Analysis of Phosphopeptide Changes as ... - ACS Publications

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Analysis of Phosphopeptide Changes as Spermatozoa Acquire Functional Competence in the Epididymis Demonstrates Changes in the Post-translational Modification of Izumo1 Mark A. Baker,*,† Louise Hetherington,† Anita Weinberg,† Nenad Naumovski,† Tony Velkov,‡ Matthias Pelzing,§ Sebastiaan Dolman,§ Mark R. Condina,§ and R. John Aitken† †

Priority Research Centre in Reproductive Science, School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW, 2308, Australia ‡ Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia § Bruker Biosciences, PTY LTD, 28 Albert Street, Preston, Victoria 3072, Australia S Supporting Information *

ABSTRACT: Spermatozoa are functionally inert when they emerge from the testes. Functional competence is conferred upon these cells during a post−testicular phase of sperm maturation in the epididymis. Remarkably, this functional transformation of epididymal spermatozoa occurs in the absence of nuclear gene transcription or protein translation. To understand the cellular mechanisms underpinning epididymal maturation, we have performed a label-free, MSbased, comparative quantification of peptides from caput, corpus and caudal epididymal spermatozoa. In total, 68 phosphopeptide changes could be detected during epididymal maturation corresponding to the identification of 22 modified proteins. Included in this list are the sodium-bicarbonate cotransporter, the sperm specific serine kinase 1, AKAP4 and protein kinase A regulatory subunit. Furthermore, four phosphopeptide changes came from Izumo1, the sperm-egg fusion protein, in the cytoplasmic segment of the protein. 2D-PAGE confirmed that Izumo1 is post-translationally modified during epididymal transit. Interestingly, phosphorylation on Izumo1 was detected on residue S339 in the caput and corpus but not caudal cells. Furthermore, Izumo1 exhibited four phosphorylated residues when spermatozoa reached the cauda, which were absent from caput cells. A model is advanced suggesting that these phospho-regulations are likely to act as a scaffold for the association of adaptor proteins with Izumo1 as these cells prepare for fertilization. KEYWORDS: sperm phosphorylation, epididymal maturation, mass spectrometry, proteomics, titanium dioxide, label-free quantitation, Izumo1

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INTRODUCTION

spermatozoa to simple balanced salt solutions results in no forward progressive movement, or even flagella beating, of any kind.8−10 In contrast, mature spermatozoa taken from cauda epididymis are highly motile when incubated in exactly the same media.10 Second, spermatozoa retained within the testis by efferent duct ligation are immotile and completely lacking in any capacity to fertilize the oocyte.11−13 Furthermore, rabbits and rats that have undergone surgical vasoepididymal bypasses of the proximal caput do not sire any litters due to major defects in sperm cell motility.14 Finally, removal of c-ros, a proto-oncogene encoding for an orphan receptor that is only found in the proximal regions of the epididymis following puberty, results in the production of infertile spermatozoa in

Spermatozoa manufactured in the testis, despite their morphological maturity, are not functionally active.1 Functionality is acquired during the post-testicular maturation of spermatozoa via mechanisms that essentially involve the acquisition, loss, or post-translational modification of proteins, since these cells are transcriptionally and translationally silent.2−6 The first step of this maturation process takes place in an organ known as the epididymis. The epididymis is a tightly coiled, highly differentiated structure that connects the efferent ducts of the testis to the vas deferens and is present in all male mammals. Several lines of evidence suggest that spermatozoa progressively acquire their fertilizing potential during epididymal descent7 and that epididymal maturation is an essential prerequisite for successful fertilization. In support of this, the translocation of immature caput epididymal © 2012 American Chemical Society

Received: May 27, 2012 Published: September 6, 2012 5252

dx.doi.org/10.1021/pr300468m | J. Proteome Res. 2012, 11, 5252−5264

Journal of Proteome Research

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association with disrupted development of the initial segment of this organ.15 Taken together, these data suggest that without epididymal maturation spermatozoa cannot undergo successful fertilization. Within the epididymal lumen, proteinaceous secretions from the epididymal epithelial cells act to extensively modify the sperm cell. Indeed, high resolution MALDI imaging8 and 2DPAGE16 of specific regions within the mouse epididymis have demonstrated discrete protein signals within each zone, suggesting that spermatozoa experience a carefully orchestrated sequence of changes in their immediate protein environment as they migrate through the epididymal lumen. To date, analysis of proteomic changes occurring in spermatozoa during epididymal maturation have demonstrated proteolytic cleavage of the MDC (Metalloprotease Disintegrin Cysteine-rich) family of proteins (fertilin-α, fertilin-β, tMDC I, tMDC II and tMDC III),17,18 testase 1 (ADAM 24)19 basigin20 and PH20.21−24 Furthermore, by taking spermatozoa from the caput and caudal regions of CD1 breeder mice and labeling sulfhydryl containing proteins using the CyDye (2D DIGE), investigators identified three basic categories of enzymes that changed in their thiol status during epididymal maturation. These included a series of chaperones (Heat Shock Proteins 2, 5, 9), structural proteins (AKAP4, actin, AKAP3) and metabolic enzymes (succinate coenzyme A, ligase, aldose A, F1 ATPase, enolase, NADH dehydrogenase).25 Our specific interest lies in the identification of phosphorylation changes occurring within the spermatozoa during epididymal sperm maturation, and clearly, regulation of protein phosphorylation plays a key role in this process. For example, inhibition of the serine/threonine phosphatase isoform 1, gamma 2, stimulates motility in otherwise normally quiescent caput epididymal spermatozoa.26 Furthermore, addition of a cell permeable analogue of cAMP causes different patterns of tyrosine phosphorylation within spermatozoa from different regions of the epididymis,27 suggesting that maturation of this pathway occurs during epididymal descent. An initial attempt to identify phosphoprotein changes in spermatozoa during epididymal transit involved enriching preparations for phosphopeptides using titanium dioxide (TiO2) and then performing label-free proteomic analysis to compare the caput and cauda sperm cell peptides.28 Although several peptide changes were observed, many of these could not be interpreted due to low abundance or inconclusive MS/MS spectra. Here, we employed a different strategy to overcome this shortcoming by utilizing an optimized uHPLC (ultra high pressure liquid chromatography) system coupled to a high capacity 3D ion-trap MS system, capable of both collision induced (CID) and electron transfer (ETD) dissociation. CID and ETD fragmentation are complementary approaches, which when combined improve overall peptide characterization (sequence annotation) from MS/MS spectra.29 Within the analysis we have also included, for the first time, spermatozoa from the corpus region of the epididymis in order to gain a more comprehensive understanding of the progression of posttranslational changes occurring during maturation. We can now report the identification of 68 phosphopeptides, corresponding to 22 protein changes during epididymal transit. Many of these proteins are unique and include the first reports of both the presence and the phosphorylation of the sodium-bicarbonate cotransporter as well as the phospho-regulation of Izumo1.

