Label-Free Quantitation of Phosphopeptide Changes During Rat

Label-Free Quantitation of Phosphopeptide Changes During Rat Sperm Capacitation Mark A. Baker,*,†,‡ Nathan D. Smith,‡ Louise Hetherington,‡ Kristy Taubman,‡ Mark E. Graham,§ Phillip J. Robinson,§ and R. John Aitken†,‡ The ARC Centre of Excellence in Biotechnology and Development, Priority Research Centre in Reproductive Science, School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW, 2308, Australia, and Cell Signaling Unit, Childrens’ Medical Research Institute, The University of Sydney, Westmead, NSW, 2145, Australia Received June 10, 2009

Before fertilization can occur, ejaculated mammalian spermatozoa must undergo a maturation process known as capacitation, which is dominated by post-translational modifications, particularly phosphorylation. Despite its biological importance, characterization of those proteins targeted for phosphorylation during capacitation remains ill-defined. Here, we report the isolation and purification of 288 phosphorylated peptides from rat spermatozoa using titanium dioxide columns in combination with nanoflow mass spectrometry. This equated to 120 identified phosphorylated proteins present in pure populations of spermatozoa. The MS survey scans of replicate titanium dioxide eluates, derived from both noncapacitated and capacitated sperm lysates, were then compared in silico using a virtual 2D PAGE format and DeCyderMS software. This analysis found 15 differentially phosphorylated proteins during capacitation. Included in this list were sperm qualifiers such as Izumo, a known sperm-oocyte fusion protein. To demonstrate that this label-free quantitative approach to phosphoprotein analysis was viable, we measured the enzymatic activity of 5′-nucleotidase, the phosphorylation status of which changed during capacitation. The results revealed, for the first time, that 5′-nucleotidase activity is up-regulated as sperm capacitate. This change, together with the other protein identifications reported in this study, constitute important new leads in elucidating the biochemical mechanisms by which spermatozoa attain a capacitated state. Keywords: Sperm phosphorylation • Capacitation • Mass spectrometry • Proteomics • Spermatozoa • proteomics • LC-MS • phosphorylation • label-free quantitation • capacitation • 5′-nucleotidase

Introduction Following ejaculation, spermatozoa must undergo a series of biochemical transformations, collectively termed “capacitation”, in order to gain the ability to fertilize the egg.1 This time-dependent process takes place in vivo within the female reproductive tract and is central to the study of infertility,2,3 the development of novel approaches to male contraception4 and the design of improved media for in vitro fertilization.5 An ability to induce capacitation in vitro by incubating spermatozoa in defined culture media has permitted analysis of the molecular mechanisms that underpin this process.6,7 In this context, it should be noted that capacitation occurs in the complete absence of gene transcription;8 rather, it is the posttranslational modification (PTM) of key proteins that controls * To whom correspondence should be addressed: Discipline of Biological Sciences, Faculty of Science and Information Technology, University of Newcastle, Callaghan, NSW, 2308, Australia. Phone: +61-2-4921 7880. Fax: +61-2-4921 6308. E-mail: [email protected]. † The ARC Centre of Excellence in Biotechnology and Development, University of Newcastle. ‡ Priority Research Centre in Reproductive Science, University of Newcastle. § Childrens’ Medical Research Institute, The University of Sydney.

718 Journal of Proteome Research 2010, 9, 718–729 Published on Web 11/30/2009

this process and endows spermatozoa with the ability to bind to, and fertilize, the oocyte.9 Our current understanding of capacitation suggests that this cAMP-dependent process is mediated by an unusual bicarbonate- and calcium-regulated soluble adenylate cyclase (sAC).10 Functional disruption of this gene, or its end product, leads to male infertility associated with the impairment of sperm movement and a failure to undergo capacitation.9,10 The pivotal role of sAC is reflected in a progressive rise in intracellular cAMP levels during capacitation, in concert with a concomitant rise in cytosolic calcium concentrations.11 In turn, intracellular cAMP is known to dissociate the regulatory subunits of protein kinase A (PKAr) from their corresponding catalytic subunits (PKAc), releasing the latter in an active state and promoting a marked increase in serine/threonine based phosphorylations in capacitating spermatozoa.12-14 For the most part, these PKA targets remain ill-defined, although one signal transduction pathway has been clearly elucidated. Upon addition of a cell permeable derivative of cAMP to spermatozoa, a sudden global increase in tyrosinephosphorylation has been observed.12 By performing 2D gel analysis, and overlaying silver stained gels with anti-phospho10.1021/pr900513d

 2010 American Chemical Society

Protein Phosphorylation during Capacitation tyrosine Western blot spots, Ficarro et al. have characterized a number of the proteins that become tyrosine phosphorylated under these circumstances.15 The tyrosine phosphorylation of these proteins appears to have major biological significance, because inhibition of tyrosine kinase activity or disruption of the sperm specific PKAcRII that drives tyrosine phosphorylation effectively suppresses the capacitation process.13 Since PKA is a serine/threonine kinase, it cannot, of its own accord, be responsible for the increased tyrosine phosphorylation observed in capacitated spermatozoa. Recently, we have presented evidence that pp60c-src is the intermediate tyrosine kinase responsible for mediating the action of PKA on tyrosine phosphorylation during sperm capacitation.14 Despite the elucidation of this specific pathway, the other phosphorylation events involved in controlling capacitation have never been systematically explored. The ability to perform high-throughput proteomic analyses using mass spectrometry is a powerful tool for the characterization of post-translational events seen in many biological systems. Interest in characterizing the precise identity and location of amino acids undergoing phosphorylation has inevitably led to the development of a variety of procedures for the isolation of phosphorylated proteins and peptides. These methodologies include immunoprecipitation,16 covalent linking of phosphopeptides to beads,17 strong cation exchange,18 and immobilized metal affinity chromatography,19 all with their apparent advantages and disadvantages.20 Recently, metal oxides, including titanium dioxide (TiO2)21 and zirconium dioxide20 have shown great promise for the rapid, efficient isolation of near pure phosphopeptides. In light of these developments, we have used a phosphoproteomic approach, based upon a TiO2-mediated enrichment of phosphopeptides, to gain further insights into the molecular mechanisms controlling sperm capacitation. In these studies, nanoflow reversed phase chromatography, directly coupled to an ion trap or quadrupole time-of-flight mass spectrometer, was used for protein identification. To further enhance the ion trap phosphopeptide identification data, we evaluated the differential expression of phosphopeptides before and after capacitation using DecyderMS (v. 2.0) software. This program was employed to create a virtual 2D image of the survey scan, by plotting peptide retention time against m/z, and then statistically evaluating which peptides changed in relation to sperm capacitation. The MS/MS spectrum of the selected peptides was then used to determine their amino acid sequence and assign the site of phosphorylation within that sequence. Here, we report the existence of 288 unique phosphopeptides in rat spermatozoa, leading to the identification of 120 phosphoproteins. Following DeCyderMS analysis, 57 peptides were found to change significantly (P < 0.05) during capacitation, and of these, 45, 11, and 9 passed the false discovery rates (FDR) of Benjamini-Hochberg (B-H),22 Benjamini-Liu (B-L)23 and Bonferroni,24 respectively. These changes give us totally new insights into the mechanisms of sperm capacitation.

