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INTRODUCTION |
After ejaculation, sperm are able to move actively but lack
fertilizing competence. They acquire the ability to fertilize in the
female genital tract in a time-dependent process called capacitation (1). Capacitation is accompanied by a cAMP-protein kinase-dependent increase in tyrosine phosphorylation of a
subset of proteins (2, 3). Because a protein kinase cascade is involved
in the regulation of the sperm fertilizing ability, it is important to
characterize the proteins that undergo phosphorylation and examine how
these changes relate to capacitation.
Post-translational protein phosphorylation by protein kinases plays a
role in many cellular processes including transduction of extracellular
signals, intracellular transport, and cell cycle progression. The use
of two-dimensional gel electrophoresis followed by tandem mass
spectrometry (MS/MS)1
provides a comprehensive approach to the analysis of proteins involved
in cell signaling (4). Specifically, changes in tyrosine phosphorylation can be monitored using two-dimensional gel
electrophoresis followed by Western blot analysis with
anti-phosphotyrosine (
-PY) antibodies (5). Proteins that
undergo changes in tyrosine phosphorylation during cellular processes
can be then isolated from a complementary gel and sequenced by MS/MS.
In the present study, we have used this approach to identify several
sperm proteins that undergo tyrosine phosphorylation during capacitation.
Identification of the site at which a particular protein is
phosphorylated is important in identifying the physiologically relevant
protein kinase involved in a particular pathway. In addition, to
understand the function of a phosphoprotein, phosphorylation sites are
excellent candidates for site-directed mutagenesis. Determination of
individual phosphorylation sites in vivo often requires the
purification to homogeneity and/or mutation analysis of the
phosphoprotein. Recently, several methods for the selective detection
and enrichment of phosphopeptides have been developed (6-8); however,
most of them have been applied only on a protein-by-protein basis. In
the present work, we have used Fe3+-immobilized metal
affinity chromatography (IMAC) prior to MS/MS to enrich digests for
peptides containing phosphoamino acids. To increase the selectivity of
the IMAC column for phosphopeptides, we have used a recently developed
modification of this technique in which acidic residues are converted
to methyl esters to block their binding to iron before IMAC is employed
(9). Using this methodology, 5 sites of Tyr, 56 of Ser, and 2 of Thr
phosphorylation have been characterized.
Although the combination of IMAC and MS/MS is ideally suited for the
characterization of phosphorylation sites on proteins in complex
mixtures, it is also important to determine which sites are
phosphorylated in response to a particular stimulus. In the present
paper, we have used differential isotopic labeling to quantify
phosphorylation of defined sequences and to determine which sites
suffer increased or decreased phosphorylation during human sperm
capacitation. The evaluation of differential phosphorylation, added to
the knowledge of the exact phosphorylated sequence, goes beyond the
sperm capacitation field and could be used to understand signaling
mechanisms in a multiplicity of cell systems.
Identification of the tyrosine phosphorylation sites of AKAP-3 and
AKAP-4 confirmed the previous findings using a two-dimensional gel
approach, suggesting that these proteins are tyrosine phosphorylated in
a capacitated sperm population. Nevertheless, because of the relative
abundance of tyrosine-phosphorylated phosphopeptides compared with
phosphoserine phosphopeptides, most of the tyrosine-phosphorylated sites remained unknown. Among them, sites in the NSF homolog VCP remained unidentified. Because VCP is associated with a role in membrane fusion in other cell types and because several aspects of
sperm physiology required membrane fusion events, VCP was chosen for
further investigation. Anti-VCP antibodies confirmed that this protein
is tyrosine phosphorylated during capacitation. In addition,
immunolocalization of VCP was determined and showed different
localization patterns before and after human sperm capacitation.
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EXPERIMENTAL PROCEDURES |
Preparation of Spermatozoa--
The basic medium used for all
experiments was modified human tubal fluid (Irvine Scientific, Santa
Ana, CA). Semen specimens were obtained by masturbation (approved by
the UVA Human Investigation Committee). Individual semen samples were
allowed to liquefy at room temperature (0.5-3 h), and mature sperm
were purified by Percoll (Amersham Biosciences) density gradient
centrifugation as described previously (5). Sperm presenting > 90% motility were treated immediately to obtain a noncapacitated
population or capacitated at 37 °C in 5% CO2 incubator.
After overnight capacitation, sperm were concentrated by centrifugation
at 20,000 × g for 2 min at room temperature and then
washed once in 0.5 ml of phosphate-buffered saline (PBS) at room
temperature. The sperm pellet was then resuspended in the appropriate
buffer depending on the experiment. For two-dimensional gel analysis,
sperm were resuspended in Celis buffer (10). For immunoprecipitation
and/or for direct MS/MS, cells were resuspended in 0.1% SDS, 25 mM Tris-HCl, pH 7.5, and boiled for 5 min, centrifuged at
20,000 × g for 5 min, and the supernatant recovered.
Two-dimensional Gel Electrophoresis and Western Blot--
Sperm
were routinely solubilized in a lysis buffer consisting of 2% (v/v)
Nonidet P-40, 9.8 M urea, 100 mM
dithiothreitol, 2% (v/v) Ampholines, pH 3.5-10, and as protease
inhibitors 2 mM phenylmethylsulfonyl fluoride, 5 mM iodoacetamide, 5 mM EDTA, 3 mg/ml
1-chloro-3-tosylamido-7-amino-2-heptanone, 1.46 µM
pepstatin A, and 2.1 µM leupeptin. 5 × 108 cells/ml were solubilized by constant shaking at
4 °C for 60 min. Insoluble material was removed by centrifugation.
Isoelectrofocusing was performed in 15 × 0.15-cm acrylamide rods,
using either the gel composition proposed by Hochstrasser et
al. (11) or that proposed by Celis et al. (10). The
carrier Ampholine (Amersham Biosciences) composition was 20% pH 7, 20% pH 7-9, and 60% pH 3.5-10. 65 µl of sperm extract (~0.15 mg
of protein) was applied per rod. The tubes were filled gently
overlaying the sample with a buffer containing 5% Nonidet P-40, 1%
Ampholines, pH 3.5-10, 8 M urea, and 100 mM
dithiothreitol. Focusing was conducted using voltage stepping: 2 h
at 200 V, 5 h at 500 V, 4 h at 800 V, 6 h at 1,200 V,
and 3 h at 2,000 V. Two-dimensional SDS-PAGE was carried out in
0.15-cm thick, 16 × 16-cm linear gradient gels (7.5-15%) in a
Protean II xi Multi-Cell apparatus (Bio-Rad). Silver staining was
performed according to Hochstrasser et al. (11). Electrotransfer and Western blots were carried out as described previously (2).