Article

EXPERIMENTAL SECTION

Materials and Reagents

Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia) at the highest research grade available. Albumin and ammonium persulfate were obtained from Research Organics (Cleveland, OH); Dglucose, sodium hydrogen carbonate, sodium chloride, potassium chloride, calcium chloride, potassium orthophosphate and magnesium sulfate were all analytical reagent grade, purchased from Merck (BDH Merck, Kilsyth, VIC, Australia); ultrapure water, sodium sulfate, 2,5-dihydroxybenzoic acid (DHB) and zinc sulfate were from Fluka (Castle Hill, NSW, Australia); chloroform, methanol and formaldehyde were purchased from Fronine (Riverstone, NSW, Australia) at the highest purity available. Tris was purchased from ICN Biochemicals (Castle Hill, NSW, Australia) and acrylamide was from Biorad (Castle Hill, NSW, Australia). Quantum Scientific (Pierce, Milton, QLD, Australia) supplied the phosphatase inhibitors and BCA assay kit, while TiO2 was collected from a disassembled column (Titansphere, GL Sciences Inc., Tokyo) and the trypsin was from Promega (Annandale, NSW, Australia). Preparation of Rat Spermatozoa

Institutional and NSW State Government ethical approval was secured for the use of Wistar rats in this research program. Adult rats (∼8 −10 weeks) were asphyxiated and the epididymides were removed. Pure suspensions of spermatozoa were obtained from the caudal region of the epididymis by retrograde flushing, as previously described.30,31 The rat spermatozoa were gently isolated into BWW (Biggers Whitten and Whittingham) media and allowed to disperse for 10−15 min. The samples were then centrifuged and washed three times (800× g, 3 min) to pellet the spermatozoa and separate these cells from any residual epididymal plasma. Caput and corpus-derived spermatozoa were isolated by finely slicing zones 1−3 with a surgical-grade scalpel. The sperm were then gently teased out into BWW media. Due to epithelial cell contamination, the sample was then processed through a 50% Percoll solution as described elsewhere.4 The pellet was inspected and only samples that contained a maximum of 1 round cell per 100 000 spermatozoa were used. Approximately 400 μL of lysis buffer consisting of 4% (w/v) CHAPS, 7 M urea, 1/100 dilution of Halt phosphatase inhibitor (Pierce) and 2 M thiourea in water was added to 1 × 107 sperm cells. The thiourea aided the extraction of proteins from both caput and caudal spermatozoa, since significant cysteine cross-linking occurs during epididymal transit. For this reason, silver staining and immunoblot analysis against α-tubulin were used to ensure equal protein loading. The samples were lysed for 1 h at 4 °C with constant rotation, then centrifuged (16 000× g, 15 min, 4 °C) and the supernatant was transferred to a new Eppendorf tube. A protein estimation was subsequently performed using a 2D quant kit (G.E. Healthcare, Castle Hill, NSW, Australia) and 150 μg of protein precipitated using methanol/chloroform as described elsewhere.32 Trypsin was then added in a ratio of 50:1 (protein/trypsin) in 25 mM ammonium bicarbonate containing 1 M urea, with constant shaking overnight at 37 °C. The following morning the sample was centrifuged (16 000× g, 15 min) at 4 °C; the supernatant was removed and stored at −80 °C until needed. 5253



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Phosphopeptide Enrichment

tion (STY). It should be noted that both deamidation and oxidation are in vitro artifacts that can be introduced as a result of sample handling. Mass Spectrometric Data Interpretation. The derived mass spectrometry data sets on the 3D-trap system were combined into protein compilations using the ProteinExtractor functionality of Proteinscape 2.1.0 573 (Bruker Daltonics, Bremen, Germany), which conserved the individual peptides and their scores, while combining them to identify proteins with much higher significance than achievable using individual searches. In order to exclude false positive identifications, peptides with Mascot scores below 25 were rejected without further consideration. Each individual spectrum was then loaded into the software “data analysis” (Bruker Daltonik, Bremen, Germany), where the y and b (CID) or c and z ion (ETD) series were manually validated on a residue by residue basis. For positive phosphopeptide identification we looked for the ion series to be present on the most abundant peaks present in the fragment ions. The mass error between any residue had to be less than 0.15 Da and a majority of the peak had to be accounted for on the basis on internal fragments, or other ion series (−17, −18 Da) of a typical CID or ETD spectrum. In the case of phosphoserine and phosphothreonine-containing peptides, the neutral loss of phosphoric acid initiated by CID was also used to confirm the number of phosphate groups in the assigned sequence. Peptides in which the intact phosphorylated residue was found in the tandem mass spectra were assigned. Additionally, as ETD fragmentation maintains the phosphorylated residue within the spectrum, this can be used to confirm the phosphorylated residue. If a phosphopeptide assignment was not possible, the peptide was reported as containing one or more phosphate groups (p) followed by the peptide sequence in parentheses.

Purification and enrichment of phosphopeptides from the tryptic digest was performed by a similar method to that previously described.33 Tryptic peptides were diluted 5-fold in DHB buffer [350 mg/mL DHB, 80% (v/v) ACN (acetonitrile), 2% (v/v) TFA (trifluoroacetic acid)] and applied to dry TiO2 beads (200 μg). The sample was then washed 1× in DHB buffer before being washed 3× with wash buffer [80% ACN (v/ v), 2% TFA (v/v)] to remove the DHB. The sample was then directly eluted using elution buffer (elution buffer consisted of 25 μL 2.5% ammonium hydroxide, pH ≥ 10.5, immediately neutralized with 0.3 μL formic acid at a final concentration of 1.2%). All buffers used ultrapure water and were made fresh on the day of experimentation. The eluates, typically in 25 μL, were used immediately. Mass Spectrometry

LC−MS. For all experiments, an Ultimate 3000 ultra high pressure liquid chromatography system (Dionex, Castle Hill, Sydney) was used equipped with a ternary low pressure mixing gradient pump (LPG-3600), a membrane degasser unit (SRD3600), a temperature controlled pulled-loop autosampler (WPS-3000T) and a temperature controlled column oven with flow manager (FLM-3100). The LC experiments were performed using the “pre-concentration” setup under the following conditions: Nanocolumn C18 PepMap100, 75 μm ID × 150 mm, 3 μm, 100 Ǻ ; mobile phase A: 99.9% water +0.1% FA (formic acid); mobile phase B: 20/80 water/ACN (v/v) + 0.08% FA; flow rate nanocolumn, 400 nL/min; gradient, 2− 40% B over 45 min, 90% B for 5 min, 4% B for 30 min; loopsize: 5 μL; injection volume, 4 μL (FullLoop) by User Defined Program. The oven temperature was set to 35 °C. 3D Trap. For the CID/ETD experiments an AmaZon ETD Ion Trap (Bruker Daltonik, Bremen, Germany) was used equipped with an online-nanosprayer. A detailed description of the ETD-setup of the ion trap instrument including the generation of the reagent anion fluoranthene has been described previously.34 Fine tuning using the smart parameter setting option (SPS) for 900 m/z, compound stability 60%, and trap drive level at 100% in normal mode resulted in the following mass spectrometric parameters: dry gas temperature, 180 °C; dry gas, 4.0 L min−1; nebulizer gas, 0.4 bar; electrospray voltage, 4500 V; high-voltage end-plate offset, −200 V; capillary exit, 140 V; trap drive, 57.4; funnel 1 in100 V, out 35 V and funnel 2 in 12 V, out 3.3 V; ICC target, 500 000; maximum accumulation time, 50 ms. The sample was measured with the ”Enhanced Scan Mode” at 8100 m/z per second (which allows monoisotopic resolution up to four charge stages) polarity positive, scan Range from m/z 100−3000, 5 spectra averaged and rolling average of 2. The ETD reaction time was set to 100 ms using a reactant ICC of 500 000 allowing a maximum accumulation time for the reactant ion of 50 ms. The “Smart Decomposition” was set to “auto”. Acquired ETD/CID spectra were processed in DataAnalysis 4.0, deconvoluted spectra were further analyzed with BioTools 3.2 software and submitted to Mascot database search (Mascot 2.2.04, Swiss-Prot database (535 698 sequence sequences; 190 107 059 residues, release date 18/4/2012). The species subset was set at Rodents, parent peptide mass tolerance for CID was 0.3 Da and for ETD, 0.5 Da. For MS/MS tolerance, CID was 0.5, ETD was 1.5 Da; enzyme specificity trypsin with 2 missed cleavages considered. The following variable modifications have been used: Deamidated (NQ), Oxidation (M), Phosphoryla-

