Proteomic approach in the identification of fertility ... - Fertility and Sterility

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Nov 14, 2011 - Utleg AG, Yi EC, Xie T, Shannon P, White JT, Goodlett DR, et al. ... Hu Q, Noll RJ, Li H, Makarov A, Hardman M, Graham Cooks R. The Orbitrap:.
Proteomic approach in the identification of fertility pattern in seminal plasma of fertile men Domenico Milardi, M.D., Ph.D.,a Giuseppe Grande, M.D.,b Federica Vincenzoni, B.D.,c Irene Messana, B.D.,d Alfredo Pontecorvi, M.D.,b Laura De Marinis, M.D.,b Massimo Castagnola, B.D.,c and Riccardo Marana, M.D.a International Scientific Institute ‘‘Paolo VI,’’ b Department of Endocrinology, and c Institute of Biochemistry and Clinical Biochemistry,  Cattolica del S. Cuore, Rome; and d Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy Universita

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Objective: To identify a panel of common seminal proteins in human seminal plasma by fertile men that might be involved in successful reproduction. Design: Experimental study. Setting: University hospital. Patient(s): Five fertile men who conceived within 3 months before the start of the study. Intervention(s): None. Main Outcome Measure(s): Proteomic analysis performed by an Ultimate 3000 Nano/Micro-HPLC apparatus equipped with an FLM-3000-Flow manager module and coupled with an LTQ Orbitrap XL hybrid mass spectrometer; gene ontology analysis. Result(s): From 919 to 1,487 unique proteins were identified per individual subject sample. Among these proteins, 83 proteins were present in all samples, including some proteins that might be involved in male fertility, such as semenogelin I, semenogelin II, olfactory receptor 5R1, lactoferrin, hCAP18, spindling, and clusterin. The gene ontology annotation analysis provided further information in describing common pattern in male fertility. Conclusion(s): The identification of common seminal plasma proteome in fertile men could provide better insight into the physiology of male fertility and might identify novel markers of male infertility. (Fertil SterilÒ 2012;97:67–73. Ó2012 by American Society for Reproductive Medicine.) Key Words: Semen, seminal plasma, proteomics, male fertility

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linicians usually rely on semen analysis in evaluating male fertility as it represents the surrogate measure of male fecundity in clinical practice (1, 2). However, since World Health Organization (WHO) reference values were adopted, it has become evident that a basic semen analysis is insufficient to determine the fertility status of the male partner (3–7). Additional sperm function tests, such as swelling and/or eosine test, do not provide additional information in the assessment of the fertility status (8). Recently, further tests, such as reactive oxygen species determination and chromatin fragmentation, have been proposed for research purposes (9).

New molecular insights into sperm properties that make it capable of fertilizing the egg are recently emerging. Increased knowledge of sperm or seminal proteome might allow us to identify new molecular markers of male fertility (9). In postgenomic era, proteomic technology has rapidly developed as a powerful tool in the research of human physiology (10) to identify potential novel biomarkers for diagnosis, prognosis, and therapy (11, 12). Human seminal plasma contains many proteins that are important in the capacitation of the spermatozoa, in the modulation of the immune responses in the uterus, in the formation of the tubal sperm reservoir (13, 14),

Received June 24, 2011; revised September 23, 2011; accepted October 11, 2011; published online November 14, 2011. D.M. has nothing to disclose. G.G. has nothing to disclose. F.V. has nothing to disclose. I.M. has nothing to disclose. A.P. has nothing to disclose. L.D.M. has nothing to disclose. M.C. has nothing to disclose. R.M. has nothing to disclose. Reprint requests: Domenico Milardi, M.D., Ph.D., International Scientific Institute ‘‘Paolo VI,’’  Cattolica del S. Cuore, Largo F. Vito, 1, 00168 Rome, Italy (E-mail: [email protected]). Universita Fertility and Sterility® Vol. 97, No. 1, January 2012 0015-0282/$36.00 Copyright ©2012 American Society for Reproductive Medicine, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2011.10.013 VOL. 97 NO. 1 / JANUARY 2012

