpH-RP) chromatography; tabs 4 and 5: fractionation of proteins into Triton X-100-soluble and -insoluble fractions, followed by 1D SDS-PAGE separation of proteins (20 gel slices per fraction). Coverage (protein coverage in %) and number of unique peptides mapping to missing proteins, proteins seen (yes)/not seen (not) in each of the five MS/MS datasets acquired in this study are reported. The expression annotations of the Human testis gene expression program (TGEP; 5 ) were also reported when available

Supplementary Table 3:

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(SET: proteins produced by genes specifically expressed in the testis; PET: proteins produced by genes preferentially expressed in the testis; IE: proteins produced by genes with intermediate expression in the testis; UE: proteins produced by genes with ubiquitous expression in the testis). The « testis-enriched gene » status in the Human Protein Atlas version 15 is also provided.

Supplementary Table 4:

Tab1: List of one-hit wonder missing proteins identified and selected for further spectral comparison (MS/MS) and PRM validation. Tab2: Extended list of 100 peptides selected for validation of the 36 one hit wonders and synthesized as crude labeled peptides.

Supplementary Table 5:

List of 111 PE2-5 proteins for which complete data mining was performed, showing the rationale for their prioritization for subsequent antibodybased studies. Entry accession numbers (column A) and gene names (column B) were retrieved from UniProtKB, transcript abundance was retrieved from HPA (column C), phylogenetic profiles in ciliated organisms were determined by Blast analysis on UniProtKB "Reference proteomes" (column D), knock-out mice phenotypes were retrieved from MGI (column E), associated publications that were not annotated in neXtProt were searched in PubMed (column F), and immunohistochemistry data was retrieved from HPA (column G). For all these criteria, a four-color grading system was adopted. Dark green and green cells were retained as positive criteria, red cells as negative ones. Based on these criteria, a score of relevance (high/medium/low) for the implication of the proteins in spermatogenesis has been assigned (column H). The existence of a suitable

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antibody in HPA is reported column I, and the list of proteins that were finally selected for IHC is provided in column J.

Supplementary


Jérôme Crépin

Thierry Bretheau

Daniel Caldemaison

Franck Ferrer

J Cre

 Pin{

Low cycle fatigue behaviour of β treated zirconium: partial irreversibility of twinning and consequences for damage

Keywords: Low cycle fatigue, Scanning electron microscopy (SEM), Alloys (zirconium), Mechanical properties (fatigue), Microstructure

INTRODUCTION

Zirconium is mainly used in the nuclear industry as a structural material. It is therefore necessary to guarantee the integrity and safety of the structures composed of this material under service conditions. For that purpose, the improvement and optimization of the mechanical behaviour of zirconium is required, especially in the ®eld of the fatigue of welded joints. In this research direction, our interest has focused on the reversibility of twinning mechanisms linked with prescribed cyclic strain at room temperature and on the consequences for damage and rupture. This study is based on the study of twinning mechanisms at dierent scales (from macroscopic to scanning electron microscopyÐ SEMÐscales) in relation with the overall mechanical response. Speci®cally, macroscopic tension/compression fatigue tests were performed while recording acoustic emissions to detect the inception of twinning, and in situ fatigue tests were performed inside the SEM chamber to observe the evolution of twins.

The paper is arranged as follows: Section 2 consists of a brief description of the studied material's microstructure and of its deformation mechanisms; Section 3 concerns the experimental techniques used. The experimental results are then presented in Section 4 followed by a closing discussion in Section 5.

MATERIAL

Grade 702 zirconium contains 0.5% (in weight) foreign elements listed in Table 1. This material has undergone an a 4 b 4 a cycle at a slow cooling rate 1158=s inducing a bainitic transformation from the high temperature b phase (body centred cubic) to the low temperature a phase (hexagonal close packed). The resulting WidmanstaÈ tten microstructure, presented in Fig. 1, consists of colonies 1500 mm grain size) of parallel laths with the same crystallographic orientation. The lath boundaries are ``walls'' constituted of precipitates of betagenic { To whom all correspondence should be addressed. elements (Fe, Cr, Ni) [1]. The alphagenic elements such as Sn and O remain in the solid solution (a phase) [2].

The texture is assumed to be isotropic because of the b treatment undergone by the material.