Statistical Analysis

Processed LC−MS data was analyzed using Profile Analysis (Bruker Daltonik, Bremen, Germany), whereby normalization based on the total peptide ion counts (sum of bucket values in analysis) was performed to compensate for intensity differences between elution profiles. The peptide counts were based upon the intact peptide, as opposed to spectral counts which use daughter ions for quantification. The program integrates the designated area under the curve based upon the MS-scan for each individual peptide and assigns an intensity value (note: if a peptide has multiple charge states, the sum of all the states are collated). Peptides within different runs were matched based upon m/z, charge and elution times. Peptide matching was manually confirmed using the survey view to ensure accuracy. Students t-tests were then performed for each group (n = 20, caput vs corpus vs cauda). As label-free quantification based on MS-survey scan is a relatively new concept, there is no standard protocol by which the data should be interpreted. Therefore, we have reported peptides with t-test results indicating that a change occurred during epididymal maturation with a probability of p < 0.05.When a total change in the phosphopeptide was found (presence or absence), the p-value was not reported since it would be infinity. SDS-PAGE and Immunoblotting

Mouse spermatozoa were solubilized in 125 μL of rehydration buffer (4% CHAPS, 2 M thiourea, 8 M urea) and following centrifugation (15 000× g, 20 min) the supernatant added to a 7 cm, 3−10 linear IPG strip (GE Healthcare). The strips were covered with mineral oil (Sigma) and left overnight. Isoelectric 5254



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focusing using a IPGphor (Amersham Biosciences) with the following settings: Peltier plate 20 °C, 50 uA/strip, gradient 500 V, 10 Vh, gradient 4000 V, 5600 Vh, step and hold 5000 V for 2500 Vh. The IPG strips were immediately placed into equilibration buffer [30% (v/v) glycerol, 2% (w/v) SDS, 6 M urea with trace amounts of bromophenol blue] supplemented initially with 0.5% (w/v) DTT for 15 min at room temperature (reduction) followed by 2.5% iodoacetamide (alkylation of cysteine) in fresh equilibration buffer for 15 min at room temperature. IPG strips were then placed on top of 10% polyacrylamide gels. The second dimension SDS-PAGE was then carried out using 25 mA constant current per gel. The proteins were then transferred onto nitrocellulose hybond super-C membrane (GE Healthcare, Castle Hill, Australia) at 350 mA constant current for 1 h. The membrane was blocked for 1 h at room temperature with tris-buffered saline [TBS-T; 0.02 M Tris, pH 7.6, 0.15 M NaCl, 0.1% (v/v) Tween-20] containing 5% (w/v) skim milk powder. The membrane was then incubated for 2 h at room temperature using 1:1000 dilution of a monoclonal antibody against Izumo1 (Santa Cruz) in TBS-T containing 5% (w/v) skim milk. After incubation, the membrane was washed 4 × for 5 min with TBS-T, and then incubated for 1 h at room temperature with goat antimouse antibody [1/3000; in TBS-T containing 5% (w/v) skim milk]. The membrane was washed 3× using TBS-T, and the presence of horse radish peroxidase conjugate was detected using an enhanced chemiluminescence (ECL) kit (GE Healthcare) according to the manufacturer’s instructions.

Phosphopeptide Comparisons from the Caput, Corpus and Caudal Spermatozoa

Figure 1. Survey view and total ion chromatogram of TiO2 enriched phosphopeptides. Rat spermatozoa taken from the (A) caput, (B) corpus or (C) cauda epididymides were washed, pelleted and lysed before being precipitated (MeOH/CHCl3). The samples were then digested overnight using trypsin (50:1, w/w). Approximately 10 μg of enriched phosphopeptide was loaded onto C-18 columns and eluted over time using acetonitrile according to the Experimental Section. The upper images represent the survey view of the eluting peptides, with their corresponding m/z (y axis) and elution time (x-axis). The lower images represent the total ion chromatograms.

Sperm cell maturation within the epididymal environment appears to be dominated by post-translational modifications to existing proteins. Of the many such modifications reported, phosphorylation appears to play a major role.27 In order to gain an overall understanding of the changes within spermatozoa during epididymal transit, we isolated (99.99% pure) sperm cells from the caput, corpus and caudal regions of this organ. Sperm isolated from these regions demonstrate extreme physiological differences. For example, spermatozoa from either the caput and corpus regions are immotile when placed in isosmotic media, while the cauda-derived sperm cells instantaneously initiate forward progressive motility. Interestingly, at the phosphopeptide level, both caput and corpus spermatozoa presented similar phosphopeptide profiles. However, the phosphopeptide profile of caudal spermatozoa was quite distinct. Figure 1 illustrates 3 representative “survey scans” (upper images; survey scans represents the m/z of each full length peptide together with the retention time) alongside the total-ion chromatogram (TIC; lower images) of TiO2enriched peptides generated from (Figure 1A) caput, (Figure 1B) corpus and (Figure 1C) caudal epididymal spermatozoa. Although only 1 biological replicate is shown in Figure 1, 20 individual samples were analyzed for each region of the epididymis in order to conduct statistically valid label-free quantification. By using the peptide mass and retention time, subsequent quantification at the MS-level is possible. Identification and quantification of peptides were performed in separate LC runs (MS-only for quantitation) of the sample. Performing separate