Image Analysis of Two-dimensional Gels--
Gel electrophoresis
was performed concurrently to ensure equivalent electrophoretic
conditions. Gels were stained with silver (5) or electroblotted to
polyvinylidene difluoride membrane and probed with
-PY (clone 4G10,
Upstate Biotechnology). The silver-stained gel and x-ray films (short
and long exposure of ECL) were scanned at 300 dpi using a desktop
Hewlett Packard scanner. Digitized images were overlaid in Adobe
Photoshop 6.0 using different percents of transparency. Using known
"landmarks" such as fibrous sheath proteins and tubulins, the
silver image was aligned with the ECL images. After marking all
reactive spots on the ECL image with arrows, the ECL image was hidden
and the arrows identified corresponding silver-stained spots. These
spots were then cored from the silver-stained gel and submitted for
mass spectrometry analysis.
Immunofluorescence Microscopy--
Noncapacitated and
capacitated sperm were air dried onto slides, washed three times with
PBS, permeabilized with methanol, washed with PBS, and then blocked
with 10% normal goat serum in PBS. Incubations were then carried out
with
-PY or
-VCP/p97 antibodies (1:250) diluted in PBS with 1%
normal goat serum, washed, and incubated with FITC-conjugated
F(ab')2 fragments of donkey
-mouse IgG (1:200) or donkey
-rabbit IgG (1:200) (Jackson ImmunoResearch) in normal goat serum in
PBS. Slides were washed with PBS and mounted with Slow-Fade Light
(Molecular Probes, Eugene, OR). Sperm were observed by differential
interference contrast and epifluorescence microscopy using a Zeiss
Axiophot microscope (Carl Zeiss, Inc. Thornwood, NY). Results depicted
in Fig. 1 represent the mean ± S.E. of three independent
experiments with triplicate determination. Statistical differences
between the groups were determined using Student's t test,
comparing the capacitated with the noncapacitated population after
arcsin transformation (12).
Preparation of Samples for MS/MS and for
Immunoprecipitation--
Sperm were directly treated or capacitated
for 18 h as described above; 1 × 108 sperm were
centrifuged, washed in PBS, and then boiled in lysis buffer containing
150 mM NaCl, 0.1% SDS, and 25 mM Tris-HCl, pH 7.5, for 5 min. This treatment was efficient in solubilizing
tyrosine-phosphorylated proteins. Proteins solubilized by this method
were subsequently treated with trypsin or V8 protease (Glu-C) and
tryptic peptides analyzed by MS/MS.
This solubilization procedure was also used for immunoprecipitation
with
-PY (4G10). Briefly, 10 µg of
-PY antibody was added to
the suspension and incubated at room temperature for 1 h. Then 100 µl of a 30% suspension of protein A-Sepharose (PAS) was added and
incubated further for 1 h. The PAS was then washed five times by
repetitive centrifugation with lysis buffer. Finally the washed PAS
beads were resuspended in 1 mM phenyl phosphate and
incubated further for 1 h. The suspension was then centrifuged and
the remaining supernatant divided; 90% of the sample was then used for
MS/MS, and the remaining supernatant was analyzed with
-PY and
-VCP/p97.
-AKAP-3 immunoprecipitation was performed using 5 µl
of
-AKAP-3 serum (13) in a similar manner. In the experiments
depicted in Fig. 2,
-PY and
-AKAP-3 immunoprecipitates were
boiled in sample buffer (14) and analyzed by Western blot using
-PY
RC20 (Transduction Laboratories),
-VCP, and
-AKAP-3 antibodies.
Peptide Synthesis--
Peptides were synthesized as described
(15). Resins, Fmoc
(N-(9-fluorenyl)methoxycarbonyl)-protected amino acids, and
phosphorylated amino acids were purchased from Calbiochem. Peptides
were purified by RP-HPLC, and sequences were verified by MS/MS experiments.
Preparation of Peptide Methyl Esters--
Digests were
evaporated to dryness. Methanolic 2 N HCl or DCl was
prepared by adding 160 µl of acetyl chloride (Aldrich) to 1 ml of
anhydrous d0-methyl alcohol (Aldrich) or
d3-methyl d-alcohol (Aldrich)
dropwise with stirring. After 5 min, 200 µl of the reagent was added
to lyophilized peptide mixtures. This solution was incubated at room
temperature for 2 h and lyophilized. If necessary, the procedure
was repeated to obtain full conversion of carboxyl groups to methyl
esters. Lyophilized peptide methyl esters were reconstituted in 0.1%
acetic acid (standard protein digests) or 1:1:1 acetonitrile, methanol,
and 0.1% acetic acid (sperm protein digests).
IMAC--
IMAC was performed as described previously (16) with
modifications (9). IMAC columns were constructed by packing 8-cm POROS
20 MC (PerSeptive Biosystems, Framingham, MA) into fused silica
microcapillaries (360 × either 200- or 100-µm inner diameter; Polymicro Technologies, Phoenix, AZ). Metal ions were removed from the
column by washing with 50 mM EDTA. Excess EDTA was removed by rinsing with water. The column was then activated with 100 µl of
100 mM FeCl3. Excess iron atoms were removed by
rinsing with 20 µl of 0.1% acetic acid. Peptide mixtures, pH
3.5, were loaded onto the IMAC column at a rate of 1 µl/min.
Nonbinding peptides were removed by rinsing with 25 µl of
acetonitrile/water/acetic acid (25/74/1 v/v/v) containing 100 mM NaCl (Aldrich). The IMAC column was re-equilibrated with
10-20 µl of 0.1% acetic acid and was then connected to a second
fused silica column (360 × 75-µm inner diameter or 360 × 100-µm inner diameter, fused silica). Phosphopeptides were eluted
with 5 µl (360 × 75-µm inner diameter IMAC column) or 8 µl
(360 × 100-µm inner diameter IMAC column) of 50 mM
Na2HPO4, pH 9.0. The reversed phase column was
then disconnected and rinsed with 0.1% acetic acid to remove salts
before subsequent MS analysis.
General LC/MS Parameters--
All HPLC experiments
employed a gradient of 0-60% B in 40 min (unless noted otherwise)
composed of solvent A (0.1 M acetic acid) and solvent B
(70% acetonitrile with 0.1 M acetic acid). All
microcapillary column connections were made with 1 cm of 0.152 × 0.03 cm inner diameter Teflon tubing (Zeus, Orangeburg, SC). In
experiments performed with IMAC enrichment, peptides were loaded onto
an IMAC column and selectively eluted to a C18 microcapillary column (see above). In experiments performed without IMAC enrichment, peptides were loaded directly onto a C18 microcapillary column.