Three major deformation mechanisms are activated under tensile conditions at room temperature: 10 10h1 210i prismatic slip, 10 12h10 1 1 i and 11 21h11 2 6 i twinning depending on the colony crystallographic orientation with respect to the tensile axis. Prismatic slip is either homogeneous or concentrated in coarse slip lines along the lath boundaries [2]. This last mechanism is most frequently observed.

EXPERIMENTAL TECHNIQUES

The samples are cut along the former rolling direction, and their dimensions for macroscopic and ``in situ'' fatigue tests are: 8 mm diameter, 20 mm gauge length and 4.5 mm diameter, 8 mm gauge length, respectively. Low cycle fatigue tests under total strain control are performed at room temperature and at a constant nominal strain rate of 10 À3 /s in symmetrical conditions R e e min =e max À1); the total strain amplitude ranges between 0.20 and 0.80%. The dierent tests start either in tension (type A) (Fig. 2) or in compression (type B). In the following, the ®rst half of the cycle is called ``part 1'' and the second ``part 2''. The dierent variables x s, e , ... associated with part 1 and part 2 are de®ned as x part1 , x part2 , respectively.

To locate, identify and follow the occurrence and evolution of the various deformation mechanisms, a Raith tension/compression loading stage located inside the scanning electron microscope is used with a5Â 10 À4 =s strain rate [START_REF] Doquet | Proceedings of the Fourth International Conference on Biaxial and Multiaxial Fatigue[END_REF] under stress control conditions. These tests start in tension for a ratio R s s min =s max À1 and are performed at room temperature under vacuum 110 À4 Pa).

All the samples are mechanically and electrolytically polished [1]. Gold microgrids (1 mm 2 ) with 5 mm pitch deposited on the sample surface give access to qualitative and quantitative characteriz- ation of the local deformation mechanisms [START_REF] Allais | [END_REF]. The local crystallographic orientation of 1 mm 3 crystal domains is determined by electron backscattering diraction (EBSD). This technique makes it possible to identify the eective active slip and twinning systems as well as to estimate their Schmid factor.

A twin formation mechanism corresponds to an instantaneous reorientation of a certain crystal volume by homogeneous shear; the potential twins in zirconium are listed in Table 2. The shear amplitude is determined by the crystallographic relationship between the two undistorted planes (K 1 and K 2 ) and takes dierent values for dierent twinning systems. The acoustic signal produced by a twin is related to the shear amplitude permitting the classi-®cation of the twinning mechanisms. As mentioned in Ref. [START_REF] Roget | Technique de l'inge nieur[END_REF], dierent global variables can characterize an acoustic emission such as the maximum amplitude (V c ), the average amplitude value (V m ), the eciency value or r.m.s. value (V r.m.s. ) or the energy (E ). The de®nitions of these variables are as follows:

V m t 1 T tT t
Vt dt with T 1±10 ms V 2 t dt:

V
The emission shape factor DV c =DV r:m:s: , de®ned by the ratio of the maximum amplitude to the eciency value of the signal is a pertinent signature of a twinning system. Detection is performed with a piezo-electric sensor with a 20 kHz ®lter and a sampling period of 100 ns during type A and B tests on an Instron machine at a nominal 10 À4 /s strain rate.

4. RESULTS

Macroscopic characterization

Most of the fatigue tests start in tension (type A) (Fig. 2). The evolution with strain of the macroscopic and microscopic characteristics [isotropic hardening (R ) and kinematical hardening (X ), friction stress (t f ) and back stress (t b )] is determined in accordance with the methods of Cottrell [START_REF] Cottrell | Dislocations and Plastic Flow in Crystals[END_REF], Kuhlmann-Wilsdorf and Laird [START_REF] Kuhlmann-Wilsdorf | [END_REF] and Lemaitre and Chaboche [START_REF] Lemaitre | MeÂcanique des mateÂriaux solides[END_REF]. The de®nitions of the dierent variables introduced above, based on Ref. [START_REF] Kuhlmann-Wilsdorf | [END_REF] and for symmetrical loading conditions are:

t f t max t t t s =2 t b t max À t t t s =2 with t t t s t s part1 À t s part2 =2 and t max s max À s min =2 X t b R t f À k with k the initial yield stress:
Note that t s part1 and t s part2 are determined on the basis of a deviation from linearity with a coecient d 10 À4 (Fig. 2).