LC runs for quantitation and identification allows higher sample amounts to be loaded for LC−MS/MS (to improve identification rates) while loading moderate sample amounts (which is optimal for quantitation as you minimize column overloading and chromatographic peak tailing) for the MS-only runs. For accurate quantification, the number of high quality MS spectra per chromatographic peak should be maximized. Also, undertaking separate LC−MS/MS analysis after LC−MS quantitation ensures MS/MS spectra of only significantly regulated peptides (which can be at low abundance and would therefore be missed in data-dependent autoMS(n) LC−MS/ MS runs) are acquired. To demonstrate this principle of labelfree analysis and reveal how peptides change during epididymal maturation, extracted ion chromatograms (EIC) are shown on two peptides for m/z values of 768.4 (Figure 2A) and 651.8 Da (Figure 2B). By overlaying 4 of the 20 biological replicates from the cauda (black) and the caput and corpus samples (blue and red respectively), it becomes evident that both peptides are higher in spermatozoa derived from the caput/corpus regions of the epididymis and absent when the sperm cell transits to the cauda (Figure 2). The inset shows the annotated MS/MS spectrum for m/z 768.4 (Figure 2A inset) and 651.8 (Figure 2B inset). The peptide sequences match to Fatty Acid Binding Protein 9 (FABP9; Figure 2A) and Outer Dense Fiber 1 (ODF1; Figure 2B). Table 1 provides information on the identification of peptides that undergo a significant (p < 0.05) change in this study. Column 1 lists the identification of the protein (ID).

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RESULTS

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Figure 2. EIC and tandem MS of phospho-peptides derived ODF1 and TLBP. TiO2 peptides were prepared and run on LC−MS according to Experimental Section. An EIC of m/z (A) 768.4 and (B) 651.8. from time 26−36 and 16−21 min, respectively. The caput samples are shown in blue, corpus in red and cauda in black (N = 4 biological replicates shown, N = 20 actually run). The inset shows the annotated spectrum for (A) LVSpSENDFENYVR and (B) KYpSYMNICK .

Where appropriate, the statistical information is given in column 2 (p-value). Columns 3−5 represent the “fold-change” observed for each peptide. As this is a three-way analysis (caput vs corpus vs cauda), the change in peptide intensity (foldchange), is presented as normalized data. For example, the first peptide present in Table 1 (LVSSENFENYVR, MS/MS number 1) is present in caput, corpus and caudal spermatozoa (column 3−5). However, this peptide increases 2.65-fold (column 5) in the cauda-derived spermatozoa, compared to the caput and corpus (both of which have been given an arbitrary value of 1). This 2.65-fold difference is significant when the cauda sample is compared to both the caput and corpus samples (p = 0.0018). The “−” symbol indicates when a peptide was not found in the sample, (for example, MS/MS number 14, cauda). Under these circumstances, the normalized fold change is depicted as “∞”. In the case of MS/MS number 14, the peptide is present in the caput and corpus, but completely absent in the cauda. However, there is no significant difference between the amount of peptide present in the caput and corpus regions. In addition to the fold change, the observed mass in which the MS/MS data was taken (column 6), the difference in mass between the observed and theoretical mass (column 7), charge (column 8), gene symbol (column 9) and peptide (column 10) are given. The boldface resides within the peptide indicate the unambiguous post-translational modifications, which, besides phosphorylation (STY), include oxidation (M) and deamidation (NQ). In the case of an ambiguous phosphorylation site, the peptide was reported as containing one or more phosphate groups (as denoted by a “p” or “pp” etc.) followed by the peptide sequence in parentheses. Finally,

the Mascot score for either the CID (column 11) or ETD (column 12) and the MS/MS file number that matches with Supplementary data 1 (Supporting Information) (column 13) are shown. Every individual tandem mass spectrum together with the residue annotation is also documented Supplementary data 1 (Supporting Information). An example of the changing peptides from m/z 640−670 Da using the survey view scan can be seen in Figure 3. Peptides that change are circled and the amino acid sequence from the peptide is given on the right-hand side. A changing peptide comes from ODF1 with the sequence KYSYMNICK, appearing either as a phosphopeptide (green), with methionine oxidation (orange and black) and/or aspartic acid deamidation (purple; note, the carbamidomethylation was intentionally ignored as iodoacetamide was used). This peptide significantly decreased in intensity during epididymal maturation (see also Figure 2A). Along a similar line, peptides derived from FABP9 (pink circle), A-kinase anchoring protein 4 (AKAP4, brown circle) and the ATP binding cassette F, all decrease in intensity during epididymal maturation. Phosphorylation of Izumo1 during Epididymal Transit

Izumo1 is a novel member of the immunoglobulin superfamily (IgSF), and the only molecule in sperm cells that has been definitively identified as being essential for sperm-egg fusion.35 The extracellular region of Izumo1 lacks sequences like those found in viral fusion peptides, and as such, it is hypothesized that Izumo1 is unlikely to be inherently fusogenic. A more likely possibility is that Izumo1 serves as an adhesion molecule, mediating cell−cell adhesion.36 In our label-free phosphopeptide analysis, we found the Izumo1 was differentially 5256


ID

5257

5′ Nucleotidase

Outer Dense Fiber 1

Spermatogenesis Associated 18

A−Kinase Anchoring Protein 4

Testis Lipid Binding Protein

caput 1 1 ∞ 1 1 3.09 1.88 2.74 2.59 1.72 5.58 7.04 4.35 ∞ 1.99 8.89 2.31 − − ∞ − 1 1 − ∞ ∞ 2.45 2.45 2.45 2.45 2.45 2.45 2.45 ∞ ∞ 1 1 1 − −

p-value

00018 0.01859 0.0018 0.0455 0.00586 0.035 0.00458 0.00489 0.00021 0.00323 0.002 0.0015 6.03 × 10−11 n/a 0.00002 0.00002 0.00003 n/a n/a n/a n/a 0.32281 0.02932 n/a n/a n/a 0.0074 n/a 0.00002 000002 0.00002 0.00001 n/a n/a n/a 0.00006 8.33 × 10−10 2.00 × 10−4 n/a n/a

1 1 ∞ 1 1 3.09 1.88 2.74 2.59 1.72 5.58 7.04 4.35 ∞ 1.99 8.89 2.31 ∞ ∞ ∞ − 1 1 − ∞ ∞ 2.45 2.45 2.45 2.45 2.45 2.45 2.45 ∞ ∞ 1 1 3.5 ∞ ∞

corpus 2.65 1.18 − 2.74 3.33 1 1 1 1 1 1 1 1 − 1 1 1 − − − ∞ 1.42 1.2 ∞ − − 1 1 1 1 1 1 1 − − 3.95 3.73 3.5 − −

cauda

normalized fold change

768.9 808.8 768.8 680 888.5 865.35 792.95 759.9 617.8 717.6 549.8 589.8 653.8 625.9 512.3 768 699 701.7 559.3 740.4 813.25 1030.5 1031 634.3 804.8 608.42 644.1 644.3 765 651.8 652.2 652.8 764.9 804.9 608.69 495.9 665.3 567.8 1075.93 1043.87

observed mass

Table 1. Significant Phosphopeptide Changes during Epididymal Maturation

z 2 2 2 3 2 2 3 2 2 3 2 2 2 2 3 2 3 3 2 3 2 2 2 3 2 2 2 2 2 2 2 2 2 2 3 2 2 2 2 2

