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Team Quantitates Phosphorylation Sites in Mice Sperm; May Lead to Male Contraceptive, Rxs


A team of investigators have used differential isotope labeling and mass spectrometry to identify 55 unique in vivo sites of phosphorylation and quantitated phosphorylation for 42 different phosphopeptides in murine sperm.

The work, which could provide a basis for the development of a male contraceptive and new drug therapies, "represents the first effort to determine which specific protein phosphorylation sites change their phosphorylation status in vivo as a result of the mammalian capacitation process," the researchers said.

In addition, "the methodology presented [in the study] goes beyond the field of reproductive biology and could be used to understand signaling mechanisms in a wide array of biological systems," according to the team, whose work appears in a study in the Journal of Proteome Research.

It is well established that mammalian sperm cannot fertilize an egg immediately after ejaculation, but instead has to wait for a time in the female reproductive tract during which physiological changes make the sperm capable of fertilization.

In earlier work, some members from the JPR team showed that this process, called "sperm capacitation," is associated with an increase in protein phosphorylation. That research also demonstrated that the increase in phosphorylation and the overall process of capacitation is regulated by a cAMP-dependent pathway.

However, little is known about the protein targets of the phosphorylation cascade or of the kinases and phosphatases that help regulate sperm function, the authors said in the JPR study.

But "with the current tools that we have — with the isotopic labeling, with the immobilized metal affinity chromatography for phosphopeptide enrichment, with the tandem mass spectrometry — we're able to understand the capacitation process at the molecular level, and that's where the strength of the research [described in the JPR study] lies," the study's corresponding author, Mark Platt, told ProteoMonitor this week.

Platt, an assistant professor of chemistry and chemical biology at Rensselaer Polytechnic Institute, stressed that the study does not identify phosphorylated proteins, but rather is meant to observe the relative quantitation of phosphorylation on specific amino acids within those proteins.

And though the study looked at sperm capacitation in mice, Platt said that the methods described in the study have applications beyond the development of a male contraceptive, and could help develop new therapies for a broad range of diseases.

The majority of current drugs are targeted to proteins, but inactivating an entire protein often leads to unwanted side effects. "With this information, instead of having to inactivate the entire protein, what we could do is … develop a drug that [would] prevent a specific site on that protein from being phosphorylated, perhaps leaving the other functions of that protein undisturbed," he said. "It's really molecular medicine that we're getting at."

In their study, Platt and his co-researchers set out to use a global approach to compare the phosphorylation status of sperm incubated in conditions that either support or do not support capacitation.

Previous work using 2D-PAGE, anti-phosphotyrosine antibody labeling, and tandem mass spectrometry identified three sites of tyrosine phosphorylation on the sperm tail proteins AKAP3 and AKAP4, but the sensitivity of the approach was limited by the 2D-PAGE separation, the researchers said.

In particular, the losses resulting from additional handling of the sample and the fact that the proteins had to be visualized before they were excised prevented MS/MS identification of the phosphorylation sites except for the most abundant proteins.

The approach described in the JPR paper, the researchers said, works around these limitations by avoiding 2D-PAGE separation completely. Because they were interested in the "global identification of capacitation-induced phosphorylation changes," they also chose not to use antibodies because anti-phosphoserine and anti-phosphothreonine antibodies "have not yet achieved sufficient selectivity."

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Instead, they used a workflow based on differential isotopic labeling coupled with IMAC-based phosphopeptide enrichment and analysis on an LTQ-FT hybrid linear ion trap Fourier transform mass spec from Thermo Fisher Scientific to measure the changes in protein phosphorylation resulting from the capacitation process.

In addition, they used the Fisher esterification reaction "in which acidic residues are converted to their corresponding methyl esters" to prevent binding of non-phosphorylated peptides to the IMAC resin, increasing selectivity for phosphopeptides.

Then, by carrying out Fisher esterification with either deuterated or non-deuterated reagent, they were able to calculate the relative phosphorylation "for phosphopeptides found in both the capacitated and non-capacitated sperm samples," they said in their study.

Their approach was designed specifically to identify in vivo sites of phosphorylation rather than using cells treated with kinase activators/phosphatase inhibitors or cells modified to overexpress specific proteins. That capability, Platt said, is a "consequence of the higher sensitivity of the instrumentation," including the mass spec and the chromatographic separation, which incorporated an in-house developed microcapillary column.

Using their approach on mice, the researchers compared phosphorylation levels for capacitated and non-capacitated sperm samples, and were able to identify 44 peptides that exhibited differential phosphorylation on 59 specific amino acids.

The relative abundances of 42 of the 44 phosphopeptides were then determined. Of the other two phosphopeptides, one appeared only in the capacitated sample, and the other appeared only in the non-capacitated sample.

In the study, the researchers said that several peptides showed only minor variance between capacitated and non-capacitated samples, but others were detected at increased levels in capacitated samples that "strongly" suggested that the phosphorylation of these sequences resulted from the capacitation process.

One such phosphopeptide was LIpSSeN-FeNYVR from fatty acid binding protein 9. Also known as PERF15, it is a testis-specific protein that is the major protein component of the sperm structure known as perforatorium. The researchers found capacitation-dependent phosphorylation of the protein on the Ser3 residue.

Because the researchers found another fatty acid binding protein family member — the 422 protein — to be regulated by phosphorylation, they said "it is tempting to speculate that similar changes in phosphorylation, occurring as a result of capacitation, may be functionally relevant for PERF15."

In other cases, some peptides were more abundant in the non-capacitated samples, suggesting that these sequences undergo dephosphorylation during sperm capacitation, they added.

Phosphorylation can lead to capacitation, but dephosphorylation can also do the same thing, Platt said. "I'm sure it's much more complex than a single phosphorylation site," he said. "It's a concerted series of reactions: dephosphorylation of some proteins [and]phosphorylation of others leading to these signal transduction cascades that eventually result in capacitated sperm."

The study corroborated earlier findings by members of the team that proline-directed phosphorylation is upregulated in mouse sperm as a result of the capacitation process. In the current study, about half of the peptides found to be phosphorylated during the capacitation process correspond to proline-directed phosphorylation sites, although more work needs to be done to understand the role and relevance of proline-direced phosphorylation in mouse sperm capacitation, they wrote.

The authors said their analysis has been limited, but called their results "encouraging." They added that they believe that more phosphorylation sites will be identified "as a comparison of the high-resolution MS spectra identified several differentially labeled peak pairs."

Development of software to simplify the analysis is being undertaken, they said, and the use of electron-transfer dissociation as an alternative to collisionally activated dissociation is being explored.

Platt and Pablo Visconti, an associate professor of veterinary and animal sciences at the University of Massachusetts at Amherst, are doing follow-up work and using ETD to recreate the initial results, and hope to go beyond the 55 phosphorylation sites identified in the current study "into the hundreds if not thousands of sites of phosphorylation," Platt said. "Based upon the data that we currently have, we've already got some good targets to look at."

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