It appears that hardening is mainly kinematical, since the isotropic contribution is negligible (Fig. 3). This hardening remains constant with the number of cycles (N ) for a given strain amplitude (De/2), but increases with amplitude. By contrast, it is found that friction stress t f remains stable with N Fig. 2. Fatigue test parameters (type A test). d is a parameter characteristic of the deviation from linearity of the unloading curve; it allows an unambiguous value for the yield stress t s to be given. The frictional stress t f and the back stress t b are derived from t s [START_REF] Kuhlmann-Wilsdorf | [END_REF]. For type B test, parts 1 and 2 are reversed. and is independent of the strain amplitude. Moreover, Fig. 4 allows the symmetry of the stress response for a symmetric applied strain to be checked De=2 0:8%). The ®rst cycle shows, either for type A or B test, that s part2 is always higher than s part1 with a stress ratio of 0.95; this ratio increases for type A tests but, after a maximum for the second cycle, decreases for type B tests. The cycles tend to symmetrize (ratio of 1) after the ®rst 10 cycles. This means that besides the ®rst cycle the stress response of the material is exactly the same for type A and B tests s compression > s tension ). Consequently, it is concluded that there is no memory eect of the test onset s part2 > s part1 ). In Fig. 5, Q represents a typical plot of the amplitude (V c ) vs the eciency value (V r.m.s. )o f acoustic emissions for a grade 702 a zirconium, with small grain size (15 mm). Since only Type I twins are activated, the acoustic emissions recorded during the test and characterized by their shape factor (DV c /DV r.m.s. ) (Table 3) can be associated with the activation of this mechanism and will be called ``Type I''. In the same ®gure, r represents the same plot for b treated zirconium; in this case, two dierent types of acoustic emissions (two dierent shape factors) are recorded. Each signal can be associated with a unique twinning mechanism as mentioned in Table 3. Consequently, the acoustic signals associated with the 11 21h11 2 6 i twinning system are called ``Type II'' in the following. 4.2.2. Tension/compression fatigue tests performed on b treated zirconium. For De=2 0:80%, the shape factor for each event, recorded in tension and in compression, is reported in Fig. 6 for a type B test. It shows that there are two dierent types of events corresponding to Type I and II emissions and occurring either in tension or in compression. Moreover, the number of events per cycle does not remain constant with the number of cycles (N ) (Fig. 7). First it increases during the ®rst cycles, and then decreases continuously. Second, there are more events in tension than in compression during the ®rst cycles, but the values are equal for larger N.

The critical stress for the inception of the ®rst acoustic emission of each cycle, tends to increase with N [Fig. 7(b)]. This is in agreement with Fig. 7(a) since the number of events decreases with N. Finally, it can be noticed that only for the ®rst cycle, the inception stress for the ®rst acoustic signal in compression is lower than in tension; after that, the eect is reversed. This property is consistent with Fig. 4, where s tension > s compression for the ®rst cycle of type B test. That inception stress, equal in compression and tension, is in agreement with the fact that the number of events is the same in tension and compression after about 10 cycles, when the cycles symmetrize.

For De=2 < 0:30%, only 10 12h10 1 1 i twins are observed in tension and in compression, and only Type I acoustic signals are recorded. This is because under these conditions, the critical stress for the inception of type II twinning is never reached [2].

Microscopic characterization.

A type A test performed inside the SEM allows the observation of the sample surface under loading conditions (for s 260 and À260 MPa from the ®rst cycle to rupture (377 cycles). The corresponding total strain amplitude De/2 is approximately 1%.

The deformation mechanisms are identical to those in monotonic tension [2]: 10 10h1 210i pris-matic slip, 10 12h10 1 1 i and 11 21h11 2 6 i twinning. Their activations depend on the colony orientation. Moreover, if a fatigue test is stopped after a certain number of complete cycles, only a few twins are observable. In contrast, if the test is stopped after half a cycle, for instance if only the tension part is completed, many twins are observed as for the tests under monotonic conditions.