Δm/z (ppm) −52 77 94.82 36.19 −26.47 36.84 −20.38 20.74 −573.11 39.51 52.64 100.51 27.89 27.87 27.83 64.33 18.34 570.78 −42.08 12 85 61.85 −53.4 35.82 −500.1 116.42 41.57 551.4 60.3 29.54 58.3 54.56 63.54 30.21 89.72 53.4 51.55 68.85 49.11 4 −65

match TLBP TLBP TLBP TLBP TLBP AKAP4 AKAP4 AKAP4 AKAP4 AKAP4 AKAP4 AKAP4 AKAP4 AKAP4 AKAP4 AKAP4 AKAP4 AKAP4 AKAP4 AKAP4 SPT18 SPT18 SPT18 SPT18 ODF1 ODF1 ODF1 ODF1 ODF1 ODF1 ODF1 ODF1 ODF1 ODF1 ODF1 5NT1B 5NT1B 5NT1B 5NT1B 5NT1B

peptide LVSSENFENYVR LVSSENFENYVR LVSSENFENYVR VACLIKPSVSISFNGER p(SLITFEGGS)MIQIQR pp(SLTSAERVS)EHLK RPEDQSQDSTEMDFISGMK GYSVGDLLQEVMK NQSLEFSAMK ppp(KIASEMAHEAVELTSSEMRG) ISPSTDSLAK ISPSTDSLAK DSKEFADSISK NQSLEFSAMK GYSVGDLLQEVMK GYSVGDLLQEVMK p(QQMCPKDSKEFADSISK) p(IASEMAHEAVELT)SSEMR LSSLVIQMARK p(GLMVYANQVASDMMVSVMK) LAQSESFLSLQDK NGSAISLLAAEEEINQLK NGSAISLLAAEEEINQLK RNSERPQDWSNYEK DVTYSYGLGSCVK LYCLRPSLRSLER KYSYMNICK KYSYMNICK p(DVTYSYGLGSCVK) KYSYMNICK KYSYMNICK KYSYMNICK DVTYSYGLGSCVK DVTYSYGLGSCVK LYCLRPSLRSLER7 YSKESLDAEKR YSKESLDAEK pp(HAITIAVSSR) pp(LASEMAHEAVELTSSEMRG) plASEMAHEAVELTSSEMRG) 51.1 36.9 44.5

53 26.6 41.3

49.0 29

40.3

49 55.6 45.9 57.7

53

47.8 39.1 30

52 69

45

58 40.2

Mascot score (CID)

53.4 38.8 48.6

61.6

61.6 34.4 25.5

53.4

76.5 42

44.8 64.9

55.5 82

35.8 31.9 50.4

51.7

79.7 63.1

Mascot score (ETD)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 10 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

suppl. one MS/MS file

Journal of Proteome Research Article


5258

Testes development−related NYD− SP18

G−protein coupled receptor 64 precursor (Epididymis−specific protein 6) Ornithine Decarboxylase Antizyme 3

Testis Specific Serine Kinase 1 Electrogenic sodium bicarbonate cotransporter 1 Glyceraldehyde−3−phosphate dehydrogenase, testis−specific

Uncharacterized protein C7orf31 Spermatogenesis Associated 19 Protein phosphatase 1 regulatory subunit 7

Uncharacterized Protein C10orf62

Sperm Acrosome Membrane Associated 1

A−Kinase Anchoring Protein 3

Izumo1

Dynein Intermediate Chain 1 cAMP−dependent protein kinase type II−alpha regulatory subunit Saccharopine dehydrogenase

ID

Table 1. continued

corpus − ∞ 1 − − − − 11 ∞ ∞ ∞ 1 1 − − ∞ ∞ 1 − ∞ ∞ − − ∞ ∞ ∞ ∞ ∞

caput − ∞ 1 − − − − 11 ∞ ∞ ∞ 1 1 − − ∞ ∞ 1 − ∞ − − − ∞ ∞ ∞ ∞ ∞

0.00006 n/a n/a n/a n/a 0.00004 n/a n/a n/a 0.19679 0.00007 n/a n/a n/a n/a 0.002 n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a

p-value n/a n/a

− − −

∞ ∞ − −

4.02 ∞ ∞ ∞ ∞ 1 − − − 1.89 14.3 ∞ ∞ − − 2.1 ∞ − −

∞ −

cauda

normalized fold change

827.33 787.3609 639.77

859.4 858.4 561.7 521.8

502.2 502.3 809.9 734.6 787.9 638.7 866.399 1071.44 742.69 1084.7 1058 610.004 750.3837 778.4 389.7 742.7 887.3 817.1 660.9

711.3 751.9

observed mass

−10.7 5.22 24.25

611.24 543.40 133.49 130.87 2 2 2

2 2 2 2

3 2 2 3 3 3 2 2 2 3 3 3 2 3 2 3 2 3 3

−405.91 47.77 50.54 −492.37 −416.63 134.77 85.57 30.9 173.05 99.92 34.72 131.95 773.05 68.41 41.05 −56.6 40.52 69.73 31.2

z 2 2

23.21 112.68

Δm/z (ppm) match

OAZ3 OAZ3 DUF3699

G3PT G3PT GPR64 GPR64

SCPDH SCPDH IZUM1 IZUM1 IZUM1 IZUM1 AKAP3 AKAP3 AKAP3 Spacal SACA1 C10ORF62 C10ORF62 C7orf31 SPATA19 PP1R7 PP1R7 TSSK1 SL4a4

DNAI1 KAP2

peptide

ppp(SRPSLYSLS)YIKR pp(SRPSLYSLS)YIKR pp(AGSSVLDLSNR)

pp(RVPTPNVSWDLTCRL) pp(RVPTPNVSWDLTCRL) RKTSIQDLRS RKTSIQDLRS

p(SVSNLKPVPVIGSK) p(SVSNLKPVPVIGSK) NSNVENKTSAAEFK LSQAEFHTDp(SS)DKVEEADN Lppp(SQAEFHTDSS)DKVEEADN SKNSNVENKTSAAEFK VIVSHNLADTVQNK p(SLSTVASELVNETVSACSKN) DKSESYSLISMK.S YKDSTSLDQSPTDIPVHEDDALSEWNE YKDSTSLDQSPTDIPVHEDDALSEWNE HGEGGVALHRDSFASK ISGTSVSKEMQR p(DMGSLFDLHSLPK) SVSHLR pp(HGGGIVADLSQQSLKDGVER) GAGQQQSQEMMEVDR TFCGSAAYAAPEVLQGIPYQPKV KERISENYSDK

DELVAGSQESVK MFGSNLDLLDPGQ

25.8 35 36.3

42.1 38.9

40.8 52.2 34.6

41.4

47.5 56.6 36.5 37.4 49.9

35.4 35.1 58.1

30.4

Mascot score (CID)

32 28.2

33

25.3

59.3 20.5

59.2

42.2 45.6

50.5

Mascot score (ETD)