and finally in both the sperm–zona pellucida (ZP) interaction and in the sperm and egg fusion (15, 16). The complex content of seminal plasma allows the successful fertilization of the oocyte by the spermatozoa. Some of the seminal proteins are secreted by the testis, epididymis, and male accessory glands such as seminal vescicle, prostatic ampulla, and bulbourethral glands. Apart from these organ-specific proteins, human seminal plasma is rich in other proteins, whose origin and function are not completely clear. Before the proteomic era, twodimensional gel electrophoresis associated with immunostaining was used in combination with mass spectrometric (MS) identification of protein spots to visualize the whole seminal proteome (17). Subsequently, the separation by gel electrophoresis (two- and onedimensional) and identification by both matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography tandem mass spectrometry 67

ORIGINAL ARTICLE: ANDROLOGY (LC-MS/MS), permitted proteomic technology to identify more than 100 protein and peptide components of normal human seminal plasma (18). Utleg et al. (19) reported the identification of 139 proteins by human prostasomes, by using gas phase fractionation and microcapillary high-performance liquid chromatography (HPLC)-tandem mass spectrometry (mLC-MS/MS). Wang et al. (20) reported the identification by LC-MS/MS analysis of 45 up-regulated proteins and of 56 down-regulated proteins in the asthenospermic group compared with the control subjects. In 1999, Makarov invented a new type of mass analyzer, the Orbitrap, which in 2005 was regarded as a tool for proteomics research by Hu (21). Orbitrap is a novel mass analyzer that features high resolution (up to 150,000) with high mass accuracy (2–5 ppm), a mass-to-charge range of 6,000 and a dynamic range greater than 103 (21, 22). High mass accuracy of the Orbitrap considerably contributes to the amount of acquired data and analytic approaches when compared with low-resolution instruments (23). Pilch and Mann (24) reported for the first time the identification of 923 seminal proteins in one subject by LTQ-FT mass spectrometer using a bottom-up approach. Subsequently, Drake et al. (25) reported the identification of 916 unique proteins in 9 samples of expressed prostatic secretion by the MudPIT approach and by high-mass accuracy Orbitrap-MS. More recently, Batruch et al. (26) identified more than 2,000 proteins, using the offline MudPIT approach and high-resolution MS, in a pool of seminal plasma samples by five controls. They also compared this protein set with the proteome of pooled seminal plasma samples by five patients after undergoing vasectomies. The aim of the present study is to analyze human seminal plasma proteome of fertile men, by LTQ-Orbitrap mass spectrometer, in order to identify a panel of common seminal proteins in fertile men.

MATERIALS AND METHODS Detailed materials and methods description is reported in the expanded materials and methods section (Supplemental Data, available online). The design of the study was approved by our Institutional Review Board.

Subjects Five fertile men, whose partners were pregnant when the study was started, participated in this study. None had a history of fertility problems. All female partners conceived within 3 months before the start of the study. All subjects gave informed consent according to the guidelines of the Declaration of Helsinki.

Hormonal Study A blood sample was collected at 8 AM to determine levels of T, E2, dihydrotestosterone (DHT), sex hormone-binding globulin (SHBG), LH, FSH, free T3, free T4, TSH, PRL, and insulin-like growth factor 1 (IGF-1). 68

Semen Analysis and In-solution Digestion In all patients, a standard semen analysis was performed, assessing the ejaculate volume and pH, sperm count, percentage of sperm motility and morphology, according to WHO (1999) classification. Liquified semen samples were then centrifuged at 9,200  g for 20 minutes to obtain the seminal plasma. After the centrifugation, an aliquot was checked under microscope to confirm that no spermatozoa were present. Seminal plasma was divided in 0.5-mL aliquots, and immediately frozen at 80 C until analysis. An aliquot of each seminal sample was subjected to in solution digestion protocol, as reported in Supplemental Data.