LC/MS Parameters on the LCQ Quadrupole Ion Trap Mass
Spectrometer--
Peptide mixtures were analyzed as described (16,
17). A C18 microcapillary column containing peptides of interest was connected to an analytical column with an integrated ESI emitter tip
(1-5-µm diameter). Peptides were gradient eluted into an LCQ quadrupole ion trap mass spectrometer (spray voltage = 1.6 kV). All MS/MS scans (both targeted and data-dependent) were
performed with an isolation window of 3 Da (precursor
m/z ± 1.5 Da). For data-dependent analyses, the dynamic exclusion option was
selected with a repeat count of 1, a repeat duration of 0.5 min, and
exclusion duration of 1 min.
LC/MS Parameters on the Fourier Transform-Ion
Cyclotron Resonance (FT-ICR) Mass Spectrometer--
Peptide mixtures
were also analyzed on a home-built FT-ICR mass spectrometer (17). C18
microcapillary columns containing the peptide of interest were
connected to analytical columns with integrated ESI emitter tips.
Peptides were eluted into the mass spectrometer with the above
gradient. Full scan mass spectra (m/z 300-5,000)
were acquired at a rate of ~1 scan/s. Mass resolving power ranged
from 5,000 to 10,000.
Phosphoproteome Analysis of Capacitated Sperm Total Protein
Digests--
Aliquots containing 700 µg and 2 mg of capacitated
sperm total protein were digested with trypsin and Glu-C (1:20
enzyme:substrate ratio), respectively, for 18 h at 37 °C. Both
digests were performed in 0.1 M ammonium acetate, pH 8.5. Peptides resulting from each digestion were incubated twice with
methanolic HCl as described. The resulting peptide methyl esters were
loaded onto activated IMAC columns (360 × 200-µm inner
diameter, fused silica). Peptides were gradient eluted into the LCQ ion
trap mass spectrometer with an HPLC gradient (0-60% B in 40 min;
60-100% B in 5 min).
Analysis of Immunoprecipitated Sperm Protein
Digests--
Aliquots containing 50 µg of immunoprecipitated sperm
proteins were digested separately with trypsin and Glu-C (1:20
enzyme:substrate ratio) for 18 h at 37 °C in 100 mM
ammonium bicarbonate, pH 8.5. From 5 to 45 µg of the resulting digest
was desalted and analyzed by IMAC/RP-HPLC/MS as described above with or
without prior methyl ester modification.
Post-IMAC Dephosphorylation of Peptides--
Alkaline
Phosphatase (AP) columns were constructed by packing 360 × 200-µm inner diameter fused silica microcapillaries with 12 cm of
immobilized calf intestine AP (matrix F7m; 50-µm polyvinyl spheres)
from Mobitec (Marco Island, FL). Columns were first rinsed with 20 µl
of reaction buffer (supplied by the manufacturer; 50 mM
Tris HCl, 0.1 mM ZnCl2, 1 mM
MgCl2, pH 8.0). The AP column was then connected to an IMAC
column charged with the phosphopeptides of interest. A C18
microcapillary column (360 × 100-µm inner diameter packed with
6 cm of 5-20-µm irregular C18 particles) was connected to the AP
column. Phosphopeptides were eluted from the IMAC column by rinsing
with 10 µl of modified reaction buffer (same as above except that the
pH was raised to 9.0 with NH3 and EDTA). The IMAC column
was disconnected, and the AP/C18 column array was rinsed with 15 µl
of 0.1% acetic acid. Finally, the C18 column was disconnected and
rinsed with 0.1% acetic acid before MS analysis of dephosphorylated peptides.
Standard Protein Digests for Phosphopeptide
Quantitation--
Standard protein solutions for phosphopeptide
quantitation were made from stock solutions of
-casein (50 pmol/µl). The indicated amount of protein was added to 100 mM NH4HCO3, pH 8.5 (500 µl, final
volume). To each solution was added 1 µg of trypsin (in a volume of 2 µl). Solutions were incubated for 18 h at 37 °C, and the
resulting peptides were converted to the corresponding methyl esters as
described above. Derivatized peptides were dissolved in 500 µl of
0.1% acetic acid. IMAC/RP-HPLC/MS/MS analyses of derivatized digests
were performed on a quadrupole ion trap and a FT-ICR mass spectrometer
as described above.
Global Quantitation of Phosphopeptides from Capacitated and
Noncapacitated Sperm Total Protein Digests--
Approximately 800 µg
each of capacitated and noncapacitated sperm total protein was digested
with trypsin (1:20 enzyme:substrate ratio) for 18 h at 37 °C.
The protein digest of capacitated sperm was treated with
d3-methanolic DCl, whereas the protein digest of
noncapacitated sperm was treated with
d0-methanolic HCl. Peptide methyl esters were
dissolved in 1:1:1 acetonitrile/methanol/0.1% acetic acid, and 100 µg of each digest was loaded onto a 360 × 200-µm inner
diameter IMAC column. Phosphopeptides were eluted to separate 360 × 100-µm inner diameter C18 columns and gradient eluted into the
mass spectrometer. In another experiment, equal portions of each digest
were combined, and 40 µg of total peptide was loaded onto the IMAC
column and gradient eluted into a FT-ICR instrument.
Data Base Searching of Phosphopeptide MS/MS Spectra
and Sequence Assignments--
MS/MS spectra were matched to sequences
in various protein data bases using SEQUEST (18). Spectra from sperm
protein analyses were searched against the nonredundant protein data
base (NRPD) from NCBI and with a subdata base of proteins from NRPD
which contained the phrase testis-specific. In these
searches, differential modification of 80 Da to Ser, Thr, and Tyr
residues was selected. For searches of MS/MS spectra recorded during
analysis of peptide methyl esters, a differential modification of 14 Da
to Glu and Asp acid and a static modification of 14 Da to the C
terminus were also selected. Rapid identification of phosphopeptide
candidates from the 1,000 MS/MS spectra acquired during a typical HPLC
gradient was accomplished with an in-house computer program (neutral
loss tool) (16). This program screens MS/MS spectra for losses
characteristic of phosphorylated peptides (98, 49, and 32.6 Da from
single, double, and triple charged precursor phosphopeptides,
respectively). Neutral loss of phosphoric acid from the peptide
precursor mass is a common feature of ion trap MS/MS spectra (19). For
all phosphotyrosine-containing peptides and one peptide containing two
phosphoserine residues, synthetic peptide MS/MS spectra matched
experimentally obtained MS/MS spectra, confirming our assignments. For
all sequences reported, spectra were manually validated and contained
sufficient information to assign not only the sequence but also the
site of phosphorylation (unless otherwise noted).