66 67 68

62 63 64 65

43 44 45 40 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61

41 42

suppl. one MS/MS file

Journal of Proteome Research Article



Article

abundant in caput-derived spermatozoa. This suggests that Izumo1 is heavily modified during epididymal transit. To confirm this, 2D-SDS-PAGE followed by immunoblot analysis was performed using an anti-Izumo1 antibody on both caput- (Figure 6A) and cauda- (Figure 6B) derived spermatozoa. While the caput sperm extract demonstrated one dominate band at the predicted molecular weight (43 kDa) and pI (5.5) for Izumo1 (Figure 6A), the caudal spermatozoa demonstrated at least one additional isoform (Figure 6B) that focused toward the positive region. This would suggest that Izumo1 undergoes post-translational modification during epididymal maturation, becoming more negatively charged. To confirm phosphorylation of Izumo1, the cauda-derived spermatozoa were treated with alkaline phosphatase. On 1DSDS-PAGE (Figure 6C), the treated sample (T) ran further into the gel compared to the boiled Alkaline phosphatase control (C). This phenomenon is seen when proteins contain multiple negative charges and repel the SDS that cause it run at an apparent higher mass than a protein of the same size would normally.37. On 2D-PAGE, Izumo1 in the caudal epididymal sperm sample treated with alkaline phosphatase appeared as a smear, as compared with 2 semidiscrete spots (Figure 6D) when the same sperm sample was treated with the boiled (inactive) phosphatase (Figure 6E). This result may reflect the underlying glycosylation status of Izumo1 when the phosphate groups were removed. Taken together with our phosphoproteomic analysis, the data strongly suggest that protein phosphorylation is occurring on Izumo1 during epididymal maturation to generate a quaternary phosphorylated protein comprising S446 and three phosphorylations on either S366, T372, S374 or S375. Such events may be beneficial for the role of Izumo1 during sperm-egg fusion.

Figure 3. Survey view demonstrating matching of peptides, peptide changes during epididymal maturation and identification. TiO2 peptides were prepared and run on LC−MS according to Experimental Section. A small snapshot of the peptides eluting from m/z 640−670 is shown for 1 run from the caput (top), corpus (middle) and cauda (bottom) samples. Peptides that have undergone a significant change are circled. These were targeted for MS/MS acquisition, and the interpretation of the spectrum and amino acid resides are presented and color coded for each (circled) peptide. The circled peptides also contain the identification of the protein.

phosphorylated. The EIC of the four changing peptides from Izumo1 with mass (A) 809.9, (B) 734.6, (C) 638.7, and (D) 787.9 are shown in Figure 4. The corresponding annotated MS/MS data files can be found in Figure 5A−D. While three of the four peptides were found predominately in the caudal spermatozoa only, the forth (m/z 638.6, Figure 4C), was more

Figure 4. EIC of phosphopeptides derived from Izumo1 during epididymal transit. TiO2 peptides were prepared and run on LC−MS according to Experimental Section. (A−D) An EIC of m/z 809.9, 734.6, 638.3, and 787.9 all correspond to peptides derived from Izumo1. The caput samples are shown in blue, corpus in red and cauda in black (N = 4 biological replicates shown, N = 20 actually run). 5259



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Figure 5. Annotated MS/MS of phosphopeptides derived from Izumo1 during epididymal transit. TiO2 peptides were prepared and run on LC−MS according to Experimental Section. (A−D) Annotated MS/MS spectrum showing the different ion series for the parent masses m/z 809.9, 734.6, 638.3, and 787.9, all of which are derived from Izumo1.

Figure 6. 1D and 2D anti-Izumo1 Western blots depicting the phosphorylation status of Izumo1 during epididymal transit. Sperm taken from the (A) caput or (B) cauda were lysed and subject to 2D-PAGE followed by immunoblot blot transfer according to the Experimental Section. The blot was probed with anti-Izumo1 antibody. Note the additional Izumo1 charge isomer in the caudal epididymal spermatozoa (arrowed). (C) Caudal derived sperm lysate was treated with (lane 2) and without (lane 1) alkaline phosphatase. The position of the molecular weight markers are shown on the left-hand side. (D) Nontreated caudal sample and (E) alkaline phosphatase treated sample were then run into 2D-PAGE.

■

DISCUSSION In this study, we have examined and quantified the expression of TiO2 enriched phosphopeptides in spermatozoa recovered from distinct regions of the epididymis. This design has permitted us to visualize the global trends that take place in spermatozoa at the phosphoproteomic level as these cells engage the process of post−testicular maturation and acquire functional competence.

peptide. Although at times both CID and ETD spectra were obtained for a given peptide (e.g., MS/MS 14), this was the exception. Figure 7 shows a Venn Diagram that illustrates the cross over between CID and ETD. A staggering 56% of peptides were found by CID only, 41% by ETD and only 3% of peptides found by both technologies (Figure 7). The number in parentheses indicates the percentage of the total number of peptides with unambiguous phosphorylation sites for each fragmentation technology. These data clearly suggest that CID and ETD are complementary in nature for the detection of phosphopeptides.

CID vs ETD Comparison

Of particular interest from a proteomic perspective was the comparison between CID and ETD fragmentation, in order to identify and characterize phosphopeptides of interest. Table 1 demonstrates which fragmentation was used to identify the 5260



Article

A second protein shown to undergo a significant change during epididymal maturation, is the electrogenic sodium bicarbonate cotransporter 1, (SLc4a4), which was found to undergo tyrosine phosphorylation during epididymal transit (MS/MS number 60). In 2003, Demarco et al. predicted that a Na+-dependent bicarbonate (HCO3−) cotransporter is present in sperm cells.43 While it has been known for some time that high concentrations of bicarbonate exist in the seminal plasma and female reproductive tract secretions when compared to epididymal fluid,44 it was unclear how the HCO3− became incorporated into the sperm cell, in order to stimulate soluble adenylyl cyclase (ADCY10), a major regulator of sperm capacitation.45,46 Demarco et al.43 demonstrated that through sequential replacement of the buffer with various salts, that a HCO3− cotransporter probably exists in these cells and plays a significant role in inducing hyperpolarization of the sperm plasma membrane during capacitation. The present study not only confirms the presence of Slc4a4 in spermatozoa for the first time, but also reveals that this transporter is tyrosine phosphorylated in the corpus region of the epididymis (MS/ MS number 70). Given the fact that ion transporters play an indispensable role in sperm maturation, we hypothesize that SL4a4 is likely to be involved very early in the capacitation process. Upon ejaculation, mammalian spermatozoa experience a significant elevation in the amount of extracellular Na+, from 30 mM in the cauda epididymis to 100−150 mM in the seminal plasma.47 At this moment, SL4a4 may exploit the rise in extracellular Na+ as a signal to cotransport one Na+ ion together with three HCO3− ions across the plasma membrane. The epididymis may be preparing SL4a4 for this early event through the phosphorylation site identified in this study. Transient protein phosphorylation during epididymal maturation may be important in establishing protein−protein interactions needed to create a functionally competent channel poised to become activated at the moment of ejaculation HCO3− together with Ca2+ are known to stimulate ADCY10 which, in turn, leads to a rise in [cAMP]I and the initiation of capacitation. Supporting this notion, replacement of extracellular HCO3− with HEPES inhibits capacitation in many species,48−51 suggesting that this cation is actively transported into the sperm cell. Furthermore, addition of 4,4′-Diisothiocyano-2,2′-stilbenedisulfonate (DIDS) and/or 4-acetomido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS), known inhibitors of SL4a4, inhibits capacitation.6 However, these data need to be interpreted carefully since both DIDS and SITS are not specific but will also act on Cl−/ HCO3− antiporters. Although two types of Slc4a4 knockout mice have been produced, these animals cannot confirm the involvement of SL4a4, because they die before reproductive age as a consequence of severe acidemia;52,53 thus only a spermspecific conditional knockout of this transporter will definitively establish its role in capacitation.