Proteomic Analysis The samples obtained from the digestion procedure were resuspended in aqueous trifluoroacetic acid and analyzed with an Ultimate 3000 Nano/Micro-HPLC apparatus equipped with an FLM-3000-Flow manager module, coupled with an LTQ-Orbitrap XL hybrid mass spectrometer. High-resolution MS spectra were collected in data-dependent acquisition mode. The three most intense multiple-charged ions were selected and fragmented by using collision-induced dissociation and spectra were recorded in the Orbitrap.

Data Analysis Tandem mass spectra were analyzed by the Thermo Proteome Discoverer 1.2 program, using SEQUEST cluster (University of Washington, Seattle, WA, licensed to Thermo Electron Corp.) as a search engine against uniprot-taxonomy-9606 human protein database. Protein information obtained by SEQUEST was then analyzed using the software tool for researching annotations of proteins (STRAP), a user-friendly, open-source C# application, to annotate each protein according to the gene ontology (GO) system. STRAP automatically obtains GO terms associated with proteins in a proteomics results identification list using the freely accessible UniProtKB and EBI GOA databases. Summarized in an easy-to navigate tabular format, STRAP includes meta-information on the protein, in addition to complimentary GO terminology (27). When an individual protein was known to be assigned to more than one GO annotation, all of the annotations were counted nonexclusively.

RESULTS All subjects had hormonal values within the normal range, as follows (mean  SD): T, 4.80  1.9 ng/mL; E2, 27.1  10.0 pg/mL; DHT, 0.37  0.1 ng/mL; SHBG, 32.1  7.9 nmol/L; LH, 4.7  2.1 MUI/mL; FSH, 3.4  1.8 MUI/mL; free T3, 2.32  1.0 pg/mL; free T4, 13.2  2.3 pg/mL; TSH, 1.82  1.41 mUI/dL; PRL, 11.4  6.2 ng/mL; IGF-1, 197  40 ng/mL. Seminal parameters were within the WHO (1999) reference values, as follows (mean  SD): volume, 2.6  1.2 mL; pH, 7.6  0.3; sperm number, 77  40 million/mL; progressive motility, 54.3%  5.3%; normal morphology, 37.9%  5.6%. Protein identification criteria resulted in the identification of 919 to 1,487 unique proteins per individual subject sample. Of these proteins, 83 proteins were present in all samples. The entire proteomic list of common proteins is reported VOL. 97 NO. 1 / JANUARY 2012

Fertility and Sterility®

TABLE 1 Panel of common seminal proteins in all fertile men. UNIPROT

Gene

MW (kDa)

IP

Protein name

Q9P0K7 O75179 A6QL64 Q8N2N9 Q5JPF3 Q12791 Q8N3K9 P49913 Q5VT06 P10909 Q5T655 Q6ZRK6 P53420 P08603 P32320 Q5H9S7 Q68CQ4 P25686 Q9UFH2 Q03001 Q5XPI4 O95071 P23769 Q92817 Q13227 A0PJZ3 Q9NZI5 Q9P2D3 Q96RW7 P39880 Q16543 O43314

RAI14 ANKRD17 ANKRD36 ANKRD36B N/A KCNMA1 CMYA5 CAMP CEP350 CLU CCDC147 CCDC73 COL4A4 CFH CDA DCAF17 DIEXF DNAJB2 DNAH17 DST RNF123 UBR5 GATA2 EVPL GPS2 GXYLT2 GRHL1 HEATR5B HMCN1 CUX1 CDC37 PPIP5K2

110.0 274.0 217.3 153.5 199.6 137.5 448.9 19.3 350.7 52.5 103.4 124.1 163.9 139.0 16.2 58.7 87.0 35.6 507.5 306.6 148.4 309.2 50.5 231.5 36.7 51.0 70.1 214.9 599.7 153.3 44.4 138.0

6.21 6.52 8.66 8.85 7.83 7.06 4.78 9.41 6.33 6.27 8.41 5.58 8.62 6.61 6.92 7.01 5.88 5.95 5.72 6.32 6.74 5.85 9.31 6.96 9.52 9.77 6.73 7.42 6.40 5.64 5.25 8.06