Twinning is partially reversible (Fig. 8). Indeed, twins appear during part 1 of the cycle (tension), tend to disappear during part 2 (compression) and reappear during the next cycle. The consequence of such behaviour is illustrated in Fig. 9: damage is located at the twin boundaries and can be observed after only 25 cycles [Fig. 9(a)]. Its evolution leads to a crack located on the twin boundaries [Fig. 9(b)]. The undeformed microgrids, in the vicinity of the cracks, indicate that this damage mechanism remains highly localized. However, it is possible to observe some extrusions in the vicinity of the twins [Figs 9(a) and(b)].

Moreover, in the case of fatigue tests performed at De=2 < 0:30%, the fracture surface presents some areas (several hundred micrometres wide, typically the grain size) without any dimple or fatigue striation (Fig. 10). They can be quali®ed as ``quasi-cleavage'' areas; they correspond to a crack propagation along a twin boundary. This phenomenon is also observed for De=2 > 0:50%, but in this case, the contribution of twinning to the global deformation becomes less important than the contribution of slip. Prismatic slip localized along the lath boundaries is dominant, as for monotonic tensile tests. A previous study [2] has shown that this localized prismatic slip results in the localization of damage and crack propagation along the lath boundaries (Fig. 11).

DISCUSSION

Macroscopic mechanical behaviour

For low cycle fatigue tests performed at room temperature and in the range of 10 À4 < _ e < 10 À3 =s, there is no isotropic hardening in large b treated zirconium grains (Fig. 3). This is in agreement with the results obtained by Robinet [START_REF] Robinet | TheÁ se de doctorat[END_REF], on small grain zircaloy-4 110 mm), which does not allow the activation of twinning mechanisms.

Therefore it can be concluded that there is no sig-ni®cant isotropic hardening in zirconium for all grain sizes. This result con®rms what is generally assumed for prismatic glide and justi®es neglecting the contribution of twinning to the macroscopic isotropic hardening.

Microscopic mechanical behaviour

The kinematical part is the major component of the hardening behaviour (Fig. 3) and is equivalent at the microscopic scale to the internal back stress t b , that can be interpreted as being the origin of the Bauschinger eect often observed in zirconium and zirconium alloys [START_REF] Christodoulou | [END_REF]13]. Indeed, this eect is linked to the existence of long range interactions generally explained either by a ``composite eect'', due to the interactions between dislocation walls of high dislocation density separated by soft areas of low dislocation density [14], or by a ``second phase eect'' resulting from the interactions between sec- ond phase particles and matrix. But, in the case of anisotropic materials with very few deformation systems like Zr, another explanation can be proposed: the back stress t b could be due to incompatibilities between the grains of the polycrystal as mentioned by Hutchinson [15]. t b is directly proportional to the grain size and linked to the crystallographic texture (disorientation between the grains). This last assumption was already suggested by MacEwen et al. [13] in the case of zirconium alloys.

In our study, transmission electron microscopy (TEM) analysis performed on samples deformed at De=2 0:80% after 700 cycles allows the conclusion that there is neither interaction between the dislocations and the precipitate networks (localized on the lath boundaries) nor occurrence of dislocation cells. Consequently, the kinematical hardening is probably due to the polycrystal eect. Furthermore, the macroscopic behaviour shows that there is no evolution of the hardening eect for cyclic loading. From this global characterization, it can also be noted (Fig. 3) that the friction stress t f , taken as equivalent to the critical stress for dislocation displacement, is consistent with the critical resolved shear stresses of the deformation mechanisms 1100 MPa already estimated in Ref. [2] at the SEM scale. This value seems to remain stable with the cycles; it implies once more that there is no sig-ni®cant hardening eect.

Nevertheless, even if any major intrinsic hardening mechanism is detected, it seems that at the onset of the tests, the stress response is not totally symmetric (s part1 /s part2 ) for a symmetric De/2 imposed strain. This eect disappears after a few cycles. A tentative explanation is given in the following.