Experimental Section Materials. Unless otherwise stated, all chemical s were purchased from Sigma-Aldrich at the highest research grade available. Albumin and ammonium persulfate were obtained from Research Organics (Cleveland, OH); D-glucose, sodium hydrogen carbonate, sodium chloride, potassium chloride, calcium chloride, potassium orthophosphate and magnesium sulfate were all analytical reagent grade, purchased from Merck

research articles (BDH Merck, Kilsyth, Australia); ultrapure water, 2,5-dihydroxybenzoic acid (DHB) sodium sulfate, barium hydroxide 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 from ICN Biochemicals (Castle Hill, NSW, Australia) and acrylamide from Biorad (Castle Hill, NSW, Australia). The 96 well plates were from Greiner BioOne (Interpath Services, Heidelberg West, Vic., 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 weeks) were asphyxiated and the epididymides were removed. Pure suspensions of spermatozoa were obtained from the caudal region of the epididymis by back flushing, as previously described.14,25,26 Spermatozoa were gently isolated into BWW (Biggers, Whitten and Whittingham) medium25 containing 3 mg/mL BSA and allowed to disperse for 10-15 min to produce sperm suspensions in a noncapacitated state. To reliably and robustly drive the spermatozoa into a capacitated state, they were treated with 1 mM pentoxifylline and 1 mM dibutryl cAMP and incubated for 90 min. There were no significant differences in the motility of the spermatozoa before (89.3 ( 1.7%) and after (88.7 ( 1.3%) the induction of this capacitation process. Although there are alternative, valid strategies for creating populations of noncapacitated spermatozoa, including the omission of bicarbonate and albumin,15 our strategy was to recover these cells shortly after dilution as, arguably, one of the most physiological means of delivering populations of uncapacitated cells (see Discussion). Once the capacitated and noncapacitated sperm population had been prepared, they were centrifuged (800g for 3 min) to pellet the spermatozoa and separate these cells from any residual epididymal plasma. The pellet was then resuspended in BWW solution without BSA and this process was repeated 3 times. Approximately, 400 µL of lysis buffer consisting of 4% CHAPS, 7 M urea, 1/100 dilution of Halt phosphatase inhibitor (Pierce, Castle Hill, Australia) and 2 M thiourea in water was added to 1 × 107 sperm cells. The sample was lysed for 1 h at 4 °C with constant rotation, then centrifuged (16 000g for 15 min at 4 °C) and the supernatant transferred to a new Eppendorf tube. A protein estimation was subsequently performed using a 2D quant kit (G.E. Healthcare, Castlehill, Australia) and 150 µg of protein was precipitated using methanol/chloroform as described elsewhere.26 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, 1 U DNase was added for 1 h at 37 °C, following which, the sample was immediately centrifuged (16 000g for 15 min) at 4 °C and the supernatant taken and transferred to a 96 well Greiner plate to avoid the losses found in Eppendorf tubes due to peptides adhering to the plastic. Protein Estimation. Following methanol/chloroform precipitation27 of an aliquot corresponding to an equal amount (approx 5 µg) of protein from each sample, the precipitate was resuspended in 1% SDS, and run into a 10% SDS PAGE gel. The gel was silver stained as previously described27 and visually inspected to ensure the quantification had been accurate. Journal of Proteome Research • Vol. 9, No. 2, 2010 719

research articles Finally, to ensure the proteins in the original sample were fully digested, we loaded equal amounts of tryptic lysate from each sample onto a 10% SDS-PAGE. These samples were placed into bromophenyl-blue-free loading buffer. The gel was run half way, using prestained molecular mass markers as a guide, in order to visualize the peptides following silver staining. Western Blot. Anti-phosphotyrosine Western blot analysis was performed as described elsewhere.14 Phosphopeptide Enrichment. Purification and enrichment of phosphopeptides from the tryptic digest was performed by a similar method to that previously described.21 Tryptic peptides were diluted 5-fold in DHB buffer [350 mg/mL DHB, 80% acetonitrile (ACN), 2% trifluoroacetic acid (TFA)] and applied to dry TiO2 beads (200 µg). The sample was then washed 1× in DHB buffer, before being washed 1× with wash buffer (80% ACN, 2% TFA) to remove the DHB. The sample was then directly eluted into a 96 well plate using elution buffer (elution buffer consisted of 25 µL of 2.5% ammonium hydroxide, pH g 10.5). The sample was immediately neutralized with 0.3 µL of formic acid (1.2% final concentration). All buffers used ultrapure water and were made fresh on the day of experimentation. The eluates, typically in 25 µL, were then dried in a vacuum concentrator and resuspended in 10 µL of 0.1% formic acid. Mass Spectrometry. Separation of tryptic peptide mixtures was achieved by nanoscale reversed phase high pressure liquid chromatography (HPLC), in combination with online electrospray ionization (ESI)-MS. Primarily, the mass spectrometric analysis was performed on an LTQ linear ion trap system equipped with an ETD source (Thermo-Finnigan, San Jose, CA), although a QSTAR XL quadrupole time-of-flight MS (Applied Biosystems) was used to supplement the ion trap data (see below). Prior to ion trap analysis, a nano-MDLC system (Ettan MDLC, GE Healthcare, Castle Hill, Australia) was used for HPLC separation, employing a linear gradient of 0-60% buffer B (84% ACN, 0.1% formic acid) over 50 min. The C18 column system consisted of a trap (300 µm inner diameter × 5 mm length from Agilent Technologies, Palo Alto, CA) and a separation column (75 µm inner diameter × 150 mm length). For online coupling, a nano ion spray source was used, equipped with a New Objective ESI needle (10 µm silica tip). The needle voltage was 1.5 kV in positive ion mode. The scan cycle consisted of a survey scan (mass range 500-2000 amu) followed by MS/MS of the six most intense signals in the spectrum with an exclusion list for ion signals set to 25 s after one occurrence. For CID analysis, we used normalized collision energies set to 26 or 35; q ) 0.18 with an activation time set to 30 ms and the isolation width set to m/z 1.0. For ETD analysis, isolation width was set to m/z 3.0, collision energy of 35, q ) 0.250 with an activation time set to 100 ms. Analysis was performed in either CID only, ETD only or CID/ETD (ETCaD) modes. MS3 was used on either the neutral loss scanning event from the MS2 scan (-17,32,49 or 98 Da) and as an alternative, MS3 was performed on the top 3 most intense peaks found in the MS2 scan event. To enhance the phosphorylation data, the same TiO2 enriched spermatozoa samples were also analyzed using a nanoHPLC system (LC Packings Ultimate HPLC system, Dionex, Netherlands) connected online to a QSTAR XL quadrupole time-of-flight MS. The chromatography and gradient system was exactly as described previously28 except that the gradient from 10% phase B to 50% phase B was extended to 48 min in total. Data files in the Analyst QS 1.1 wiff format (Applied Biosystems) were converted to dta format using WIFF to dta version 1.1.11.29 720