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RESULTS |
Characterization of Proteins That Undergo Tyrosine Phosphorylation
during Capacitation--
To identify the proteins that serve as
substrates for tyrosine phosphorylation during capacitation, human
sperm proteins were extracted before and after overnight capacitation
and separated by two-dimensional gel electrophoresis. As described
previously (20), after transfer of the two-dimensional gels to
Immobilon P, a capacitation-associated increase in
protein-tyrosine phosphorylation was observed by Western blot using
-PY (Fig. 1A).
Immunofluorescence experiments also showed an increase in
-PY
fluorescent staining, indicating an increase in tyrosine
phosphorylation (Fig. 1B). Before capacitation, 18% of the
sperm displayed a low intensity
-PY signal in the tail only. After
overnight capacitation, there was a significant increase in the number
of sperm that show fluorescent staining of the head. Although we did
not use an imaging program to quantify fluorescence intensity, it was
possible to observe qualitatively an increase in the fluorescence of
the tail in capacitated sperm compared with a noncapacitated sperm
population as observed in Fig. 1B.

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Fig. 1.
Analysis of the
capacitation-associated increase in protein-tyrosine phosphorylation by
two-dimensional gels and immunofluorescence. A, human
sperm were treated immediately (NON) or capacitated
overnight (CAP), and sperm proteins were analyzed using
two-dimensional gel electrophoresis, transferred to polyvinylidene
difluoride membranes, and Western blots were performed with -PY
(4G10) and developed as described. The arrow in the
right panel indicates the VCP spot. B, different
patterns of Tyr phosphorylation observed in human sperm before
(NON) and after capacitation (CAP). Human sperm
were fixed in paraformaldehyde, dried out, and immunofluorescence was
performed as described under "Experimental Procedures." Values
represent the mean ± S.E. of three independent experiments.
C, silver-stained (left panel) and parallel
Western blots (middle panel, 30-s exposure; right
panel, 5-min exposure) using -PY. Both images were overlaid
using Adobe Photoshop, and silver-stained spots exhibiting Tyr
phosphorylation also were cored and sequenced by MS/MS.
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To identify tyrosine-phosphorylated proteins, extracts from capacitated
human sperm were separated by two-dimensional gel electrophoresis. In
each experiment, two gels were run in parallel. One gel was stained
with Coomassie Blue and subsequently with silver, and the other was
transferred and probed with
-PY (Fig. 1C). Both the
silver-stained gel and the Western blot were scanned and compared.
Protein spots showing
-PY staining were excised, digested, and
sequenced. The results of this analysis are summarized in Table
I and their exact localization shown in
Fig. 1C.
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Table I
Proteins that undergo Tyr phosphorylation during capacitation
Proteins were cut out from silver-stained two-dimensional gels and
microsequenced using MS/MS. To assign a particular protein to the
respective cut band, at least five peptides from a single spot matched
the data-base sequence for the assigned protein.
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Phosphoproteome Analysis of Capacitated Human Sperm
Cells--
Identification of phosphorylation substrates using
two-dimensional gel electrophoresis and immunoblotting is a powerful
approach. However, this methodology has several limitations. First, it
is difficult to identify proteins that undergo phosphorylation on serine or threonine residues because antibodies against those phosphoamino acids are not sensitive enough to detect most proteins phosphorylated on these residues. Second, MS/MS of proteins obtained from polyacrylamide gels has detection limits several orders of magnitude higher than MS performed on proteins not embedded in gels.
Third, the use of this approach strongly suggests that a given protein
is phosphorylated on tyrosine residues; nevertheless, a full
demonstration requires the use of an independent method (e.g. cross-immunoprecipitation, direct sequencing,
mutagenesis analysis). Fourth, although in some cases it is possible to
obtain the exact site of phosphorylation of a candidate protein, in
general the phosphorylation site remains elusive because of the
aforementioned lack of sensitivity. In addition, to determine the site
of phosphorylation is, in most cases, a very important goal in a
phosphorylation study and the strongest demonstration that a protein is
phosphorylated on a particular amino acid.
As shown above, several proteins undergo tyrosine phosphorylation
during capacitation. However, no sites of phosphorylation were defined.
To understand further the role of phosphorylation in human sperm
capacitation, we have analyzed the phosphoproteome of capacitated human
sperm. Toward this goal, we have improved the enrichment of
phosphopeptides by IMAC converting acidic residues to methyl esters and
adapting this technology to the analysis of phosphorylation sites
directly from capacitated human sperm total protein extracts.
Phosphorylated peptides were identified by screening MS/MS spectra for
an abundant neutral loss of phosphoric acid from the peptide precursor
mass. This process is commonly observed in ion trap MS/MS spectra of
phosphorylated peptides (19). When peptides (1 mg) were analyzed by
this method, more than 200 distinct phosphorylated species were
detected. Manual and SEQUEST (18) interpretation of MS/MS spectra led
to the identification of 18 sites of Ser phosphorylation and a single
site of tyrosine phosphorylation on a total of 7 different proteins
(Table II, trypsin). Fragment ions in the
spectra allowed unambiguous assignment of the phosphorylation sites.
MS/MS spectra recorded on synthetic peptides confirmed the sequence
assignment of the phosphotyrosine-containing peptide (data not shown).
Although 200 phosphopeptides were detected, a majority of the spectra
(>75%) showed multiple (up to four) losses of phosphoric acid from
the precursor mass and were difficult to interpret.
Because trypsin digestion can generate phosphopeptides that are too
large or too small to be compatible with RP (C18) chromatography, another protease with a different specificity was used. V8 protease (Glu-C) cleaves substrate proteins on the C-terminal side of Asp and
Glu. Using this protease combined with IMAC and MS/MS, 40 phosphopeptides were detected. Data base searching and de
novo sequencing efforts elucidated new sites of phosphorylation
including 11 on Ser, 1 on Thr, and 2 on Tyr (Table II, Glu-C). Fragment ions in spectra allowed phosphorylated residues to be assigned unambiguously. Alternative enzymes (i.e. chymotrypsin) may
be necessary to define the entire capacitated human sperm
phosphoproteome. No nonphosphorylated sperm peptides were detected in
either analysis. This illustrates that conversion of the sample to
peptide methyl esters prior to IMAC increases significantly the
selectivity of this technique toward phosphorylated peptides.