Q6VAB6 P02788 Q16787 Q96CN5 P48059 Q8WXI7 Q9UKX2 P20929 Q8WXH0 P48681 Q9HC29 Q8NH85 O75127 Q14435 Q5GLZ8 Q7Z333 P07288 Q9HCK5 Q13948 Q5T0W9 Q9H081 P48634 Q5JSZ5 O43663 Q14690 Q8N2C7 Q15286 Q7Z5J4 A1A4S6 P04279 Q02383 Q8IVM8 O15020 Q9Y657

KSR2 LTF LAMA3 LRRC45 LIMS1 MUC16 MYH2 NEB SYNE2 NES NOD2 OR5R1 PTCD1 GALNT3 HERC4 SETX KLK3 EIF2C4 CUX1 FAM83B MIS12 PRRC2A PRRC2B PRC1 PDCD11 UNC80 RAB35 RAI1 ARHGAP10 SEMG1 SEMG2 SLC22A9 SPTBN2 SPIN1

107.6 78.1

8.69 8.12

75.9 37.2 2351.2 222.9 772.4 795.9 177.3 112.5 36.7 78.8 72.6 109.5 299.2 28.7 97.0 77.2 114.7 24.1 228.7 242.8 61.3 208.6 291.5 23.0 175.2 89.3 52.1 65.4 62.1 271.2 29.6

6.23 8.05 6.00 5.82 9.07 5.36 4.36 7.02 8.28 8.59 7.99 6.51 7.21 7.68 9.06 5.48 8.97 5.69 9.45 8.34 7.83 8.87 7.12 8.29 9.14 7.18 9.29 9.07 8.10 6.11 6.96

Ankycorbin Ankyrin repeat domain-containing protein 17 Ankyrin repeat domain-containing protein 36A Ankyrin repeat domain-containing protein 36B Ankyrin repeat domain-containing protein 36C Calcium-activated potassium channel subunit alpha-1 Cardiomyopathy-associated protein 5 Cathelicidin antimicrobial peptide Centrosome-associated protein 350 Clusterin Coiled-coil domain-containing protein 147 Coiled-coil domain-containing protein 73 Collagen alpha-4(IV) chain Complement factor H Cytidine deaminase DDB1- and CUL4-associated factor 17 Digestive organ expansion factor homologue DNAJ homologue subfamily B member 2 Dynein heavy chain 17, axonemal Dystonin E3 ubiquitin-protein ligase RNF123 E3 ubiquitin-protein ligase UBR5 Endothelial transcription factor GATA-2 Envoplakin G protein pathway suppressor 2 Glucoside xylosyltransferase 2 Grainyhead-like protein 1 homologue HEAT repeat-containing protein 5B Hemicentin-1 Homeobox protein cut-like 1 Hsp90 co-chaperone Cdc37 Inositol hexakisphosphate and diphosphoinositolpentakisphosphate kinase 2 Kinase suppressor of Ras 2 Lactotransferrin Laminin subunit alpha-3 Leucine-rich repeat-containing protein 45 LIM and senescent cell antigen-like-containing domain protein 1 Mucin-16 Myosin-2 Nebulin Nesprin-2 Nestin Nucleotide-binding oligomerization domain-containing protein 2 Olfactory receptor 5R1 Pentatricopeptide repeat-containing protein 1 Polypeptide N-acetylgalactosaminyltransferase 3 Probable E3 ubiquitin-protein ligase HERC4 Probable helicase senataxin Prostate-specific antigen Protein argonaute-4 Protein CASP Protein FAM83B Protein MIS12 homologue Protein PRRC2A Protein PRRC2B Protein regulator of cytokinesis 1 Protein RRP5 homologue Protein unc-80 homologue Ras-related protein Rab-35 Retinoic acid-induced protein 1 Rho GTPase-activating protein 10 Semenogelin I Semenogelin II Solute carrier family 22 member 9 Spectrin beta chain, brain 2 Spindlin-1