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Baker et al. DecyderMS Analysis. LTQ files of the survey scan performed in profile mode were imported into DecyderMS (v 2.0). Following visual interpretation of a complete DecyderMS scan, the image was cropped before the appearance of the first peptide and following the appearance of the last eluted peptide. Typical cropped images covered retention times of 25-55 min. These images were then used for peptide analysis in the pepdetect module. The standard filters were used with the following modifications: signal-to-background threshold set to 10, with background subtracted quantification checked, charge assignment was required, with limited charge assignment from two peaks; peptides below a signal-to-noise ratio of 4 and an unspecified charge, together with overlapping peptides were removed. A typical run consisted of a peak width of 0.7 min. Following detection, every peptide was manually inspected for accuracy of charge and correct peak assignment. In cases of incorrect charge assignment, the m/z was manually modified. Peptides were manually adjusted in cases of incorrect detection, or added in cases where the program had clearly missed the peptide. Once the set of assignments had been made for all images, they were imported into the pep-match module, the retention times aligned using the standard parameters, and the peptides matched using the aligned retention times. Matching tolerances were 1.5 min elution time and a m/z of 0.5 Da. Statistical Analysis. Following importation into DecyderMS, the program normalized the data based upon total peptide counts to compensate for intensity differences between the elution profiles. The peptide counts were based upon the intact peptide (in contrast to spectral counts which use the daughter ions). The program integrates the designated area under the curve based upon the MS-scan for each individual peptide and assigns a Log2 intensity value (if a peptide had multiple charge states, the sum of all the states was collated). Peptides within different runs were matched based upon m/z, charge and elution times. Peptide matching was manually confirmed using MS/MS spectral comparison. Students t-test were then performed for each group (noncapacitated state, n ) 6 vs capacitated, n ) 5, due to poor needle spray on one run). For statistical evaluation, we only took into account those peptides that were matched in at least 9 of the 11 profiles. In this instance, 154 peptides were detected. As label-free quantification is a relatively new concept to mass spectrometry, there is no standard protocol by which the data should to be interpreted. Therefore, we have reported all 154 peptides with t-test results indicating that a change occurred during capacitation with a probability of P < 0.05. Also, we applied three FDR algorithms as described;24 these included B-H,22 B-L23 and Bonferroni24 corrections. Those peptides significant for each correction are clearly labeled. Mass Spectrometric Data Interpretation. The derived mass spectrometry data sets were converted to generic format (*.dta) files using the Bioworks Browser (v3.3.1). These files were then searched against the IPI database (v3.36 containing 42 688 proteins) using the Bioworks (v3.3.1) search algorithm, TurboSequest (v3.3.1; Thermo-Finnigan). The species subset was set at Rattus norvegicus, the number of allowed trypsin missed cleavages was set to 1.0, while oxidation of methionine and phosphorylation of serine, threonine and tyrosine were selected. Peptide tolerance was set to 1.0 and intensity threshold was set to 100. The parent ion selection was set to 1.4 with fragment ion set to 0.7. For the global analysis of phosphopeptides, the following filters were set for every peptide: minimum Xcorr for +1 and +2 peptides g2.9, and +3 peptides

Protein Phosphorylation during Capacitation g3.2. Peaklists were generated using the TurboSequest (v3.3.1) algorithm. Most importantly, from these peaklists, every spectrum that passed the above criteria was manually validated to ensure the correct assignment of the y and b ion series in the case of CID or z and c ions for ETD, and to ensure that the sequence was not redundant, thus, justifying final inclusion in our list. To ensure that a true phosphorylation site had been observed, we looked for the characteristic neutral loss for serine/threonine residues. Accurate mass could be determined using the QSTAR. In cases of phosphorylation ambiguity, where the spectra did not permit assignment, we have shown the potential phospho-sequence in parentheses. After analyzing the peptide differences in DeCyderMS (using the survey scan), the program then readily identifies the appropriate MS/MS spectrum for those peptides changing between the noncapacitated and capacitated state. These individual spectra can then be run through TurboSequest and again manually validated for the correct assignment of ions to ensure their validity. In the case of 3 peptides identified for AKAP4, online MASCOT searches (http://www.matrixscience.comH) were used, with the same modifications set out for Sequest. 5′-Nucleotidase Assay. Rat spermatozoa were collected in noncapacitated or capacitated states as previously described. The cells were immediately washed 3× (2500g for 3 min) using BWW. Following the final centrifugation, the supernatant was removed, and the cells lysed [50 mM Tris-HCl, pH 8.0, 0.5% (v/v) Triton X-100 with Halt-phosphatase inhibitor] for 30 min at 4 °C with constant rotation. The sample was then centrifuged (15 000g for 5 min) and the supernatant taken for further analysis. 5′-nucleotidase activity was measured using the fluorescent based procedure described elsewhere.30 Briefly, 200 µM of the fluorescent product, 1,N6-etheno-AMP, was added to 6.6 µg of protein in a total of 1 mL (50 mM Tris, pH 8.0). The sample was then incubated at 37 °C for 1 h, following which, termination occurred upon addition of 100 µL of 0.15 M zinc sulfate together with 100 µL of 0.15 M barium hydroxide. The samples were incubated on ice for 10 min, centrifuged (6500g for 10 min) and the fluorescence of the supernatant recorded (ex, λ275; em, λ410). The activity was expressed as the change in AMP/(min/µg protein).

Results In a recent publication, Larson et al. have described the purification of highly enriched phosphopeptides from a 5-protein mixture using TiO2.21 To ensure this procedure was robust, we first confirmed the ability of our methodology to purify and identify the phosphorylated peptides in a standard monophosphopeptide R-casein digest (data not shown). We then turned to semicomplex protein mixtures. The known phosphopeptidecontaining proteins R-casein and β-casein were diluted with nonphosphorylated protein digests of BSA, β-lactoglobulin and carbonic anhydrase in different molar ratios of 1:1, 1:2 and 1:100. These semicomplex samples were then enzymatically digested overnight and subjected to TiO2 enrichment (see Experimental Section). To visualize the data, DeCyderMS, which plots the m/z of peptides detected in the survey scan against the elution time from the nanoflow RPC, was used to create a virtual 2D image of the peptide map. An example of an image of the full peptide mix (Figure 1A), and the peptide mix following TiO2 elution (Figure 1B), together with the ion current of the survey scan is shown. The number and complexity of peptides derived from the 5 protein mixture (Figure 1A) is greater than that following phosphopeptide enrichment.

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Figure 1. DeCyderMS imagess of peptides before and after TiO2 purification. Approximately 1 µg of R-casein, β-casein, BSA, β-lactoglobulin and carbonic anhydrase was mixed, precipitated and digested with trypsin. (A) The sample (1 µL) was then run through RPC coupled to an LTQ MS and visualized using DeCyderMS. This program plots the mass-to-charge ratio (m/z) against time. (B) The sample was prefractionated using TiO2, prior to running through RPC/LTQ MS. Inset: Demonstration of a blank run over the same time period.