IMAC Analysis of Immunoprecipitated Protein Digests--
IMAC/MS
analyses of total protein extracts facilitated the discovery of three
sites of tyrosine phosphorylation. Western blotting (Fig. 1), however,
suggests that many more proteins are tyrosine phosphorylated. In an
effort to identify sites of tyrosine phosphorylation selectively, total
capacitated human sperm protein extracts were immunoprecipitated with
-PY antibodies (clone 4G10). Elution was performed using 1 mM phenyl phosphate to ensure selective elution from the
-PY antibody after precipitation with PAS. Immunoprecipitated proteins were digested separately with trypsin or Glu-C and analyzed by
IMAC and MS/MS. De novo and SEQUEST interpretation of the
data identified many new sites of phosphorylation, including 24 on Ser,
1 on Thr, 1 on Tyr, and 1 ambiguous site (phosphoserine or phosphothreonine) (Table II, IP).
Post-IMAC AP Treatment of Phosphopeptides--
Many MS/MS spectra
recorded during IMAC analysis of sperm peptides had peaks at
m/z values corresponding to multiple losses of
phosphoric acid from the precursor mass. Such spectra contain few ions
indicative of amino acid sequence because amide bond cleavage cannot
effectively compete with gas phase dephosphorylation during MS/MS. To
assess whether phosphate removal prior to MS analysis would facilitate
peptide identification, we converted sperm tryptic peptides to methyl
esters. After IMAC enrichment, phosphopeptides were eluted on-line to
an AP column before capture on C18
particles and MS/MS. For comparison, a similar analysis was performed
without AP treatment. These parallel
analyses each detected several peptides (Tables III and IV). The
MS/MS spectrum of SVESVK, recorded during analysis of AP-treated
peptides, contains peaks at m/z values
corresponding to y4, b4, and b5 ions, whereas the phosphorylated
analog, pSVEpSVK, does not (Fig. 2,
A and B, respectively). This illustrates that
phosphate removal prior to MS analysis aids in peptide sequence
determination. Although sequences of dephosphorylated peptides
themselves cannot reveal where or to what extent a peptide is
phosphorylated, complementary information provided by phosphopeptide
MS/MS spectra obtained through parallel analysis without AP treatment
may provide enough information to assign the phosphorylation sites.
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Table III
Phosphopeptide sequences derived from interpretation of
MS/MS spectra recorded during a second analysis of
IMAC-enriched tryptic peptides
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Fig. 2.
On-line post-IMAC AP treatment of
phosphopeptides. MS/MS spectra of a peptide IMAC enriched from
trypsin-digested sperm total protein extract without (A) and
with (B) on-line AP treatment are shown. Predicted masses
for ions of type b and y are shown above and below the sequences,
respectively. Those observed are underlined. Loss of the
elements of phosphoric acid is denoted by . Loss of water is denoted
by *.
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The peptides PLASSPPR and VSGSSQSPPNLK were also detected after AP
treatment. These peptides are derived from PLApSpSPPR and VSGpSSQpSPPNLK observed in analyses of trypsin-digested sperm proteins
(see Table II). Not all phosphorylated peptides detected without AP
treatment were detected after AP treatment. This is probably the result of incomplete elution of phosphopeptides from the
IMAC column in the AP experiment because phosphate was not included in
the IMAC elution buffer to avoid inhibition of the AP enzymatic
activity. In contrast, the use of the AP column allows analysis of
peptides not amenable to positive ion MS in their phosphorylated form.
For example, the peptide SPSAPPAKPPSTQR, detected during MS analysis of
dephosphorylated sperm peptides (Table IV), was not found in samples
without AP treatment. Retention of this peptide by IMAC indicates that
this stretch of amino acids was phosphorylated within the parent
protein (AKAP-4). The phosphorylated form of this peptide may not
ionize well in the positive ion mode, preventing the identification of
this peptide during analysis performed without the use of AP. These
results suggest that dephosphorylation of phosphopeptides after IMAC
enrichment prior to MS analysis is a useful tool to derive sequences of
multiple phosphorylated peptides.
Quantitative Phosphorylation Analysis: Protein Standards--
We
have identified 56 sites of phosphorylation in several capacitated
human sperm proteins. Although this information describes phosphorylation in sperm on a global scale, it would be useful to
discern sites of phosphorylation induced during capacitation. This goal
could be achieved by comparison of the ratio of any particular
phosphopeptide present in digests of proteins from capacitated and
noncapacitated sperm cells. To adapt IMAC/MS methodology to display
phosphopeptides differentially from different sperm capacitated states,
we utilized an isotopic labeling strategy (Fig.
3A). Tryptic peptides from two
samples of cells are converted to peptide methyl esters with deuterated
(d3) and nondeuterated (d0) methanol, respectively. Both samples are
then mixed in equal proportions and the mixture purified by IMAC to
ensure that only phosphopeptides were retained. Signals for
phosphopeptides present in both samples appear as doublets separated by
n(3 Da)/z (where n is the number of
carboxylic acid groups in the peptide and z is the charge on
the peptide). The ratio of the two signals in the doublet changes as a
function of expression level of the particular phosphoprotein in each
sample. Peptides of interest are then targeted for sequence analysis
subsequently performed on the ion trap instrument.

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Fig. 3.
Quantitation of phosphorylated peptides in
protein digests. A, schematic representation of the
procedure for phosphopeptide quantitation. B, ratios of
deuterated and nondeuterated peptide FQpSEEQQQTEDELQDK present in
trypsin digests of -casein. QIT, quadrupole ion trap.
C, capacitated and noncapacitated sperm total protein
extracts measured using the approach described above. D,
mass spectrum recorded during coelution of deuterated and nondeuterated
pSVEpSVK peptides. E, single ion chromatograms
(SIC; obtained by plotting the sum of the intensities of
ions within a small mass window, i.e. 418.67 ± 0.1 Da,
versus time) derived from MS analysis of differentially
methyl ester-modified tryptic sperm peptides using an FT-ICR mass
spectrometer. Double charged ions corresponding to deuterated
(d6) and nondeuterated
(d0) forms of peptides P1 (pSVEpSVK) and P2
(INApSTDpSLAK) were observed. Note that both peptides contain two
carboxyl groups so that derivatized analogs differ in mass by 6 Da.
Peptide sequences were derived from parallel MS/MS experiments
performed on an ion trap mass spectrometer.