Deformation mechanisms characterization

From Figs 5 and6, it is clear that acoustic emission reveals the existence of 10 12h10 1 1 i and 11 21h11 2 6 i twinning that are both active in tension and compression. The microscopic characterization and the observation of only a few twins after symmetrical loading allow the conclusion that the emissions correspond to the appearance and disappearance of twins, but not to the activation of two dierent types of mechanisms (tensile twins and compression twins as those reported in Ref. [START_REF] Reed-Hill | Deformation Twinning[END_REF]). Nevertheless, it is easier to activate twinning in tension than in compression (Fig. 7). Indeed, more events are detected in tension than in compression, and the critical stress for twin inception is lower in tension than in compression (except for the ®rst cycle starting in compressionÐtype B test). This conclusion does not contradict the ®nding that the stress ratio (s part1 /s part2 ), between the ®rst cycle of type A and B tests (Fig. 4), is exactly the same. So, the non-symmetric response under symmetric loading can be explained by the non-symmetric behaviour of twins (Fig. 8). Indeed, twins appear in the ®rst half-cycle and then disappear during the second half-cycle; their contribution to the macroscopic strain is less important for the reverse part of the loading so that an increment of plastic glide is needed to reach the symmetric strain amplitude. The consequence of such an increment is that s max part2 > s max part1 : After the ®rst cycle, there is no dier- Finally, the increase in the inception stress [Fig. 7(b)] and the decrease in the number of twins [Fig. 7(a)] with N mean that there is a hardening mechanism for twinning, even if the macroscopic behaviour seems to be insensitive to twinning (Fig. 3). Moreover, a preliminary study at the TEM scale reveals the occurrence of two parallel ``walls'' of dislocations (Fig. 12), corresponding to the location of former twin boundaries close to the h11 20i direction. This area (Fig. 12) has been observed under dierent diraction conditions: g 1 1 00, g 01 10, g 10 10: The walls are never out of contrast; so the wall dislocations are neither hai, nor hci, nor hc ai: Song and Gray III [16±18] have already reported the existence of such dislocation structures with a new type of stacking fault observed within deformation twins in titanium and zirconium. In their studies, the stacking faults are associated with the shuing of atoms due to twinning. In our study, more tests are required to con®rm the existence of this type of dislocations and stacking faults and eventually to understand their consequences on hardening and damage mechanisms.

Damage nucleation

Twinning mechanisms cannot be disregarded from a damage description of the low cycle fatigue. Damage appears early on at twin boundaries (Fig. 9). This phenomenon is particularly important if the microstructure consists of very large grains (500 mm) and if the strain amplitude is very small. Indeed, in that instance, the contribution of twins to deformation cannot be disregarded compared to slip. Moreover, if the material is strongly textured (rolling texture) and if the loading direction is favourable for twin activation (e.g. parallel to the transverse direction for a rolling texture), the twin contribution to damage is then increased. Finally, since twins are well known to be observed more frequently near free surfaces than in the bulk, then damage due to twins is more critical for thin structures (tubes) than for thick ones in low cycle fatigue.

CONCLUSION

The experimentation at dierent scales has revealed the role of twinning under fatigue conditions at room temperature. Twinning is a deformation mechanism admitting partial reversibility like slip. However, it seems that the irreversible part of twinning leads to the occurrence of damage localized along twin boundaries after only a few cycles for De=2 < 1%: The consequences of this reversibil- De=2 0:8%: ity are more important for low applied stress. In this case, twinning cannot be disregarded compared to slip as for monotonic tensile tests since damage on twins induces a very detrimental ``quasi-cleavage'' mechanism.

Fig. 1 .

 1 Fig. 1. Microstructure of b treated 702 zirconium after electrolytic polishing (polarized light microscopy). The white ellipse delimits a lath and the black one delimits a colony.

Fig. 3 .

 3 Fig. 3. Evolution of the isotropic hardening, kinematical hardening, back stress and the friction stress vs N for dierent total strain amplitudes: Q, De=2 0:20%; w, De=2 0:80%: ÐÐ, kinematical hardening (back stress t b ); ± ± ±, friction stress t f ;------,isotropic hardening.

Fig. 4 .

 4 Fig. 4. Comparison of the stress response symmetry. Total strain amplitude De=2 0:80%:

Fig. 5 .

 5 Fig. 5. Amplitude (V c ) vs the eciency value (V r.m.s. ) of the acoustic emission during monotonic tensile tests: Q, grade 702 a zirconium with small grain size [9]; r, b treated grade 702 a zirconium [2].

Fig. 6 .

 6 Fig. 6. Acoustic emission in fatigue: shape factors for twinning systems vs N De=2 0:80%).

Fig. 7 .