Furthermore, a completely different peptide profile was seen following TiO2 elution; in this case, specific enrichment of peptides that were previously in low abundance is evident. For comparison, the inset demonstrates a blank run. To determine the identity of those peptides seen in the TiO2 eluate, TurboSequest was used, with S, T and Y phosphorylation and M oxidation selected as variable modifications (Table 1). All MS/ MS derived spectra were then manually validated. The statistics for the peptide identifications is shown in Supplementary Table 1. With the exception of the triply and quadruply phosphorylated peptides previously reported,21 we identified all the expected phosphopeptides. An inability to detect the multiply phosphorylated peptides has been seen by others, and may be due to either the ion-trap technology used21 or the poor elution of multiply phosphorylated peptides from TiO2 columns.31 Having validated the method of phosphopeptide enrichment using TiO2, we then went on to use this approach for the achievement of two aims. First, we planned to identify as many phosphorylated proteins in rat spermatozoa as possible. Our second aim was then to characterize those peptides that changed (became phosphorylated or dephosphorylated) during rat sperm capacitation. In both cases, quantified sperm lysates (150 µg protein) of samples from both noncapacitated and capacitated rat spermatozoa were obtained and enzymatically digested before undergoing TiO2 phosphopeptide enrichment. To demonstrate the effective preparation of capacitated and noncapacitated sperm populations, the samples were subjected to Western-blot analysis using an anti-phosphotyrosine monoclonal antibody (Figure 2A). As anticipated, capacitated samples showed a dramatically increased level of tyrosine phosphorylation compared to noncapacitated samples, particularly in a cohort of proteins exceeding 55 kDa. To ensure this was not an artifact of unequal protein loading, the blot was stripped and reprobed with anti-alpha tubulin (Figure 2B). These same samples were then subject to trypsin digestion. Every effort was made to ensure that equal amounts of peptide were used for the replicate analyses (see Experimental Section). Following elution, the samples were then loaded onto a capillary C18 column and analyzed initially by the LTQ mass spectrometer. Since our first aim was to identify the presence of as many phosphorylated peptides as possible, aliquots of this sample were also run through a time-of-flight mass spectrometer (see Experimental Section). To examine every phosphopeptide present in both fractions, the data files from Journal of Proteome Research • Vol. 9, No. 2, 2010 721

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Baker et al.

Table 1. Identification of Phosphopeptides Derived from Semicomplex Mixtures protein ID

casein casein casein casein

alpha-S1 alpha-S1 alpha-S1 alpha-S1

[Bos [Bos [Bos [Bos

taurus] taurus] taurus] taurus]

casein alpha-S2 [Bos taurus] beta-casein variant CnH - bovine

peptide sequence

Alpha Casein S1 VPQLEIVPNSAEER K.DIGSESTEDQAMEDIK.Q K.DIGSESTEDQAMEDIK.Q K.YKVPQLEIVPNSAEER.L

1660.795 1863.72 1927.692 1951.95

Alpha Casein S2 TVDMESTEVFTK

1482.608

Beta Casein FQSEEQQQTEDELQDK

both mass spectrometers were initially run through TurboSequest and filtered for good MS/MS spectra (minimum Xcorr for +1 and +2 peptides g2.9, and +3 peptides g3.2). In our experience, these filter parameters generate very high quality spectra. Nevertheless, every spectrum was then manually validated before being incorporated into our final list. In all, we can report a total of 120 phosphoproteins that were present in the combined noncapacitated and capacitated spermatozoa lysates (Table 2). Supplementary Table 2 shows the peptide statistics for every MS/MS spectrum obtained from the combined mass spectrometers. For this total phosphopeptide analysis, we characterized 131 (45.5%) monophosphorylated peptides, while 118 (40.9%) and 39 (13.5%) had double and triple phosphate groups, respectively. Additionally, we detected 34 peptides (10% of total peptides identified) to be nonphosphorylated. The Supplementary III data shows the annotated (y and b, or z and c ion series) MS/MS spectra for the 288 phosphopeptides (290 spectra are given, because in two instances, we give MS2 and MS3 on the same peptide). Having performed a global analysis of phosphopeptides, our next aim was to determine how the induction of sperm capacitation changed the pattern of phosphopeptide expression by rat spermatozoa. For this purpose, we used the relatively new concept of label-free quantitation. The DeCyderMS images obtained from the TiO2 purified peptides were compared before and after attainment of a capacitated state. In this analysis, if a peptide was present in the +2 state, DeCyderMS determined

Figure 2. Anti-phosphotyrosine increase during capacitation. (A) Four samples from noncapacitated spermatozoa (Non Cap) and three samples from the capacitated cells (Cap) were loaded into an SDS-PAGE, transferred and subject to Western-blot analysis using anti-phosphotyrosine antibody. (B) The membrane was stripped and reprobed using anti-alpha tubulin as a loading control. The position of the molecular markers are shown on the left-hand side. 722


(M + H)+

2061.829

the presence of the +1 and possible additional charge states of the same peptide. The total ion count for this given peptide was then summated for all charge states and statistically compared to other images. We analyzed 6 biological replicates of noncapacitated TiO2 phosphopeptide enriched samples and 5 biological replicates of capacitated samples (note, for each biological replicate, at least one individual rat must be used). For statistical analysis, only peptides that were detected in at least 9 of the 11 survey scans were taken further, which summated to 154 peptides. Figure 3 illustrates the label-free quantitative approach for one example peptide. The global DeCyderMS virtual 2D images of the phosphopeptide profiles of noncapacitated (Figure 3A) and capacitated (Figure 3B) rat sperm samples are shown. A magnified view of peptides (m/z 800-814 Da) is shown in Figure 3C,D. Here, surrounding peptides, for example, m/z 806.5 Da (top arrow) m/z 804.5 Da (bottom arrow) demonstrate minimal variation between the noncapacitated state (Figure 3C) to the capacitated state (Figure 3D) and as such could be considered as an internal loading control. Nevertheless, the peptide at m/z 809.0 Da (blue box) is present in the noncapacitated map (Figure 3C), but is severely reduced in the capacitated map (Figure 3D). Since the experiments were run a number of times, the total peptide intensity data for all the images are shown in Figure 4A. Upon the basis of these data, Student t-tests can then be performed to generate a level of significance. Furthermore, when the total comparative data set is summated (from this analysis, 154 peptides were compared), a FDR can then be used to determine the level of significant change. Once a peptide is classified as significant (again, this is based on the MS-survey scan), then the MS/MS spectra for that particular peptide can be interpreted (Figure 4B). In the case illustrated in Figures 3 and 4, the sequence is derived from the sperm-oocyte fusion protein Izumo, with the phosphothreonine shown (Figure 4B). In total, we observed 57 peptides (P < 0.05; 45 significant using B-H FDR; 11 with the B-L correction and 9 using Bonferroni FDR) changing from the noncapacitated to the capacitated state. From this analysis, the tandem MS (or MS3 or ETD) of 22 peptides could be identified (Table 3) including 20 peptides significant according to the B-H algorithm; 7 peptides significant according to B-L and 6 peptides according to Bonferroni FDR, see also Figure 6). The intensity (2Log) values for all the peptides, together with the total FDR significance values are given in the Supplementary IV data. Detailed analysis of every peptide changing during capacitation revealed that all of them contained at least one phosphorylated amino acid. Among these changes, 11 were singly phosphorylated, 8 were doubly phosphorylated and 3 were triply phosphorylated. Interestingly, 15 phosphopeptides were more intense in noncapacitated spermatozoa, while the remaining 7 had higher intensities in