Figure 7. Venn diagram comparing CID and ETD phosphopeptide identifications. The percentage of total spectra identified in this study using either CID only, ETD only or both is shown. The number in parentheses designates the number of phosphopeptides from which we were able to obtain unambiguous phosphorylation site identification.

Epididymal Maturation “Prepares” Proteins for Capacitation

One intriguing facet of this study was that a number of the proteins found to change during epididymal maturation have previously been documented to have a major role in a secondary post-testicular maturation process, capacitation. For example, the peptide derived from FABP9, LVSSENFENYVR, was shown in this current study to be phosphorylated during epididymal transit. This suggests that the third serine in the peptide is a major site of regulation, since others have previously shown the same peptide to be phosphorylated during capacitation.38 Interestingly, quantification in the previous example was performed using deuterated and nondeuterated phosphopeptides that were enriched by immobilized metal affinity chromatography (IMAC). Similarly, A-kinase anchoring protein 4 (AKAP4) undergoes 13 phosphopeptide changes from the caput to the cauda [note, the phosphopeptide GYSVGDLLQEVMK (MS/MS number 16) change is likely to be the result of a deamidation event on an already accounted for phosphopeptide (MS/MS number 17). Two of these peptides (MS/MS 8 and 20), have also been previously reported,38 with the latter, LSSLVIQMARK, demonstrating a 4.62-fold increase in Ser3 during capacitation. This peptide is found within a region of AKAP4 involved in binding the regulatory subunits (RI and RII) of protein kinase A (PKA), which confines the holoenzyme to the midpiece and tail section of sperm cells.39,40 AKAP4 has a domain that will bind RIα and RIIα (AA 219−232) and a secondary domain that can only bind RIα (AA 335−344). Intriguingly, a phosphorylation on S226 was found that is only present in spermatozoa derived from the corpus epididymis (peptide LSSLVIQMAKR, MS/MS number 19), suggesting that this interaction is regulated during epididymal transit. Our label-free phosphoproteomics study also revealed a phosphopeptide on RIIα itself. In this case, when comparing the caput/corpus spermatozoa with the caudal-derived sperm cells, a complete loss of pS395 (MS/MS number 42) was found, demonstrating that the post-translational modification status of RIIα in caput/ corpus spermatozoa is different to sperm from the cauda. However, the significance of this regulation is unclear, considering that genetic knockout studies have shown that RIα, but not RIIα is indispensable for successful fertilization.41,42 Given the fact that AKAP4 is phosphorylated on the site at which RIIα binds, and RIIα itself is phosphorylated, it is plausible that the two “negative” charges may repel one another, thereby preventing this interaction from occurring during the early stages of epididymal transit and facilitating the maintenance of spermatozoa in a quiescent state.

Regulation of Izumo1 during Epididymal Transit

One of the major findings from this epididymal phosphoproteomics study was the post-translational phosphorylation of Izumo1. Animal knockout studies demonstrate that Izumo1 is critical for sperm-egg fusion35 and the most likely explanation, at a molecular level, is that Izumo1 functions through protein− protein interactions.36 However, the candidate egg protein(s) that Izumo1 interacts with have yet to be identified. We found Izumo1 to be multiply phosphorylated in the caudal epididymal region, as depicted in our 2D Western blot analysis, which demonstrated the presence of a second protein species cross 5261



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suggesting similar mechanisms of regulation. This site is also present in caudal spermatozoa (MS/MS 45). However, the second site, S339 is conserved in the mouse (S353), but alignment with the human sequence demonstrated no homologous region, suggesting a rodent-specific mechanism of regulation at this region for Izumo1. By using the information on the other Izumo1 peptides (MS/MS 46 and 47), it is evident that rat caudal spermatozoa have a phosphorylation event on three of four residues (ambiguous site phosphorylation), which include S366, T372, S374 or S375. Interestingly, all these amino acid residues are homologous, or conservatively substituted in the mouse. However, the human homologue only shows significant alignment with the T365 (conservatively substituted with S366 of the mouse), again suggesting a different mechanism or residue involved in human Izumo1 regulation. As for the regulation of Izumo1 phosphorylation, several unique kinases exist that are only expressed in the testis. One such family member is the testis specific kinase 6 (TSSK6). Tssk6 knockout mice are healthy and females have normal fertility.54 In contrast, Tssk6−/− males are completely infertile with reduced sperm counts, impaired motility and morphological abnormalities. Spermatozoa from the TSSK6 −/− spermatozoa cannot fuse with the egg,54,55 suggesting that Izumo1 in these animals may not normally be regulated. Hence, TSSK6 could be a direct mediator of Izumo1 function. Future studies will investigate this possibility.

reacting with the anti-Izumo1 antibody (Figure 6). This observation was ratified by the addition of alkaline phosphatase, which, upon removal of the phosphate groups, led to increased migration in SDS-PAGE (Figure 6C) and changes in 2D-PAGE mobility (Figure 6A/D versus E). Mapping of the residues demonstrated that all of the phosphorylation changes observed within Izumo1 were present in the very small cytoplasmic region of this protein. A schematic depicting the phosphorylation status of Izumo1 during epididymal development is shown in Figure 8. In terms of the biological relevance of these

Dynein Intermediate Chain 1

Figure 8. Schematic diagram representing the major post-translational phosphorylation states of Izumo1 during epididymal transit. (Top) Izumo1 from the caput possess at least two phosphorylation sites, on residues 339 and 446. While S339 appears to be lost during epididymal transit, a further three phosphorylation sites were detected in the more mature sample.