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TABLE 1 Continued. UNIPROT Q9UQE7 Q9BR01 Q15431 Q8IWB6 Q8WZ42 Q01664 Q13595 Q9BSH4 Q86WT6 Q9P275 Q6X4T0 Q5T5A4 A6NGG8 Q96IT1 Q9NU63 Q5T7W0 Q86XU0

Gene SMC3 SULT4A1 SYCP1 TEX14 TTN TFAP4 TRA2A TACO1 TRIM69 USP36 C12orf54 C1orf194 C2orf71 ZNF496 ZFP57 ZNF618 ZNF677

MW (kDa)

IP

141.5 30.2 114.1 162.6 3827.5 38.7 32.7 32.5 57.4 122.6 11.7 19.3 139.6 66.9 51.9 104.9 68.0

7.18 5.91 5.96 5.25 6.35 5.87 11.27 8.13 6.48 9.67 8.29 9.28 8.07 5.69 9.11 7.09 9.13

Protein name Structural maintenance of chromosomes protein 3 Sulfotransferase 4A1 Synaptonemal complex protein 1 Testis-expressed protein 14 Titin Transcription factor AP-4 Transformer-2 protein homologue alpha Translational activator of cytochrome c oxidase 1 Tripartite motif-containing protein 69 Ubiquitin carboxyl-terminal hydrolase 36 Uncharacterized protein C12orf54 Uncharacterized protein C1orf194 Uncharacterized protein C2orf71 Zinc finger protein 496 Zinc finger protein 57 homologue Zinc finger protein 618 Zinc finger protein 677

Milardi. Seminal proteomics and male fertility. Fertil Steril 2012.

in Table 1. For each protein we reported the following information: UNIPROT, gene name, molecular weight, isoelectric point, and description. The largest proportion of GO annotations for molecular function were ‘‘binding proteins,’’ which occurred in 54 proteins (58%). Twenty proteins were annotated as involved in catalytic activity, 10 in structural molecule activity, and 1 was involved in enzyme regulation. Other activities were reported for eight proteins. Analysis of GO cellular distribution annotations is reported in Figure 1. Biological process analysis of the proteic pattern is reported in Figure 2.

DISCUSSION The importance of knowing the composition of seminal plasma is essential in understanding the physiology of

reproduction, as seminal fluid has a crucial role in spermatozoa survival and overall fertilization success. Analysis of seminal plasma proteome readily reflects the physiological/ pathological state of the testis, epididymis, and other accessory sex glands. Therefore, alterations of human seminal proteome may explain the molecular mechanism in some cases of infertility. This study used modern and more specific methodologies for the detection of a common pattern of seminal proteins secreted in vivo in fertile subjects. The LTQ-FT mass spectrometer combines high sensitivity and fast sequencing cycles with very high mass accuracy and resolution. We identified 83 common proteins in seminal plasma in a group of fertile subjects. This is the first identification of the common pattern of seminal proteins in male fertility, using high-resolution MS. Previously Pilch and Mann (24)

FIGURE 1

Cellular distribution GO annotations of reported proteins. Milardi. Seminal proteomics and male fertility. Fertil Steril 2012.

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FIGURE 2

Biological process GO annotations of reported proteins. Milardi. Seminal proteomics and male fertility. Fertil Steril 2012.