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Protein Phosphorylation during Capacitation Table 2. Phosphorylated Proteins Present in Rat Non- And Capacitated Rat Spermatozoa IPI accession

identification

IPI00187698.2 IPI00188804.1 IPI00189904.1 IPI00190777.2

IPI accession

32 kDa protein 60S acidic ribosomal protein P2 Glycogen synthase kinase-3 alpha Uncharacterized protein C2orf57 homologue

IPI00365822.3 IPI00365910.4 IPI00365935.3 IPI00366181.2

IPI00191798.2 IPI00192187.1 IPI00193741.4 IPI00197564.1 IPI00197605.2

Testis-specific actin-related protein 3 Hypothetical protein LOC499579 Similar to RIKEN cDNA 1700052H20 RING finger protein 166 Synaptosomal-associated protein 23


IPI00198377.1 IPI00198468.2 IPI00198723.1

IPI00368107.2 IPI00368335.3 IPI00368927.4 IPI00369950.3 IPI00370215.3 IPI00370359.2 IPI00371050.3 IPI00371907.2

Vascular endothelial zinc finger 1 DDI1 homologue 1 Slingshot-like 2 Hypothetical protein LOC303534 Hypothetical protein LOC311795

IPI00372388.1 IPI00372804.2 IPI00373412.3 IPI00373487.2 IPI00382136.4 IPI00382200.3 IPI00388665.3 IPI00390795.2 IPI00390854.3 IPI00391492.3 IPI00392771.1

Cct3 T-complex protein 1 subunit gamma Saccharopine dehydrogenase Similar to CG5435-PA Tudor domain containing 3 Protein FAM70A Cb1-727 Acrosome formation-associated factor precursor Cation-transporting ATPase Ubiquitin specific protease 38 LOC497959 Similar to novel protein Izumo sperm-egg fusion protein 1 precursor

IPI00394157.3 IPI00400646.7 IPI00417373.2 IPI00464667.1

19 kDa protein Protein phosphatase inhibitor 2 Kinesin 13B HMT1 hnRNP methyltransferase-like 1

IPI00214500.1

Ropporin-1 Isoform 3 of Outer dense fiber protein 2 Solute carrier family 2, facilitated glucose transporter member 3 Glutathione transferase GSTM7-7 Sperm acrosome associated 1 58 kDa protein Dynein intermediate chain 1, axonemal Spermatogenesis-associated protein 19, mitochondrial precursor A kinase anchor protein 3 Ornithine decarboxylase antizyme 3 Checkpoint kinase Chk2 RGD1559450_predicted hypothetical protein Dynein heavy chain at 89D CG1842-PA Ubiquitin-activating enzyme E1 Glutathione S-transferase Mu 5 Uncharacterized protein C7orf31 homologue Heat shock protein HSP 90-alpha DnaJ homologue subfamily C member 5 Sperm mitochondrial-associated cysteine-rich protein A-kinase anchor protein 4 precursor 66 kDa protein Testis lipid-binding protein Isoform XB of Plasma membrane calcium-transporting ATPase 4 Aanat Serotonin N-acetyltransferase

Ninein Tumor necrosis factor, alpha-induced protein 2 Prostaglandin E synthase 3 Isoform 2 of Testicular haploid expressed gene protein Interferon-inducible GTPase 5 Kinectin 1 Serine/threonine-protein kinase PLK4 Zinc transporter SLC39A9 Isoform 1 of Uncharacterized protein C14orf166B homologue Heat-shock protein beta-9 Calmin Receptor-interacting factor 1 isoform 1

IPI00464742.2

IPI00215463.1 IPI00230883.2 IPI00324565.5 IPI00326744.1 IPI00331885.1 IPI00358174.4 IPI00358599.3

Calmodulin-like protein 3 Calmodulin-dependent protein kinase type IV Mitochondrial uncoupling protein 2 Monocarboxylate transporter 2 Sperm protein SSP3111 Sarcolemma associated protein 59 kDa protein

IPI00464806.1 IPI00471761.1 IPI00471782.4 IPI00471827.3 IPI00471857.1 IPI00476178.3 IPI00476550.4

IPI00358733.2 IPI00358771.2 IPI00359210.3 IPI00359398.3 IPI00359744.3 IPI00360072.3

Structural maintenance of chromosomes 2-like 1 RhoGEF4 CG8606-PA, isoform A Tubulin tyrosine ligase-like family, member 2 Similar to CG8312-PA, isoform A Similar to DAZ interacting protein 1 Pleckstrin homology domain containing, family H (with MyTH4 domain) member 2 Coiled-coil domain-containing protein 11 Dynein, axonemal, heavy chain 8 CAP-binding protein complex interacting protein 1 68 kDa protein

IPI00556961.1 IPI00557391.2 IPI00558444.2 IPI00558943.2 IPI00560835.2 IPI00561268.2

Isoform 1 of Spermatogenesis-associated protein 18 DnaJ homologue subfamily B member 6 Putative uncharacterized protein RGD1359156 Protein FAM71B A kinase anchor protein 10 Uncharacterized protein C9orf128 homologue Proteasome subunit alpha type-3 Propionyl-CoA carboxylase alpha chain, mitochondrial precursor 116 kDa protein Chromosome 17 open reading frame 27 RGD1564067_predicted hypothetical protein Roundabout homologue 2 precursor Cancer susceptibility candidate 5 isoform 1 Akap4 83 kDa protein

IPI00564577.1 IPI00564854.3 IPI00566094.1

105 kDa protein Similar to Protein C14orf155 Coiled-coil domain-containing protein 116

IPI00569302.3 IPI00608136.1

IPI00362771.3

Phosphatidylinositol-3-phosphate/ phosphatidylinositol 5-kinase, type III isoform 2 209 kDa protein

HoloCentric chromosome binding Protein family member Tesb protein


p21 (CDKN1A)-activated kinase 7 Hypothetical protein LOC305476 Akap2 A kinase anchor protein 2 RAD23a homologue Absent in melanoma 1 ZH11 protein Hypothetical protein LOC292078 Cldn23 Claudin 23


IPI00199482.2 IPI00199757.3 IPI00200184.1 IPI00200420.3 IPI00200836.1 IPI00201274.1 IPI00204807.3 IPI00205562.1 IPI00205592.5 IPI00205945.4 IPI00207115.3 IPI00208636.3 IPI00209486.2 IPI00210566.3 IPI00210881.1 IPI00211031.1 IPI00212365.1 IPI00212806.2 IPI00213531.3 IPI00213557.2


identification

IPI00608181.1

Isoform 1 of Thioredoxin domain-containing protein 2 Similar to Protein C6orf203 Retinol dehydrogenase 14 F-box only protein 21 Similar to FLJ44048 protein CD209 antigen-like protein B 12 kDa protein Odf2 Cenexin 2 LOC500060 Similar to testes development-related NYD-SP18 Journal of Proteome Research • Vol. 9, No. 2, 2010 723

research articles

Baker et al.