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To validate this approach, we used
-casein because tryptic digestion
of this protein produces a phosphorylated peptide, FQpSEEQQQTEDELQDK, which is detectable by positive ion electrospray. Briefly, 50 and 250 pmol of tryptic peptides from the phosphorylated protein
-casein was
converted to d0-peptide methyl esters, and 500 pmol of tryptic peptides from the same protein was converted to
d3-methyl esters. Equal portions were combined
to produce known concentrations of the d0- and
d3-tryptic peptide methyl esters from
-casein and analyzed by MS after IMAC enrichment. Because the phosphorylated
-casein peptide contained seven free carboxyl groups, the mass difference between nondeuterated (d0) and
deuterated (d21) analogs was 21 Da. Mass
spectrometry of these samples revealed
d0:d21 ratios within 11%
of the actual ratio in two separate experiments, indicating that this
method is useful for the quantitation of phosphopeptides (Fig.
3B). Subsequent MS/MS confirmed the sequence of the peptides
to be the deuterated and nondeuterated FQpSEEQQQTEDELQDK. The fact that
deuterated and nondeuterated peptides nearly coelute (deuterated forms
having a slightly lower retention time) coupled with the accurate mass
measurements (typically
20 ppm in our experiments) of FT-ICR allows
for quick correlation of related species. The number of carboxyl groups
can be calculated by Equation 1, where
mass is the mass difference
between deuterated and nondeuterated isotopic distributions of the same
charge state.
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(Eq. 1)
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The information gained by FT-ICR analysis of phosphopeptides
(accurate mass and number of carboxyl groups) can be coupled with the
complementary information provided by data-dependent or
targeted ion trap MS/MS analyses (fragment ions, sequence tags, and
minimum number of phosphorylated residues from neutral losses of
phosphoric acid) to identify peptide sequences from protein data bases rapidly.
To identify capacitation-associated sites of phosphorylation, 2 × 108 human sperm were separated in equal aliquots, one
aliquot was immediately used, the remaining sperm aliquot was
capacitated overnight, and then protein extracts were obtained. Because
sperm are unable to synthesized proteins de novo and both
samples contained the same amount of sperm, quantitation of
phosphopeptides in each sample reflected changes in phosphorylation
which occurred during capacitation. Both noncapacitated and capacitated
human sperm extracts were then digested with trypsin, and the resulting
peptides were converted to the corresponding peptide methyl esters
using d0 and d3 methanol,
respectively. Peptide pools (derived from capacitated and
noncapacitated digests) were then mixed in equal proportions, and an
aliquot was analyzed by IMAC/RP-HPLC/ESI/MS on a home-built FT-ICR
instrument. The data were examined manually to identify singlet peaks,
i.e. phosphopeptide species unique to the capacitated or
noncapacitated sample. Parallel IMAC/RP-HPLC/ESI/MS(/MS) experiments on
a quadrupole ion trap mass spectrometer were used to define the
sequences of these peptides.
Using this method, the peptide pSVEpSVK from a novel
Ca2+-binding protein, CABYR, was present in the capacitated
and noncapacitated sperm total protein digests at about the same level
(Fig. 3C). In contrast, the peptide INApSTDpSLAK, derived
from AKAP-4, was found at a level 23 times greater in capacitated sperm
digests (Fig. 4C), indicating
a capacitation-dependent phosphorylation of this peptide.
The charge state of these peptides can be determined by the difference
in mass between the C12 and C13 isotope peaks observed by MS analysis.
Because the actual mass difference between these isotope peaks is 1 Da,
the observed mass difference of 0.5 Da between peaks in the isotopic
envelopes of pSVEpSVK (Fig. 3D) and INApSTDpSLAK (data not
shown) indicates that both peptides were double charged. Using Equation 1, it was deduced that both phosphopeptides (pSVEpSVK and INApSTDpSLAK)
possessed two free carboxyl groups because double charged isotopic
envelopes corresponding to deuterated and nondeuterated analogs were
separated by 3 Da (Fig. 4D and data not shown). Total ion
chromatograms were obtained by plotting the sum of the intensities of
ions in mass spectra versus time (data not shown). Single
ion chromatograms were acquired by plotting the sum of the intensities
of ions within a small mass window (i.e. 418.67 ± 0.1 Da, versus time) derived from MS analysis of sperm tryptic
peptide methyl esters using an FT-ICR mass spectrometer (Fig.
3E).

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Fig. 4.
VCP/p97 and AKAP-3 are Tyr phosphorylated
during human sperm capacitation. A, total protein
extracts of noncapacitated (Non) and capacitated
(Cap) human sperm were separated in 10% PAGE, transferred
to Immobilon P, and Western blots were performed using -PY (clone
4G10), -VCP (S), donated by Dr. Samelson (23), and -VCP (T)
donated by Dr. Tonks (26), as indicated in the figure. B,
noncapacitated and capacitated human sperm proteins were extracted as
described under "Experimental Procedures" and then
immunoprecipitated with -PY (clone 4G10: 1:50) (left
panel) or with -AKAP-3 (21) (right panel) for 1 h. PAS was then added for another h. PAS beads were then washed five
times using the same buffer, and proteins bound to the PAS were eluted
boiling in sample buffer. Immunoprecipitated proteins were then
separated by 8% PAGE, transferred, and probed with -PY (RC20),
-VCP (S) (29), or -AKAP-3 as indicated in the figure. * indicates
the localization of the VCP band, and # indicates the localization of
AKAP-3. The band shown with the symbol ~ is likely to be an
unprocessed form of AKAP-3 because it is recognized by both -PY and
-AKAP-3 antibodies. Western blots shown in A and
B have been exposed for ~2 min in each case. The same
concentration of nonimmune antibody was unable to immunoprecipitate
VCP/p97 or AKAP-3 (data not shown).
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Approximately 500 peptide species were observed in FT-ICR MS analysis
of IMAC-enriched modified peptides. Most of these species were observed
as doublets, indicating similar levels of phosphorylation between
capacitated and noncapacitated sperm populations; 20 unique species
were differentially phosphorylated in these populations. Most of these
spectra showed neutral losses characteristic of phosphopeptides,
however, peptide sequences could not be derived. Further experiments
will define additional phosphopeptides unique to the capacitated total
protein digests.