reported the identification of 923 seminal plasma proteome in a single sample. We identified up to 1,487 unique proteins per single sample, showing a greater sensitivity of the procedure. Recently Batruch et al. (26) identified more than 2,000 proteins in a seminal plasma sample obtained after pooling different samples of five subjects. At present this is the most exhaustive list of seminal proteins, pooling interindividual differences in the seminal proteome. Our goal, instead, was to describe which proteins are common in all fertile men, obtaining the panel of proteins involved in reproduction, regardless of interindividual variability. Within the group of common proteins we reported the olfactory receptor (OR) 5R1. The G-protein-coupled ORs make up a large multigene family, whose ectopic expression has been related to testis and germ cells, where several dozen human and mouse ORs were shown to be transcribed (28, 29). The OR proteins appear to be expressed in late spermatids and on the tail midpiece of mature spermatozoa, implying that testicular ORs are involved in either sperm maturation, migration, or fertilization (30, 31). These results therefore led to the hypothesis that at least some ORs are involved in mammalian sperm chemotaxis. Evidence for the involvement of human hOR17-4 (OR1D2) (32, 33) and mouse MOR267-13 (34) in sperm chemotaxis was provided. Another hypothesis proposed that ORs linked to the major histocompatibility complex locus and expressed in testis are implicated in olfaction driven mate choice (35). Some ORs, such as the OR51E2, were also found to be overexpressed in human prostate (36, 37). In this study, we identified an OR among a common proteic pattern in fertile men, confirming the importance of ORs in sperm chemotaxis and fertility, as previously reported. Proteomic platform may give new insight in understanding the expression and role of ORs in germ cells and seminal plasma. In our study, semenogelin (Sg) I and Sg II are expressed in all samples. This was predictable as Sg is the main protein of human semen coagulum, and plays an important physiological role in the suppression of sperm motility at ejaculation. VOL. 97 NO. 1 / JANUARY 2012

With regard to the proteins identified in all patients, we identified some proteins involved in fertility and reproduction such as lactoferrin, human cationic antimicrobial protein (hCAP18), and spindlin 1. Lactoferrin was first identified in human seminal plasma in 1966 (38). Previous studies reported that it has antibacterial, antioxidative, and an immune-modulating role in seminal plasma. It is also involved in maintaining normal sperm structure and motility and modulating the composition and quality of the semen during sperm maturation and migration through the male genital tract (39, 40). It has been demonstrated that the increased lactoferrin concentration in some leukocytospermia, oligospermia, and asthenospermia is beneficial in the reduction of leukocyte concentration, in increasing sperm motility, in rescuing sperm morphology and functions and in improving the semen quality (39–41). Recently, Wang et al. (42) demonstrated that lactoferrin receptor is expressed in the testis and is anchored to the sperm membrane by glycophosphatidylinositol during spermatogenesis, being involved in an important role by binding to and mediating lactoferrin. The hCAP18 has a key role in the innate immunity of the male reproductive system. It was reported as present in the epithelium of human epydidimis, in the seminal plasma at high concentrations, and in association with spermatozoa (43). It is not unlikely that the spermatozoa transport hCAP18 with them on their way to the ovum and that the hCAP18 provides protection against microorganisms during fertilization. Recent studies suggest that spindling 1 protein is involved in spermatogenesis in the first wave of spermatocyte meiosis. In addition, the protein was also highly expressed in the cytoplasm of primary spermatocyte in adult testes. Spindlin 1 was also localized at the tail of mouse sperm and could be essential for normal sperm motility (44). In the present study we identified other proteins whose reduction in sperm messenger RNA (mRNA) expression in men was associated in a previous microarray study with teratozoospermia, such as C12orf54 CDA, TACO1, CUX1, EIF2C4, 71