Discussion

Figure 3. DeCyderMS images of TiO2-purified peptides from noncapacitated and capacitated rat spermatozoa. Rat spermatozoa were either: (A and C) lysed immediately (noncapacitated) or (B and D) placed in capacitation medium for 40 min, then subjected to lysis. The samples were precipitated, digested with trypsin and the phosphopeptides enriched using TiO2 affinity chromatography before being run through RPC coupled to an LTQ MS. A DecyderMS analysis, showing the m/z against time is shown for the complete run (A and B) and a magnified view of peptide changes (C and D).

capacitated spermatozoa, suggesting both kinase, and, for perhaps the first time according to our knowledge, phosphatase activity is occurring during capacitation. To validate these phosphorylation changes in terms of functionality, we searched the list of protein candidates for those exhibiting a biochemical activity that could be readily assessed. From the inventory of proteins undergoing a phosphorylation change during capacitation, many appeared to be structural. However, one enzyme appeared in this list, in which multiple peptide changes were observed during capacitation and which was amenable to biochemical evaluation, 5′nucleotidase. We therefore measured 5′-nucleotidease activity before and after capacitation. It should be noted that, although the IPI database reports this enzyme as “retinol dehydrogenase”, a blast search demonstrated that the enzyme of interest is in fact 5′-nucleotidase, cytosolic version 1B. The role of this enzyme is to dephosphorylate noncyclic nucleoside monophosphates to nucleosides and inorganic phosphate, thus, regulating the cellular levels of ribo- and deoxyribonucleotides. In our case, the cytosolic version of 5′-nucleotidase has a high specificity toward AMP. Therefore, we measured the level of 5′-nucleotidase activity in both noncapacitated and capacitated cells (Figure 5A). As shown, it is evident that, during capacitation, 5′-nucleotidase activity significantly decreases. Several attempts were made to raise an antibody against this enzyme, all without success; therefore, to demonstrate that the change in 5′-nucleotidase activity was not due to unequal loading, we generated the corresponding silver stained gels, an example of which is given in Figure 5B, together with a parallel anti R-tubulin blot. The totality of these data suggest that the phosphorylation changes associated with cytosolic 5′-nucleotidase down-regulate its activity during capacitation. The identification of this, and other phosphorylation changes in this study, opens up new opportunities for research into the role these molecules play in the attainment of a capacitated state. 724


Resolution of the molecular mechanisms controlling sperm capacitation is central to sperm cell biology. Although the general importance of serine, threonine32 and tyrosine12-14 phosphorylation in this process has been previously documented, accurate identification of the phosphoproteins involved in this process has received little attention. While it has been well established that tyrosine phosphorylation increases dramatically during capacitation, our analysis has highlighted the importance of proteins exhibiting changes in the phosphorylation status of serine or threonine residues. The reason for this lies in simple stoichiometry. Analysis of vertebrate cell lysate gives a phosphoamino acid ratio (pSer/pThr/pTyr) of 1800:200:1.33 This is in keeping with other reported phosphorylation data sets, which contain 90% pSer/pThr and only a few percent pTyr.34 In our data set, it is difficult to determine the phosphoamino acid ratio, since in many peptides, ambiguous site phosphorylation is reported. Nevertheless, analysis of those peptides reported as unambiguous suggests the presence of 81% pSer, 15% pThr and 4% pTyr. With this in mind, it is evident that, in order to mine the phosphotyrosine containing peptides of a given biological system, different methods of isolation will be necessary. One promising angle is that involving molecular imprinting, whereby a polymer is made that specifically detects phosphotyrosine-containing peptides.35 With this technology, it may ultimately be feasible to identify those proteins (or rather peptides) undergoing tyrosine phosphorylation during capacitation. The use of label-free based quantitation is a relatively new concept to proteomics, and as such, there is no true standard as to how peptides need to be reported. In this analysis, we have included the use of three separate FDRs, including B-H,23 B-L24 and Bonferroni22 corrections. The Venn diagram (Figure 6) outlines the number of peptides (and consequent proteins denoted in parentheses) that are significant when the different FDR algorithms are applied. The Bonferroni correction is the most conservative approach and, as such, has come under intense scrutiny with respect to its use in quantitative proteomics.36 With such strict criteria, it is more than likely that application of both the Bonferroni37 and probably the B-L corrections is leading to Type II errors. Unfortunately, no other reports exist to our knowledge that can confirm our data on the phosphopeptides changing during capacitation. Furthermore, even though we could demonstrate, for the first time, that 5′-nucleotidase does undergo a significant change in enzyme activity during capacitation (Figure 5), only two phosphorylation events occurring during capacitation in that protein (Table 3) were significant using the Bonferroni algorithm. Thus, we cannot discriminate from our current data which FDR method is the most applicable. There is one other report on LC-MS/MS analysis of phosphopeptides from noncapacitated and capacitated spermatozoa derived from the mouse.38 In it, the authors have used the t-test (P < 0.05) to report on significant peptide changes. Interestingly, comparison of the phosphopeptides found to change during capacitation in the two studies indicates that no common phosphorylation events were identified. This is probably the case for three reasons. First, the mechanisms used to create populations of uncapacitated spermatozoa in the two studies were different. In our case, we took spermatozoa shortly after they had been diluted into culture medium before significant capacitation had taken place, as judged by their tyrosine phosphorylation status. Alternatively, Platt et al.38 omitted

Protein Phosphorylation during Capacitation

research articles

Figure 4. Sequest generated MS/MS spectra. (A) The normalized Intensity (2Log) values from all replicates of the noncapacitated (red squares) and capacitated (blue cross) samples for the parent ion 809.17. (B) Shown are the y and b ion series, in the (a) +1 and (b) +2 states. The parent ion of 809.17 shows typical neutral loss peaks reminiscent of phosphorylation for the sequence, which corresponds to testis lipid binding protein. The arrows demonstrate two peptides that do not change during capacitation.

bicarbonate and albumin from the medium and incubated the cells in parallel with the capacitated sperm population. Both approaches are valid but neither is perfect. If spermatozoa are selected prior to capacitation, then the effect of incubation alone is not accommodated in the analysis. Alternatively, if vital ingredients are omitted from the medium such as bicarbonate and albumin, then secondary changes to the sperm proteome might occur as a result of failures to control, for example, intracellular pH or lipid peroxidation status. In addition, changes such as the loss of decapacitation factors from the sperm surface will still occur under these noncapacitating conditions that are, in reality, critical to the successful completion of this process.39 Such differences in experimental design have to be acknowledged and considered in the interpretation of data generated by these and similar studies. Second, it is evident that, in the mouse, the phosphorylation events occurring during capacitation appear to be different from those seen in the rat. For example, hexokinase is found to be tyrosine phosphorylated equally in noncapacitated and capacitated mouse samples.40 In contrast, this pattern of tyrosine phosphorylation is not seen in rat spermatozoa.41 Third, a comparison of different phosphopeptide enrichment methods including IMAC (used in the mouse phospho-analysis) and TiO2 chromatography (used in this analysis) has recently demonstrated that each method isolated different segments of the phosphoproteome.42 Thus, while the use of IMAC was seen to identify multiply phosphorylated peptides, TiO2 identified more singly phosphorylated peptides.31 The reason for this is that the multiply phosphorylated peptides bound to TiO2 could not be eluted readily.43 Hence, it is apparent that no single method