A phosphopeptide derived from dynein intermediate chain 1 (DNAI1) was found only in caudal epididymal spermatozoa (MS/MS 41). Interestingly, this phosphopeptide appears to be conserved among species, as the identical sites of phosphorylation were also reported in human spermatozoa using IMAC enrichment followed by LC−MS,56 suggesting a fundamental role in the regulation of sperm motility If caput/corpus spermatozoa are diluted into medium BWW, they are unable to exhibit any forward progressive movement, whereas caudal spermatozoa suddenly burst into a state of vigorous sustained motility. Although the mechanisms responsible for the regulation of motility activation are poorly understood, it is possible that this phosphorylation change on dynein is critically involved in the mechanisms by which the potential for sperm movement is acquired.57

changes, it may be significant that the localization of Izumo1 changes following the acrosome reaction from the entire head to the postacrosomal region.54 For this relocation to occur, it is likely that Izumo1 becomes associated with other proteins. It is these protein−protein interactions that might be regulated by the phosphorylation status of this molecule. In order to determine the universality of such a mechanism a CLUSTALW analysis was undertaken to align and compare the phosphorylated regions of rat Izumo1 with both mouse and human Izumo1. In this case, all of the phosphorylation changes observed in this study were present between residues 336−351 and 364−382 of rat Izumo1. The amino acid sequence of these regions, together with the mouse and human homologues sites are given shown (Table 2). On the basis of MS/MS file number 48 (Table 1), we have shown that residues S339 and S346 are more highly phosphorylated in caput/corpus epididymal spermatozoa. Interestingly, S346 aligns with mouse T359 and human S326,

■

SUMMARY In summary, we have identified 68 phosphopeptide changes that occur as sperm travel down the epididymis. The majority of the changes occur as the spermatozoa leave the corpus and enter the caudal epididymal zone. We have been able to identify 22 proteins that appear to change as spermatozoa mature within the epididymal lumen and gain the capacity to fertilize the oocyte. This analysis has provided several new leads concerning the post-translational modifications that drive this unique cell into a state of functional maturity.

Table 2. Alignment of Rat Izumo1 Phosphorylated Residues with Mouse and Human Rat: Mouse Human:

■

(336)

SKNSNVENKTSAAEFK(351).............(365) LSQAEFHTDSSDKVEEAD(382) (349) LKNASDEVKPTASGSK(364).............(376) ASQADFNSDYSGDKSEAT(395) (321) --------- DFIKSSLFGLG(331).............(364) ATDSRQQ-----------------(383)

ASSOCIATED CONTENT

S Supporting Information *

Spectra. This material is available free of charge via the Internet at http://pubs.acs.org. 5262



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(17) McLaughlin, E. A.; Frayne, J.; Barker, H. L.; Jury, J. A.; Jones, R.; Ford, W. C.; Hall, L. Cloning and sequence analysis of rat fertilin alpha and beta–developmental expression, processing and immunolocalization. Mol. Hum. Reprod. 1997, 3 (9), 801−9. (18) Frayne, J.; Jury, J. A.; Barker, H. L.; Hall, L. The MDC family of proteins and their processing during epididymal transit. J. Reprod. Fertil. Suppl. 1998, 53, 149−55. (19) Zhu, G. Z.; Myles, D. G.; Primakoff, P. Testase 1 (ADAM 24) a plasma membrane-anchored sperm protease implicated in sperm function during epididymal maturation or fertilization. J. Cell Sci. 2001, 114 (Pt 9), 1787−94. (20) Saxena, D. K.; Oh-Oka, T.; Kadomatsu, K.; Muramatsu, T.; Toshimori, K. Behaviour of a sperm surface transmembrane glycoprotein basigin during epididymal maturation and its role in fertilization in mice. Reproduction 2002, 123 (3), 435−44. (21) Zhang, H.; Jones, R.; Martin-DeLeon, P. A. Expression and secretion of rat SPAM1(2B1 or PH-20) in the epididymis: role of testicular lumicrine factors. Matrix Biol. 2004, 22 (8), 653−61. (22) Rutllant, J.; Meyers, S. A. Posttranslational processing of PH-20 during epididymal sperm maturation in the horse. Biol. Reprod. 2001, 65 (5), 1324−31. (23) Sabeur, K.; Cherr, G. N.; Yudin, A. I.; Primakoff, P.; Li, M. W.; Overstreet, J. W. The PH-20 protein in human spermatozoa. J. Androl. 1997, 18 (2), 151−8. (24) Cowan, A. E.; Myles, D. G.; Koppel, D. E. Lateral diffusion of the PH-20 protein on guinea pig sperm: evidence that barriers to diffusion maintain plasma membrane domains in mammalian sperm. J. Cell Biol. 1987, 104 (4), 917−23. (25) Ijiri, T. W.; Merdiushev, T.; Cao, W.; Gerton, G. L. Identification and validation of mouse sperm proteins correlated with epididymal maturation. Proteomics 2011, 11 (20), 4047−62. (26) Chakrabarti, R.; Cheng, L.; Puri, P.; Soler, D.; Vijayaraghavan, S. Protein phosphatase PP1 gamma 2 in sperm morphogenesis and epididymal initiation of sperm motility. Asian J. Androl. 2007, 9 (4), 445−52. (27) Lin, M.; Lee, Y. H.; Xu, W.; Baker, M. A.; Aitken, R. J. Ontogeny of Tyrosine Phosphorylation-signaling pathways during spermatogenesis and epididymal maturation in the mouse. Biol. Reprod. 2006. (28) Baker, M. A.; Smith, N. D.; Hetherington, L.; Pelzing, M.; Condina, M. R.; Aitken, R. J. Use of titanium dioxide to find phosphopeptide and total protein changes during epididymal sperm maturation. J. Proteome Res. 2011, 10 (3), 1004−17. (29) Kim, M. S.; Pandey, A. Electron transfer dissociation mass spectrometry in proteomics. Proteomics 2012, 12 (4−5), 530−42. (30) Baker, M. A.; Krutskikh, A.; Curry, B. J.; McLaughlin, E. A.; Aitken, R. J. Identification of cytochrome P450-reductase as the enzyme responsible for NADPH-dependent lucigenin and tetrazolium salt reduction in rat epididymal sperm preparations. Biol. Reprod. 2004, 71 (1), 307−18. (31) Baker, M. A.; Krutskikh, A.; Curry, B. J.; Hetherington, L.; Aitken, R. J. Identification of cytochrome-b5 reductase as the enzyme responsible for NADH-dependent lucigenin chemiluminescence in human spermatozoa. Biol. Reprod. 2005, 73 (2), 334−42. (32) Baker, M. A.; Lane, D. J.; Ly, J. D.; De Pinto, V.; Lawen, A. VDAC1 is a transplasma membrane NADH:ferricyanide reductase. J. Biol. Chem. 2004, 279, 4811−9. (33) Larsen, M. R.; Thingholm, T. E.; Jensen, O. N.; Roepstorff, P.; Jorgensen, T. J. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol. Cell. Proteomics 2005, 4 (7), 873−86. (34) Hartmer, R.; Kaplan, D. A.; Gebhardt, C. R.; Ledertheil, T.; Brekenfeld, A. Multiple ion/ion reactions in the 3D ion trap: Selective reagent anion production for ETD and PTR from a single compound. Int. J. Mass. Spectrom 2008, 276 (2−3), 82−90. (35) Inoue, N.; Ikawa, M.; Isotani, A.; Okabe, M. The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs. Nature 2005, 434 (7030), 234−8. (36) Schultz, R.; Williams, C. Developmental biology: sperm-egg fusion unscrambled. Nature 2005, 434 (7030), 152−3.

AUTHOR INFORMATION

Corresponding Author

*Discipline of Biological Sciences, Faculty of Science and Information Technology, University of Newcastle, Callaghan, NSW, 2308, Australia. Phone: +61-2-4921 7880. Fax:+ 61-24921 6308. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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