Valosin-containing Protein (VCP/p97) Is Tyr
Phosphorylated and Changes the Immunofluorescence Pattern during
Capacitation--
Because capacitation prepares sperm to undergo the
acrosome reaction, a form of regulated exocytosis, phosphorylation of
proteins involved in fusion events may regulate this process and is of particular interest for further investigation. VCP, also known as p97
(VCP/p97), has been implicated in several fusion events in yeast and
mammalian cells (21, 22). This protein appears to undergo tyrosine
phosphorylation during capacitation (Fig. 1C and
arrow in Fig. 1A). However, we were unable to
characterize the VCP site of tyrosine phosphorylation. Several
possibilities could explain the lack of phosphotyrosine phosphopeptides
from VCP. First, the VCP tyrosine phosphorylation site could be masked by the complexity of the MS/MS spectra. Second, although the global analysis of phosphorylation sites is a very powerful approach, it has
limitations. One relevant limitation is that abundant phosphopeptides will be sequenced at higher rates, masking the ability of the method to
detect nonabundant phosphopeptides. This limitation also explains why
we have detected more phosphorylated Ser residues than Tyr residues.
Third, it is also possible that the tyrosine-phosphorylated sequence of
VCP is present in a tryptic peptide that is not detectable with this methodology.
As an alternative, to confirm that VCP is tyrosine phosphorylated
during capacitation we used an independent approach. An
-VCP (S),
donated by Dr. Samelson (23) was used to analyze the presence of this
protein after
-PY immunoprecipitation of noncapacitated as well as
of capacitated sperm protein extracts. As described previously (20,
24),
-PY Western blots from total extracts show that there is an
increase in tyrosine phosphorylation after human sperm capacitation
(Fig. 4A, left panel). 20 million sperm of each
population were extracted in a SDS 0.1% buffer as described under
"Experimental Procedures" and immunoprecipitated using
-PY
(clone 4G10). The immunoprecipitates were then probed with either
-PY (RC20) or
-VCP (S) by Western blot (Fig. 4B, left two panels). Because RC20 is already labeled with
peroxidase, it does not need a secondary antibody avoiding detection of
IgG in the immunoprecipitates. This experiment shows that VCP was more
abundant in the
-PY immunoprecipitates from the capacitated population, confirming the tyrosine phosphorylation of this protein during the capacitation process observed using two-dimensional gel
analysis (Fig. 1A, see arrow in right
panel). A similar experiment was performed to confirm tyrosine
phosphorylation of AKAP-3. In this case, 2 × 107 of
either noncapacitated or capacitated human sperm were extracted and
immunoprecipitated with
-AKAP-3 (13). The immunoprecipitates were then probed with
-AKAP-3 and with
-PY (RC20) (Fig.
4B, right panels). As predicted, the AKAP-3
signal is similar in both immunoprecipitates from noncapacitated or
capacitated sperm. However, the
-PY signal is higher in the
capacitated population confirming that this protein is tyrosine
phosphorylated during capacitation.
Because VCP/p97 has been shown to change subcellular localization after
phosphorylation (25), the localization of VCP/p97 in human sperm was
analyzed before and after capacitation. To evaluate the localization of
VCP/p97, we have used two antibodies from independent sources,
-VCP
(S), donated by Dr. Samelson (23), and
-VCP (T), donated by Dr.
Tonks (26). Both antibodies recognized a single protein by Western blot
(Fig. 4A). Using these antibodies, it was possible to
observe that before capacitation VCP/p97 localized to the neck region
of human sperm. However, after overnight capacitation, the appearance
of fluorescent staining in the anterior head of human sperm was
observed (Fig. 5). It is noteworthy that
the signal in the neck decreased at the same time that the signal in
the anterior head increased. In contrast, another protein, SP-10, which
is present in the sperm acrosomal matrix, localized in the anterior
head in capacitated as well as in noncapacitated human sperm (Fig.
6). Similarly, AKAP-3 localized in the
flagellum in both capacitated and noncapacitated human sperm (Fig. 6).
Because capacitation prepares the sperm to undergo a
ligand-dependent exocytosis and VCP/p97 has a role in
membrane fusion events in other biological systems, our findings
suggest that the regulation of VCP/p97 might be a link between
capacitation and the acrosome reaction.

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Fig. 5.
Immunolocalization of VCP/p97 in human sperm
before and after capacitation. Left panels show the
immunofluorescence (IF) of air-dried human sperm at ×40
magnification using two different rabbit anti-VCP/p97 antibodies,
-VCP (S) and -VCP (T), before (Non) and after
(Cap) capacitation. The antibodies were visualized using
FITC -rabbit secondary antibody as described under "Experimental
Procedures." Right panels are the corresponding bright
fields. Insets in the upper right bright fields
show the -VCP immunofluorescence in either noncapacitated or
capacitated sperm at higher magnification (×100). Controls were
performed using normal rabbit serum.
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Fig. 6.
Immunolocalization of AKAP-3 and SP-10 in
human sperm before and after capacitation. Left panels
show the immunofluorescence (IF) of air-dried human sperm
using a rat -AKAP-3 (×40) antibody or a mouse monoclonal -SP-10
(×100) antibody, -VCP (S) and -VCP (T), before (Non)
and after (Cap) capacitation. The antibodies were visualized
using FITC -rat or FITC -mouse secondary antibody as described
under "Experimental Procedures." Right panels are the
corresponding bright fields. Controls were performed using normal rat
serum or normal mouse serum and showed no immunofluorescence staining
(data not shown).
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DISCUSSION |
The physiological changes that render mammalian sperm able to
fertilize are collectively known as capacitation. Capacitation has been
correlated with the increase in tyrosine phosphorylation of several
proteins (1). With the exception of two members of the AKAP family (13,
27) and CABYR (28), proteins that undergo tyrosine phosphorylation
during capacitation have not yet been characterized. Capacitation
prepares the sperm to undergo the acrosome reaction and also is
associated with changes in sperm motility (e.g.
hyperactivation) in a number of species (1). Therefore, one may
postulate that components of the sperm exocytotic and motility
machinery are modified during capacitation (e.g. phosphorylation of specific proteins, changes in protein localization, and/or modification of protein-protein interactions). In the present work, we have analyzed the phosphoproteome of capacitated human sperm
using 1) two-dimensional gels followed by
-PY Western blot and 2)
direct MS/MS sequencing of phosphopeptides in total protein extracts.
