Mammalian sperm are not capable of fertilization immediately after ejaculation. In 1951, Chang1
independently demonstrated that mammalian sperm require residence in the female tract for a period of time to acquire their fertilizing capacity. Following these original observations, many studies confirmed that the environment of the female tract induces a series of physiological changes in the sperm; these changes are collectively called “capacitation”. This maturational process is associated with the activation of a phosphorylation cascade in which protein kinase A (PKA), a serine/ threonine kinase, is upstream of the increase in tyrosine phosphorylation of several proteins.19
To understand, at the molecular level, the function of this phosphorylation cascade, as well as how it is regulated, it is important to identify the proteins that change their phosphorylation status during capacitation. Recently, identification of proteins that become tyrosine phosphorylated during this process has been conducted using a combination of 2D-PAGE and MS/MS.7,20
However, very little is known about proteins phosphorylated during capacitation on serine or threonine residues. In the present manuscript, a global approach has been taken to compare the phosphorylation status of sperm incubated in conditions that either support or do not support capacitation. Importantly, this study was designed to identify in vivo
sites of phosphorylation rather than using cells treated with kinase activators/phosphatase inhibitors or cells genetically modified to overexpress specific proteins.
Phosphorylation by protein kinases forms the basis of intracellular signaling networks, including transduction of extracellular signals, intracellular transport, and cell cycle progression. Among the many methods used to study protein phosphorylation, mass spectrometric identification of phosphorylation sites is preferred due to its speed and high sensitivity.21
Although methods have been developed to isolate phosphopeptides in the mass spectrometer on the basis of CAD-induced neutral losses,22
competitive ionization with nonphosphorylated peptides in the source limits the overall suitability of this method for the global identification of phosphorylation sites from complex mixtures. Instead, a majority of current methods utilize a phosphopeptide enrichment step prior to mass spectrometric analysis.23-25
In a recent study, over 2200 nonredundant phosphorylation sites were identified in Saccharomyces cerevisiae
following iron(III) IMAC-based phosphopeptide enrichment and analysis on an LTQ-Orbitrap mass spectrometer.26
Although a similar enrichment methodology was employed here, the ultimate goal of this study was not only to identify the specific sites of phosphorylation present in a capacitated sperm population, but also to compare the relative extent of phosphorylation occurring at these sites as a result of the capacitation process.
Similar to other high-throughput methodologies such as microarrays, the advantage of a global phosphoproteomics approach is that this method is unbiased and capable of generating an extensive amount of data in a relatively short time. However, more targeted information can be achieved when this technique is used to analyze the functional changes occurring in a biological process. Because different peptides have different ionization efficiencies and mass spectrometric responses, mass spectrometry is not amenable for direct quantification. However, stable-isotope analogues of a chemical entity can be compared and used for relative quantification between two or more differentially labeled populations. In proteomics, differential isotopic labeling can be achieved by chemical modification27
or by metabolic labeling.28
In the present work, the esterification of peptide carboxylic acids was used primarily to prevent nonphosphorylated peptides in the complex mixture from interfering with the IMAC-based phosphopeptide enrichment. However, by taking advantage of this chemical modification and conducting the Fisher esterification with both deuterated and nondeuterated methanol, it became possible to quantify phosphopeptide expression between the capacitated and noncapacitated samples. Signals for phosphopeptides present in both samples appeared as doublets in the MS spectra separated by n
is the number of carboxylic acid groups n the peptide and z
is the charge on the peptide). The ratio of the two signals changes as a function of the phosphorylation or dephosphorylation that occurs during the capacitation process. At the same time, peptides were isolated and fragmented using CAD in the linear ion trap to provide MS/MS spectra for subsequent phosphopeptide sequencing. This methodology allowed for the determination of capacitation-dependent changes in phosphorylation; moreover, comparison of doublets gave a quantitative estimate of the level of phosphorylation of each sequence and the ability to analyze exact phosphorylation sites. Interestingly, a phosphotyrosine containing peptide from hexokinase type I was identified and determined to be present at the same level in both sample types. This finding is consistent with previous reports3
indicating that hexokinase type I is tyrosine phosphorylated in mouse sperm.
A fortunate consequence of this approach was that the differential labeling procedure also aided in the sequencing of the phosphopeptide spectra. The fact that deuterated and nondeuterated forms of the same peptide nearly coelute (deuterated forms having a slightly lower retention time) coupled with the accurate mass measurements from the high resolution FT-ICR mass spectra allows for quick correlation of related peptide species. The use of this information in combination with the readily discernible peptide charge state allows for the number of modifications to be determined in each phosphopeptide. The knowledge of the number of acidic residues in a given peptide greatly limits the number of potentially “correct” peptide assignments and manual inspection of the deuterated and nondeuterated MS/MS spectra quickly reveals the types of ions present (b- or y-type ions), permitting further refinement. As this study required a comprehensive analysis of peptide expression in a complex mixture, all analyses were conducted on LTQ-FT hybrid linear ion trap-Fourier Transform mass spectrometer, a technology particularly well-suited to the analysis of differential peptide/protein expression.
Among the sites presenting a capacitation-associated increase in phosphorylation was the phosphopeptide LIpSSeNFeNYVR, from fatty acid binding protein 9, also known as PERF15.29,30
PERF15 is a testis-specific protein and it is the major protein component of the sperm structure known as perforatorium in the perinuclear theca, which is found between the inner acrosomal and the outer face of the nuclear envelope of the sperm head. Our results demonstrate a capacitation-dependent phosphorylation of PERF15 on the Ser3 residue. In the context of our findings, it has been shown that another fatty acid binding protein family member, the 422 protein, is also regulated by phosphorylation.31
It is tempting to speculate that similar changes in phosphorylation, occurring as a result of capacitation, may be functionally relevant for PERF15.
In the highly polarized sperm cell, various compartmentalized functions are regulated by protein kinase A (PKA) signaling. In particular, sperm capacitation has been associated with the activation of a phosphorylation cascade in which PKA, a serine/ threonine kinase, is upstream of the increase in tyrosine phosphorylation of several proteins.19
Taking this into account, the finding that several sites belonging to the sperm-specific A-kinase anchor protein 4 (AKAP4) are phosphorylated during capacitation is noteworthy. In particular, we note that the peptide LSpSLVIQMAR increased its phosphorylation by a factor of 4.62 in the capacitated sample. This peptide is part of the α helix involved in the binding of AKAP4 to PRKAR2 (RIIα)32,33
and changes in the charge density of this α helix due to phosphorylation may have consequences in the binding of AKAP4 to RII and therefore affect cAMP-dependent signaling.
Almost half of the peptides found to be phosphorylated as a result of capacitation corresponded to proline-directed phosphorylation sites. These observations are in agreement with previous work from our laboratory11
and from others.34
Among proline-directed kinases, ERK1/2 are present in mouse sperm; however, two different ERK pathway inhibitors, U0126 and PD098059, were not able to block the capacitation-associated increase in proline-directed phosphorylation.11
Further studies will be necessary to determine the role of this kinase, other proline-directed kinases, and the overall relevance of proline-directed phosphorylation in mouse sperm capacitation. Although our analysis has been limited to date, these results are encouraging and it is believed that more phosphorylation sites will be identified in the future, as a comparison of the high-resolution MS spectra identified several differentially labeled peak pairs. Toward this goal, development of software to simplify the analysis is currently ongoing and the use of electron transfer dissociation (ETD) as an alternative to CAD for phosphopeptide fragmentation is being explored. The methodology presented here goes beyond the field of reproductive biology and could be used to understand signaling mechanisms in a wide array of biological systems.