ORIGINAL ARTICLE: ANDROLOGY PPIP5K2, PTCD1, CEP350, and SYCP1 (45). The same study reported in sperm the presence of RNA for other proteins identified in our study, such as FAM83B, SPIN1, EVPL, GXYLT2, DST, DCAF17, HMCN1, CMYA5, TEX14, PRC1, RAB35, and TACO1. In the same study, an increase in translational activator of cytochrome c oxidase 1(TACO1) mRNA was observed in teratospermia. Considering the molecular function annotation of the proteins, the most abundant group include proteins involved in protein binding (58%). It may be possible that in many cases protein binding function represents an auxiliary role to the main one of that protein, which is closely linked to enzymatic or transport activity. However, some of these proteins were previously reported in seminal plasma, with a proteinbinding function, such as clusterin. Clusterin is a protein previously identified in seminal vesicle secretions (46) and related to the damaging oxidative reactions (47), protein precipitation (48), agglutination of abnormal spermatozoa (49), and control of complement-induced sperm lysis (50). Clusterin was previously described as a biomarker of in vivo fertility in stallions (51), but never reported as a marker of fertility in men. Catalytic activity was annotated in 22% of proteins. An additional 1% of proteins is classified as their regulators, implying that 23% of the seminal fertile proteomic pattern is involved in enzymatic activity. Most of these proteins, in fact, such as prostatic specific antigen (PSA), Sg I, and Sg II, are involved in the regulation, processing or degradation of seminal fluid proteins, and coagulation of semen. Both the study of the cellular localization of proteins and of the biological processes in which proteins are involved, according to GO annotations, reported an overlap among the categories. Thus, the nonexclusive assignment of the proteins may confuse the data analysis. Membrane proteins are present with a frequency of 27 annotated proteins. The high percentage of membrane proteins identified, even though seminal plasma being a secreted fluid may depend on whether some of these proteins are bound to the sperm surface during ejaculation, thus forming protein-coated layers (52). This is confirmed by the number of membrane proteins that are annotated both as membrane proteins and as extracellular or surface proteins (7 proteins). The largest group of proteins, according to GO Biological Process annotations, is composed of 40 proteins that are involved in the cellular process, followed by 31 proteins annotated as involved in regulation. According to GO annotations and STRAP elaboration, the high incidence of proteins in these annotations is justified by the presence of both enzymes involved in the regulation, processing, or degradation of seminal fluid proteins and coagulation of semen such as in the basic cellular processes. In summary, with the availability of a modern mass spectrometer like LTQ-Orbitrap, we are now able to identify hundreds and thousands of proteins in complex biological samples such as seminal plasma. We offered a proteomic approach to identify a proteic panel for male fertility by using the LTQ-Orbitrap mass analyzer. Last, we also constructed a database containing GO information. The identification of 72

common seminal plasma proteome in fertile men could provide better insight into the physiology of male fertility and might identify novel markers of male infertility.

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ORIGINAL ARTICLE: ANDROLOGY

SUPPLEMENTAL DATA EXPANDED MATERIALS AND METHODS SECTION The design of the study was approved by our Institutional Review Board.

Subjects Five fertile men, whose partners were pregnant when the study started, participated in this study. None had a history of fertility problems. All of the women conceived within 3 months before the start of the study. All subjects gave informed consent according to the guidelines of the Declaration of Helsinki.

In Solution Digestion An aliquot of each seminal plasma sample corresponding to 1 mg of total protein (as measured by Bradford assay) was mixed with 100 mM of ammonium bicarbonate at pH 8.0 and reduced with 200 mM dithiothreitol (DTT, 10 mM final; Sigma) for 5 minutes at 100 C, 15 minutes at 50 C, and alkylated with 200 mM of iodoacetamide (55 mM final; Sigma) in the dark at room temperature for 60 minutes. The samples were left to digest overnight at 37 C by adding 100 mM of ammonium bicarbonate (pH 8) with sequencing grade-modified porcine trypsin (1:50, trypsin: protein concentration; Promega). To stop the digestion, the samples were acidified with aqueous trifluoroacetic acid (TFA/H2O 0.2% vol/vol), immediately frozen, and lyophilized.