is capable of providing a whole phosphoproteome. In light of these findings, a technique known as SIMAC (sequential elution from IMAC) has now been proposed whereby multiply phosphorylated peptides are first captured on IMAC.31 The unbound fraction is then applied to TiO2 in order to capture the single phosphorylated peptides. This technique led to a synergistic result, whereby 3-fold more phosphopeptides were identified compared to when each technique was used separately.31 Although we are currently developing these methods, from the present analysis, it is evident that the use TiO2 in conjunction with a label-free software package allowed the identification of 57 peptides that were shown to undergo differential phosphorylation during capacitation based on the MS survey scan (P < 0.05). From this, 22 could be confidently identified by MS/MS, leaving 35 peptides without MS/MS interpretation. To investigate why this was the case, we looked at the 35 nonassigned spectra to determine whether a phosphate group was present on the peptide (note, since most of the data was generated on an ion-trap mass spectrometer, we could not tell the difference between phosphorylation and sulfation). In every case, we could detect a loss of 32 Da (+3 peptides) or 49 Da (+2) in the MS2 scan, reminiscent of a β-elimination of the phosphate ion from either serine or threonine (data not shown). Although we attempted MS3 and ETD on both the neutral loss fragment ion and intact peptide, respectively, we have had no further success in identifying these peptides. From the interpretable MS/MS data, 17 peptides demonstrated higher phosphorylation in the noncapacitated state, compared to 7 which showed higher phosphorylation in the capacitated state. An increase in the expression of a given Journal of Proteome Research • Vol. 9, No. 2, 2010 725

research articles

Baker et al.

Table 3. Identification of Proteins Undergoing Phosphorylation during Rat Sperm Capacitation IPI accesseion IPI00187698.2 IPI00198377.1 IPI00199757.3 IPI00200420.3 IPI00201274.1 IPI00211342.1 IPI00358599.3 IPI00367419.3 IPI00371050.3 IPI00372804.2 IPI00392771.1 IPI00464742.2 IPI00471761.1

IPI00656420.1 IPI00758442.1

a

protein identification Ppp1r7 32 kDa protein Ropporin-1 Sperm acrosome associated 1 LOC500442 Dynein intermediate chain 1, axonemal Akap3 A kinase (PRKA) anchor protein 3 Cldn23 Claudin 23 59 kDa protein Serine/ threonine-protein kinase PLK4 Hypothetical protein LOC303534 Saccharopine dehydrogenase Izumo sperm-egg fusion protein 1 precursor Spermatogenesisassociated protein 18 Putative uncharacterized protein RGD1359156 Similar to Protein C6orf203 Retinol dehydrogenase 14 (5′-nucleotidase)

peptide

av. diff. (2Log)´

t test pa

FDR (Bh)

FDR (Bl)

Bonferroni adjustment

K.HGGGIVADLpSQQpSLK.D

-2.39

3.53 × 10-3

*

8.44 × 10-3

4.63 × 10-4

3.25 × 10-4

R. p(SEQVPLS)NWAELTPELLK.V R.ERp(SEQVPLS)NWAELTPELLK.V K.App(STT)EIQSEM*SSMR.Y

2.76 4.14 -1.57

7.22 × 10-3 3.90 × 10-2 1.33 × 10-3

* *

1.07 × 10-2 1.85 × 10-2 6.17 × 10-3

5.17 × 10-4 8.02 × 10-4 4.16 × 10-4

3.25 × 10-4 3.25 × 10-4 3.25 × 10-4

-0.65

-3

*

7.47 × 10-3

4.42 × 10-4

3.25 × 10-4

3.15

3.32 × 10-3

*

7.79 × 10-3

4.49 × 10-4

3.25 × 10-4

K.KTASSQDGp(SSS)R.S R. ppp(YTPSPGPLRYTPS)PGPLR.Y R.Lp(SLSS)VLDHPFMp(SRNPST)K.S

-1.71 2.47 -1.71

2.64 × 10-5 7.41 × 10-5 3.28 × 10-4

*** *** **

1.30 × 10-3 1.62 × 10-3 3.25 × 10-3

3.38 × 10-4 3.42 × 10-4 3.66 × 10-4

3.25 × 10-4 3.25 × 10-4 3.25 × 10-4

R.VEEQDGp(SSSGLTS)SK.M

-0.86

7.98 × 10-3

*

1.20 × 10-2

5.53 × 10-4

3.25 × 10-4

1.74

3.02 × 10-2

1.69 × 10-2

7.26 × 10-4

3.25 × 10-4

K.Np(SNVENKTS)AAEFK.S

-1.42

3.41 × 10-3

*

8.12 × 10-3

4.56 × 10-4

3.25 × 10-4

R.LLPLLQp(TSFSS)LGVGK.I

-1.94

3.03 × 10-4

***

2.92 × 10-3

3.61 × 10-4

3.25 × 10-4

K.LIQApp(SEHSLQT)ALEK.H

-1.25

7.19 × 10-3

*

1.04 × 10-2

5.09 × 10-4

3.25 × 10-4

K.DADEEDp(SDEETS)HLER.S

-1.39

9.00 × 10-5

***

1.95 × 10-3

3.47 × 10-4

3.25 × 10-4

R. ppp(SPSTRAPS)VDENR.S

-1.74

8.06 × 10-6

***

6.49 × 10-4

3.29 × 10-4

3.25 × 10-4

K. pp(SPVQQPSPQT)R.T R.Lppp(STQGSQEIPVPNTDS)R.G R.YSKEpSLDAEK.R R.TIpp(TPLDSQPPTPPETEPDS)R.R R.TITPLDSQPPpTPPETEPDSR.R R.Lpp(STQGSQEIPVPNTDS)R.G

-0.81 -1.83 -1.37 -2.37 -1.33 2.54

1.04 × 10-4 5.67 × 10-4 5.57 × 10-3 8.38 × 10-3 1.06 × 10-2 1.21 × 10-2

*** * * * * *

2.27 × 10-3 4.55 × 10-3 9.74 × 10-3 1.23 × 10-2 1.40 × 10-2 1.43 × 10-2

3.52 × 10-4 3.87 × 10-4 4.93 × 10-4 5.62 × 10-4 6.14 × 10-4 6.25 × 10-4

3.25 × 10-4 3.25 × 10-4 3.25 × 10-4 3.25 × 10-4 3.25 × 10-4 3.25 × 10-4

R.DELVAGpSQEpSVK.V R.DKp (SESYSS)LISM*K.S

R.p(SVS)NLKPVPVIGSK.L

2.83 × 10

*Significant for BH; **significant of BH and BL; ***significant of BH/BL and Bonferroni.