To understand the link between capacitation and the acrosome reaction,
an increased knowledge of the mechanisms that regulate this exocytotic
event in sperm is necessary. Sperm homologs of SNARE (29) as well as
SNARE-associated proteins have been detected in sea urchin (30) and
mammalian sperm (31, 32). These observations support the idea that the
sperm acrosome reaction might be regulated in ways similar to the
exocytotic processes in somatic cells. Among the proteins that undergo
tyrosine phosphorylation during capacitation, we identified VCP/p97 in
this study. VCP/p97, a member of the AAA family (ATPases
Associated with various cellular Activities)
(33), along with NSF and the Golgi t-SNARE syntaxin 5, mediate the
fusion of Golgi membranes (21, 34). Although VCP/p97 and NSF are highly
homologous, they appear to act in distinct fusion events, presumably
because of additional specific cofactors (35). VCP/p97 has a role as a
chaperone and aids in the assembly, disassembly, and functional
operation of protein complexes. VCP/p97 undergoes tyrosine
phosphorylation during T cell activation, and although this
phosphorylation did not alter its ATPase activity (23), tyrosine
phosphorylation regulates the subcellular localization of this protein
(25). Moreover, a membrane fusion process such as the transitional
endoplasmic reticulum assembly in vitro requires tyrosine
phosphorylation of VCP/p97 (36). We have demonstrated that VCP/p97
undergoes tyrosine phosphorylation during capacitation. In addition, we
have shown that prior to capacitation VCP/p97 localizes in the neck of
human sperm, whereas after overnight incubation, immunofluorescence
experiments showed a decreased staining in the neck and the appearance
of this protein in the anterior head of capacitated sperm. At least
three hypotheses can be made to explain these different
immunofluorescent patterns. First, modifications of the sperm during
capacitation allowed
-VCP antibodies to enter the anterior head of
the sperm. Although possible, experiments showing that SP-10 can be
recognized before and after capacitation added to the observation of a
decrease in fluorescent staining in the neck of capacitated sperm argue against this explanation. Second, the epitopes recognized by two
-VCP antibodies are unmasked in the anterior head, but they are masked in the neck of capacitated sperm. Third, there is a
translocation of VCP during capacitation from the neck to the anterior
head of human sperm. Ideally this hypothesis should be tested by a direct measurement of VCP in the neck and the anterior head after separation of these subcellular compartments; however, the
impossibility of separating the neck from the anterior head prevented
us from performing this experiment. Considering the role of VCP/p97 and other members of the AAA family of phosphatases in fusion processes, these results suggest that VCP/p97 and tyrosine phosphorylation of this
protein could have a role as a link between capacitation and the
acrosome reaction. Alternatively, this protein can act as a chaperone,
bringing relevant membrane fusion proteins to the site of the acrosome reaction.
Capacitation is also linked to events that occur in the sperm
flagellum. AKAPs represent a growing family of scaffolding proteins that function to tether the regulatory subunits of protein kinase A and
other enzymes to organelles or cytoskeletal elements. These proteins
permit the precise control of signal transduction in discrete regions
of the cell (37). In the present work, we have confirmed tyrosine
phosphorylation of AKAP-3 and AKAP-4 during human sperm capacitation
and mapped eight phosphorylation sites of these proteins. Among these
sites, the AKAP-4 phosphopeptide INApSTDpSLAK was found to be 23 times
more abundant in capacitated sperm, suggesting that this
phosphorylation site might be involved in the regulation of AKAP-4
function during capacitation.
Multiple methodologies have been used to study phosphorylation.
Recently, mass spectrometry has become the preferred method to identify
phosphopeptides because of its speed and high sensitivity. Nevertheless, because phosphorylation sites are usually
substoichiometric, it has been necessary to devise new methods for
selective detection and enrichment of phosphopeptides. Recently,
although several methods have been developed, most have been applied
only on a protein-by-protein basis (8) with a few notable exceptions (38, 39). None of these methods has been used successfully to identify
phosphotyrosine residues from complex mixtures probably because of
multiple steps of derivatization that reduced the final recovery. On
the other hand, IMAC has been used previously to enrich digests for
phosphorylated peptides (6, 16); however, the selectivity of this
technique is poor because peptides containing acidic residues
(i.e. Glu and Asp) bind to the immobilized iron atoms (40).
To solve this lack of specificity, acidic residues were converted to
methyl esters, eliminating the binding of nonphosphorylated species to
the IMAC column. This procedure does not generate diasteromers and is
compatible with phosphorylated Ser, Thr, and Tyr residues. This
methodology was used successfully to map 60 sites of phosphorylation.
The sites of phosphorylation identified here defined in vivo
sites of phosphorylation resulting from normal phosphorylation events
in capacitated human sperm and were not obtained by kinase overexpression or by kinase activators or phosphatase inhibitors. Although most of the sites described in the present work have not been
observed previously, phosphorylation of the C-terminal protein
kinase A catalytic subunit peptide IRVpSINE has been demonstrated to be
necessary for the catalytic activity of recombinant protein kinase A
(41). Another observation derived from the capacitated human sperm
phosphoproteome is that four of the five tyrosine-phosphorylated sequences contained phosphoserine in the proximity of the
phosphotyrosine. This result raises the possibility that a dual
specificity kinase is involved in the capacitation process and/or that
phosphoserine is part of the substrate recognition motif for a sperm
tyrosine kinase. Because the identity of tyrosine kinases present in
sperm is at present not known, peptide sequences found to be
tyrosine-phosphorylated can be used as substrates to purify tyrosine
kinases from sperm.
After enrichment of phosphorylated peptides, the next step is to derive
their sequences and define the sites of phosphorylation. In many cases,
identification of the exact phosphorylation sequence was precluded
because of the complexity of the phosphopeptide MS/MS spectra. To
simplify interpretation of phosphopeptide spectra, we performed on-line
AP dephosphorylation after IMAC phosphopeptide enrichment. AP treatment
has been shown to enhance detection of multiply phosphorylated peptides
(42). Analysis of phosphopeptides followed by AP treatment and
reanalysis has been demonstrated as a technique for phosphopeptide
identification based on 80-Da differences (43). This method allowed
identification of one additional peptide that was not detected without
AP treatment.
In summary, 16 proteins that are recognized by
-PY antibodies in
two-dimensional Western blots were determined. From these proteins,
VCP/p97 was also detected in
-PY immunoprecipitates and showed
different immunolocalization patterns before and after capacitation. In
addition, we have successfully employed IMAC enrichment of
phosphopeptides coupled to MS/MS analysis to isolate and sequence
phosphopeptides from total protein digests of human capacitated sperm.
This is the first study in which this methodology has been used to map
phosphopeptides in a human cell type. Moreover, we have coupled this
methodology with differential isotopic labeling and IMAC enrichment to
derive quantitative information on phosphorylation events that occur
during capacitation. This technique goes beyond the field of
reproductive biology and could potentially be used to map and compare
sites of phosphorylation in a variety of biological systems.