Proteomic Analysis Hormonal Study A blood sample was collected at 8 AM for the determination of T, E2, dihydrotestosterone (DHT), sex hormone-binding globulin (SHBG), LH, FSH, free T3, free T4, TSH, PRL, and insulin-like growth factor I (IGF-I). Testosterone, E2, and PRL were assayed in duplicate by RIA with the use of commercial kits by Radim. The LH, FSH, and SHBG were assayed by immunoradiometric methods on a solid-phase (coated tube), which is based on a monoclonal double-antibody technique. Dihydrotestosterone was assayed by RIA with the use of commercial kits by Chematil. The IGF-I levels were measured by Immulite 2000 (DPC). Serum TSH, free T4, and free T3 concentrations were measured by the electroimmunochemiluminescent method (Roche, ElecsysÒSystems 1010/2010/modular analytics E170). Reference values of the studied hormones were: T, 2.5–8.4 ng/mL; E2, 10–35 pg/mL; DHT, 0.30–0.85 ng/mL; SHBG, 15–65 nmol/L; LH, 2.5–10 MUI/mL; FSH, 2.5–11 MUI/mL; free T3, 2.3–4.2 pg/mL; free T4, 8.5–15.5 pg/mL; TSH, 0.35–2.80 mIU/L; PRL, 3.5–15.5 ng/mL, and IGF-I, 80–330 mg/L. The intra-assay coefficients of variation (CV, %) were 6.1% for T, 2.3% for E2, 5.1% for DHT, 6.9% for SHBG, 5.6% for LH, 6.9% for FSH, 4.5% for TSH, 4.1% for free T4, 1.4% for free T3, 3.7% for PRL, and 6.4% for IGF-I. The interassay CV were 9.3% for T, 3.5% for E2, 8.9% for DHT, 8.5% for SHBG, 9.1% for LH, 8.4% for FSH, 3.4% for TSH, 2.9% for free T4, 3.8% for free T3, 3.1% for PRL, and 11.5% for IGF-I.

Semen Analysis and Sample Preparation for Proteomic Analysis In all patients a standard semen analysis was performed, assessing the ejaculate volume and pH, sperm count, percentage of sperm motility, and morphology, according to World Health Organization (1999) classification. Each semen specimen was analyzed at 1 hour from collection. Liquefied semen samples were then centrifuged at 9,200  g for 20 minutes to obtain the seminal plasma. After the centrifugation, an aliquot was checked under microscope to confirm that no spermatozoa were present. Seminal plasma was divided in 0.5-mL aliquots and immediately frozen at 80 C until analysis. 73.e1

The samples were resuspended in 40 mL of TFA/H2O (0.2% vol/vol) and analyzed by an Ultimate 3000 Nano/MicroHPLC apparatus (Dionex) equipped with an FLM-3000-Flow manager module, coupled with an LTQ-Orbitrap XL hybrid mass spectrometer (ThermoFisher). Separations were performed by a Supelco Discovery Bio Wide Pore C18 column (Supelco Park) (3-mm particle diameter; column dimension 1 mm id  10 cm) (Supelco), using the following eluents: (A) 0.056% (vol/vol) aqueous TFA and (B) acetonitrile:water, 80:20 with 0.05% (vol/vol) aqueous TFA. The applied gradient was linear from 0–50% of solvent B in 60 minutes, at a flow rate of 80 mL/min. The LTQ-Orbitrap mass spectrometer was operated in a data-dependent mode in which each full mass spectrometric (MS) scan (60,000 resolving power) was followed by three MS/MS scans where the three most intense multiple-charged ions were dynamically selected and fragmented by collision-induced dissociation using a normalized collision energy of 35%.

Data Analysis Tandem mass spectra were analyzed by the Thermo Proteome Discoverer 1.2 program, using SEQUEST cluster as a search engine (University of Washington, Seattle, WA, licensed to Thermo Electron Corp., San Jose, CA) against the uniprottaxonomy-9606 human protein database. Data were searched with three missed cleavages, fixed carbamidomethylation of cysteines, and the following variable modifications: oxidation of methionines and phosphorylation of threonines, tyrosines, and serines. Protein information obtained by SEQUEST were then analyzed using the Software Tool for Researching Annotations of Proteins (STRAP), a user-friendly, opensource C# application to annotate each protein according to Gene Ontology (GO) system. STRAP automatically obtains GO terms associated with proteins in a proteomics results identification list using the freely accessible UniProtKB and EBI GOA databases. Summarized in an easy-to-navigate tabular format, STRAP includes meta-information on the protein in addition to complimentary GO terminology (26). When an individual protein was known to be assigned to more than one GO annotation, all of the annotations were counted nonexclusively. VOL. 97 NO. 1 / JANUARY 2012