 7 Fig. 7. Acoustic emission analysis for type B uniaxial fatigue test De=2 0:80%). (a) Number of events per cycle vs N. (b) Average critical stress for the inception of the ®rst two events vs N.

Fig. 8 .

 8 Fig. 8. The reversal of two 11 21h11 2 6 i twins during type A fatigue test. The lath boundaries are vertical; the dots are part of a microgrid (vertical stress axis): (a) s 260 MPa, two twins have nucleated; (b) s À260 MPa, the twin traces almost disappear.

Fig. 9 .

 9 Fig. 9. Damage and extrusions. (a) Same grain as in Fig. 8: damage and extrusions located at the horizontal twin boundaries N 25). (b) Crack propagation along the damaged twin boundaries N 377; broken specimen).

Fig. 10 .

 10 Fig. 10. A quasi-cleavage zone in the fracture surface.

Fig. 11 .

 11 Fig. 11. Cracks propagating along localized prismatic slip bands as under monotonic tensile test[2].De=2 0:8%:

Table 1 .

 1 Chemical composition of grade 702 zirconium

	Elements	C	H	O	N Cr Fe Ni	Sn
	Analysis (wt p.p.m.) 58 4±7 1400 33 240 760 50 2280

Table 2

 2

	Z 2	Shear magnitude

. Twinning elements in pure zirconium

[START_REF] Reed-Hill | Deformation Twinning[END_REF]

First undistorted plane, K 1 Twinning shear direction, Z 1 Second undistorted plane, K 2

Table 3 .

 3 Shape factors DV c /DV r.m.s. of twinning systems for tensile tests performed at room temperature

		Grain size (mm)	f10 12gh10	1	1 if 11	21gh11	2	6 i
	Grade 702 b treated zirconium	500	5.05 220%)	1.07 24%)
	Grade 702 a treated zirconium	15	3.50 21%)±

AcknowledgementsÐThe authors wish to thank the CEA/ CEREM for supplying both materials and ®nancial support, M. Terrien (LMS) for his assistance with the acoustic emission and J. Tchir (LMS) for his contribution to the study of the macroscopic fatigue behaviour.


Mohand Lounes Bentaha

Olga Battaïa
email: battaia@emse.fr

Alexandre Dolgui
email: dolgui@emse.fr

A Stochastic Formulation of the Disassembly Line Balancing Problem

Keywords: Sustainable Development, Disassembly Line Design, Stochastic Programming, L-shaped Method

The disassembly line balancing problem is studied under uncertainty. Disassembly task times are assumed random variables with known probability distributions. An AND/OR graph is used to model the precedence relations among tasks. The goal is to assign the disassembly tasks to workstations while respecting precedence and cycle time constraints. The objective is to minimize the total line cost including the incompletion cost arising from task incompletion within the cycle time. A stochastic linear mixed integer programming formulation is developed.

Introduction

The growing amount of postconsumer products poses challenges for business and society at large [START_REF] Güngör | Issues in Environmentally Conscious Manufacturing and Product Recovery: A Survey[END_REF]. To decrease the amount of waste to be sent to landfills, more and more manufacturers turn to end-of-life processing of products [START_REF] Güngör | A Solution Approach to the Disassembly Line Balancing Problem in the Presence of Task Failures[END_REF]. The selective separation of desired parts and materials, executed by disassembly, is a mandatory step before recycling or remanufacturing [START_REF] Güngör | Disassembly Line in Product Recovery[END_REF]. As a consequence, disassembly systems tend to play an important role in industry. Since their design results in complex optimization problems, including disassembly planning, balancing and sequencing, efficient mathematical tools are needed in order to improve their performances and their cost effectiveness. Such tools must take into account the high degree of uncertainty in the structure and the quality of the products to be disassembled because of varying conditions affecting them before they arrive in a disassembly system. This paper deals with the uncertainty related to the disassembly task times and proposes a new mathematical formulation for the stochastic disassembly line balancing problem (DLBP) as well as an exact method to solve it efficiently.