Figure 5. Decrease in 5‘-nucleotidase activity during capacitation. (A) Spermatozoa were taken in either a noncapacitated (Non) or capacitated (Cap) state and immediately lysed (0.1% Triton X-100, 50 mM Tris-HCL, pH 7.4). Following lysis, the samples were quantified and 6.6 µg of protein was used to measure 5‘-nucleotidase activity. The results represent the average and standard deviation of 3 independent samples. (B) Silver stained gel (top) together with anti R-tubulin Western blot to demonstrate even loading. Approximately 6.6 µg of protein from either a noncapacitated spermatozoa (Non) or the capacitated spermatozoa (Cap) of each of the independent samples was loaded onto a 10% SDS-PAGE with subsequent silver staining or Western blotting.

phosphopeptide during capacitation might appear to suggest that the addition of a phosphate group had occurred. Never726


theless, it is possible that, for example, a doubly phosphorylated peptide, not detected (or sequenced) in the noncapacitated

Protein Phosphorylation during Capacitation

Figure 6. Venn diagram summarizing false discovery rates. Following DecyderMS analysis, those peptides with statistical significance for t-test (P < 0.05) together with a summary of the peptides (and consequent proteins denoted in parentheses) passing the B-H, B-L and Bonferroni corrections are shown.

sample, has lost a phosphate group, thereby generating a new phosphopeptide which appeared in the capacitated sample. Although far from definitive, manual inspection of the 35 peptides that we could not interpret did demonstrate 3 peptides with a precursor mass consistent with an additional phosphate, when compared to those peptides seemingly phosphorylated during capacitation; these derived from Ppp1r7, C6orf203 and Saccharopine dehydrogenase. Indeed, phosphopeptides from the latter two that had increased during capacitation did contain a serine that, potentially, could have been phosphorylated in the noncapacitated state. These data indicate that, although phosphopeptides appear in the capacitated samples, their arrival might not have been attributable to just kinase action but may, in fact, have been due to phosphatase activity. Similarly, a decrease in the expression of a given phosphopeptide from the noncapacitated to the capacitated state might suggest one of two things: that additional phosphate(s) have been added due to the activation of protein kinases or that a phosphate group has been removed as a consequence of enhanced phosphatase activity. Of the 15 proteins shown to undergo a significant phosphorylation change, only three have previously been reported as being involved in the function of capacitated spermatozoa. These include Ropporin44,45 Izumo46 and AKAP3.47,48 Ropporin is a structural protein, containing a 32 amino acid stretch (from amino acids 12-43) that forms an RIIR binding domain.44,45 This domain is responsible for binding to the regulatory subunit of protein kinase A (PKA).45 PKA is a tetramer composed of two catalytic and two regulatory subunits (PKAc and PKAr, respectively); in the presence of cAMP, the PKAr and PKAc subunits dissociate, allowing phosphorylation of target proteins to occur. Targeted disruption of the sperm-specific PKAcRII gene results in infertile mice, with a reduced capacity for activated motility and no capacity for hyperactivated motility.13 Interestingly, we could show a phosphorylation event occurring on Ropporin, at amino acid 58, just after the RIIR binding domain, suggesting that phosphorylation of Ropporin may help to regulate the binding of the regulatory PKA subunit. The second protein shown to be previously involved in the functionality of capacitated spermatozoa is Izumo. Null mutation of the Izumo gene generates a very clean phenotype, with the

research articles spermatozoa unable to fuse with the oocyte.46 The possibility that this protein might exist in multiple phosphorylated states has, however, not previously been described. This finding may have clinical importance, as a recent analysis of infertile patients whose spermatozoa failed to fuse with the oocyte demonstrated that Izumo was indeed present in their spermatozoa.49 Thus, while the physical absence of this particular protein could not explain the lack of sperm-oocyte fusion in these patients, the present results raise the possibility that deficiencies in the phosphorylation state of Izumo could be involved in the etiology of this condition. Identification of the enzymes responsible for this phosphorylation event might, therefore, be of interest in resolving the cause of this particular form of infertility. The final protein previously reported to be involved in capacitation is AKAP3. AKAPs represent a growing family of scaffolding proteins that tether the regulatory subunits of PKA as well as other enzymes to cytoskeletal components.50 In doing so, AKAPs permit regional control of signal transduction pathways within cells. Previous work has demonstrated that AKAP3 (as well as AKAP4) undergoes tyrosine phosphorylation during human sperm capacitation with 8 phospho-specific sites identified.15 The interaction between the pSer and pTyr sites on AKAP3 during capacitation now need to be further elucidated. To validate our data, and demonstrate whether or not labelfree quantitation is valuable in a biological context, we sought to demonstrate that the significant phosphorylation events we detected during capacitation were associated with a detectable change in biochemical activity. Unfortunately, for most of the identified proteins, no assays were available. Nevertheless, from our analysis, one enzyme which allowed us to monitor biochemical activity was retinol dehydrogenase. Homologous database searches demonstrated that the description of “retinol dehydrogenase” was an error in nomenclature on behalf of IPI, and in fact, this protein is truly a 5‘-nucleotidase, cytosolic version 1B. The role of the latter is to dephosphorylate noncyclic nucleoside monophosphates to nucleosides and inorganic phosphate, thus, regulating the bioavailability of riboand deoxyribonucleotides. In our case, the cytosolic version of 5‘-nucleotidase has a high specificity toward AMP. By measuring the activity of 5‘-nucleotidase in both noncapacitated and capacitated spermatozoa, we could show that the level of enzyme activity significantly decreased during capacitation. The involvement of adenosine as a second messenger molecule involved in regulating the availability of cAMP during sperm capacitation has been suggested previously.51,52 A proposed mechanism for the involvement of this molecule has been put forward whereby adenosine stimulates adenylyl cyclase activity (thus increasing cAMP levels) in noncapacitated spermatozoa, but is inhibitory in capacitated spermatozoa.51 Such a mechanism would ensure that the cell achieves a measure of homeostasis with respect to cyclic nucleotide availability, avoiding an overabundance of cAMP that might prematurely trigger acrosomal exocytosis. Our finding that 5‘nucleotidase activity is higher in noncapacitated cells (and as a consequence would produce more adenosine, thus, stimulating adenylyl cyclase), and lower in the noncapacitated state, fits this model perfectly. In summary, we have identified 120 phosphoproteins that are associated with rat spermatozoa. Of these, we have been able to identify 15 proteins that appear to change as spermatozoa engage the process of capacitation and gain the capacity to fertilize the oocyte. Of course, this list is not definitive for a Journal of Proteome Research • Vol. 9, No. 2, 2010 727

research articles variety of reasons including the fact that our lysis method may not have solubilized all of the proteins present in the spermatozoa and our TiO2 columns would not have captured all of the phosphoproteins liberated from these cells. Nevertheless, this analysis has provided several new leads concerning the post-translational modifications capable of driving this unique cell into a state of functional maturity. Abbreviations: PTM, post-translational modification; sAC, soluble adenylyl cyclase; PKA, protein kinase A; AKAP3, a kinase anchoring protein 3; BWW, Biggers, Whitten and Whittingham medium; cAMP, adenosine 3′,5′-cyclic monophosphate; DHB, dihydroxybenzoic acid; M, methionine; MDLC, multidimensional liquid chromatography; TiO2, titanium dioxide; FDR, False discovery rate; B-H, BenjaminiHochberg; B-L, Benjamini-Liu.

Supporting Information Available: Tables showing the statistics for the peptide identifications, the peptide statistics for every MS/MS spectrum obtained from the combined mass spectrometers, and the intensity (2Log) values for all the peptides, together with the total FDR significance values. The annotated (y and b, or z and c ion series) MS/MS spectra for the 288 phosphopeptides. This material is available free of charge via the Internet at http://pubs.acs.org.

Baker et al.

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PR900513D

Journal of Proteome Research • Vol. 9, No. 2, 2010 729

Label-Free Quantitation of Phosphopeptide Changes During Rat

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