Problem Formulation

The Disassembly Line Balancing Problem considered here aims to assign a given set of disassembly tasks I 1,2, … , to an ordered sequence of workstations ,~ , , 0, , [START_REF] Dolgui | Supply Chain Engineering: Useful Methods and Techniques[END_REF], [START_REF] Sarin | A Methodology for Solving Single-Model, Stochastic Assembly Line Balancing Problem[END_REF][START_REF] Silverman | A Cost-based Methodology for Stochastic Line Balancing with Intermittent Line Stoppages[END_REF]. The task times of set I are modeled by random vector , ,…, varying over a set Ξ in given probability space Ξ, , . Random variables , are assumed to be mutually independent. Let , . The precedence relations among tasks are given by an AND/OR Graph (AOG), [START_REF] Altekin | Profit-Oriented Disassembly-Line Balancing[END_REF], [START_REF] Koc | Two Exact Formulations for Disassembly Line Balancing Problems with Task Precedence Diagram Construction Using an AND/OR Graph[END_REF], see Fig. 1. Each subassembly of the product to be disassembled is represented by an auxiliary node A , in the AOG. Each disassembly task gives a basic node B , Two types of arcs define the precedence relations between the subassemblies and the disassembly tasks. AND-type arcs (in bold) dictate the normal precedence relation. OR-type arcs (remaining arcs) permit the selection of any of the successors. A dummy task is introduced into the precedence graph as a sink node, see the figure below.

Fig. 1. AND/OR precedence graph

The following notations have to be introduced.

∑∑ S 1 (2) ∑ 1, (1)
∑∑ S ∑∑ P , \ 0 (3)
∑ S ∑∑ P , \ 0 , (4)
∑ 1 (6) ∑ ∑ , (5)
∑ C , (7)
, (8) 0
, 0,1 , ,

, (10) 0
The objective function includes fixed and recourse costs, where stands for the expected value with the respect to the distribution of the random vector :

∑ ∑ d (12)
Note that the integral [START_REF] Sarin | A Methodology for Solving Single-Model, Stochastic Assembly Line Balancing Problem[END_REF] makes the model nonlinear one. Constraints (1) ensure the value of to be when dummy task is assigned to station . Constraint (2) imposes the selection of only one disassembly task (ORsuccessor) to begin the disassembly process. Constraint set [START_REF] Birge | Stochastic Programming Computation and Applications[END_REF] indicates that a task is to be assigned to at most one workstation. Constraints (4) and ( 5) define OR-and AND-precedence relations, respectively. Constraint [START_REF] Güngör | A Solution Approach to the Disassembly Line Balancing Problem in the Presence of Task Failures[END_REF] imposes the assignment of the dummy task to one station. Constraints (7) ensure the precedence relations for dummy task . The constraints [START_REF] Güngör | Disassembly Line in Product Recovery[END_REF] force the respect of the cycle time limitations. Sets ( 9)-( 11) represent the trivial constraints. Let { | constraints ( 1)-( 7), ( 9)-( 10) are satisfied} and 1,2, … , L ,L . If has a finite discrete distribution , , , 0, ( is the realization probability of of ), then the model presented is an ordinary linear program with a so-called dual decomposition structure.

Deterministic Equivalent (I')

min L s.t. C , , , 0, ,
Depending on the number of realizations of , i. e. L , this linear mixed integer program, may become very large in scale, but its particular block structure can be exploited by specially designed algorithms such as the L-shaped method, [START_REF] Ahmed | The Sample Average Approximation Method for Stochastic Programs with Integer Recourse[END_REF], [START_REF] Birge | Stochastic Programming Computation and Applications[END_REF][START_REF] Birge | L-shaped Method for Two-stage Problems of Stochastic Convex Programming[END_REF], [START_REF] Kall | Stochastic Programming[END_REF], [START_REF] Ntaimo | Fenchel Decomposition for Stochastic Mixed-Integer Programming[END_REF] which will be developed in the next section.

Solution Method

The L-shaped Method The main idea of the L-shaped method is to approximate the nonlinear term in the objective function of the two-stage stochastic problems [START_REF] Birge | Stochastic Programming Computation and Applications[END_REF]. Assume a finite realizations set Ξ of the stochastic vector such as |Ξ| L. The L-shaped method for the DLBP can be written as follows.

L-shaped Algorithm

Step 0. Set 0.

Step 1. Set 1 . Solve the following LP: min

s.t. , 1, … , (13)
, 1, … , (14)
binary, 0

Let , be an optimal solution.

Step 2. For 1 ,…,L solve the following LP: min

…, 1 , until for some the optimal value 0 . In this case, let be the associated simplex multipliers, define and in order to generate a constraint called a feasibility cut of type [START_REF] Silverman | A Cost-based Methodology for Stochastic Line Balancing with Intermittent Line Stoppages[END_REF]. Set 1, add constraint type [START_REF] Silverman | A Cost-based Methodology for Stochastic Line Balancing with Intermittent Line Stoppages[END_REF] and return to Step 1. If for all , 0 , go to Step 3.

Step 3. For 1,…,L , solve the LP: min q 0 Let be the simplex multipliers associated with the optimal solution of problem above and define and .

Let

. If , stop; is an optimal solution. Else, generate a constraint called optimality cut of type (14), set 1, add constraint type (14) and return to Step 1. This method approximates ∑ ∑ d using an outer linearization.

Two types of constraints are sequentially added:

• feasibility cuts (13) determining | ∑ ∞ ;

• optimality cuts (14), which are linear approximations to ∑ .

Example

The method presented has been applied to the compass example illustrated in Fig. 2.

It is made of seven components: (1) wheel, (2) left leg, (3) right leg, (4) left fixation screw, (5) lead, (6) tip and ( 7) right fixation screw. The AOG for this example is shown in Fig. 1. The input data for the DLBP is presented in Table 1. The L-shaped method was implemented in Microsoft Visual C++ 2008. ILOG CPLEX 12.4 was used to solve the model on a PC with Pentium(R) Dual-Core CPU 2.30 GHz and 3Go RAM. The optimal solution contains 2 workstations. Task 5 is assigned to the 1 st workstation and tasks {8,10} to the second one. The total line cost is 5.342 when the recourse cost is 0.242. The resolution time was 58 s. The overall idle time of the disassembly line, if mean time of each task selected is considered, is 0.10.

Fig. 2. The Compass example

Conclusion and Perspectives

The disassembly line balancing problem was formulated under uncertainty. A twostage stochastic linear mixed integer program with fixed recourse was developed to solve it. The formulation presented can be easily adapted for the stochastic assembly line balancing problem as well. Then, the model can be considered for the disassembly task times given by, for example, triangular distribution. Further research work should be done in order to refine the proposed mathematical model and solution method in order to approach real-world problems where uncertainty does not only concern the disassembly task times but the quantity and the quality of the inputs and outputs of the disassembly line as well.

  The objective considered in this paper is to minimize the line cost including fixed workstation operating costs and recourse costs caused by exceeding . A recourse variable , , measures the amount of time exceeding if there is any. The following model is used for the problem presented.

		: Random task time of B ,	;
	P S	: Predecessors index set of A , : Successors index set of A ,	i.e. P , S	|B precedes A ; |A precedes B .
	Decision Variables
		1 if task from is assigned to workstation , 0 otherwise.
		1 if sink task is assigned to workstation , 0 otherwise.
		if	1,
		0 otherwise.
	Stochastic MIP Formulation (SMIP I)
		min
	s.t.
			,

Parameters 1,2, … , , disassembly tasks' index set, ; 1,2, … , , workstations' index set, ; 0,1, … , 1 , index set of the AND/OR precedence graph's auxiliary nodes, ; A : Auxiliary node of the AND/OR graph, ; B : Disassembly task, ; : The AND/OR graph's sink node, 0; : Fixed cost per unit time of operating the workstations; : cost per unit time of exceeding , ; : Cycle time, 0;

Table 1 .

 1 Input data

	Task			Subassembly Component
	1 2 3 4 5	0.21 0.21 0.50 0.21 0.50	0.05 0.05 0.10 0.05 0.10	1:5 1:3,6,7 2,4,5 1:3 2,4,5/3,6,7	6;7 4;5 1;3 4;5 1	|Ξ| 1024 1,2,3 0,1, … ,5 5 7,
	6 7	0.21 0.50	0.05 0.10	1:3 3,6,7	6;7 1;2	0.51
	8	0.21	0.05	--	2;4;5
	9	0.50	0.10	--	1;2;3
	10	0.21	0.05	--	3;6;7

Acknowledgement. This work was partially supported by Research Cluster on Operational Research (GDR RO) of the National Centre for Scientific Research, France.