Comprehensive Identification of Phosphorylation Sites in Postsynaptic Density Preparations

Abstract

In the mammalian central nervous system, the structure known as the postsynaptic density (PSD) is a dense complex of proteins whose function is to detect and respond to neurotransmitter released from presynaptic axon terminals. Regulation of protein phosphorylation in this molecular machinery is critical to the activity of its components, which include neurotransmitter receptors, kinases/ phosphatases, scaffolding molecules, and proteins regulating cytoskeletal structure. To characterize the phosphorylation state of proteins in PSD samples, we combined strong cation exchange (SCX) chromatography with IMAC. Initially, tryptic peptides were separated by cation exchange and analyzed by reverse phase chromatography coupled to tandem mass spectrometry, which led to the identification of phosphopeptides in most SCX fractions. Because each of these individual fractions was too complex to characterize completely in single LC-MS/MS runs, we enriched for phosphopeptides by performing IMAC on each SCX fraction, yielding at least a 3-fold increase in identified phosphopeptides relative to either approach alone (SCX or IMAC). This enabled us to identify at least one site of phosphorylation on 23% (287 of 1,264) of all proteins found to be present in the postsynaptic density preparation. In total, we identified 998 unique phosphorylated peptides, mapping to 723 unique sites of phosphorylation. At least one exact site of phosphorylation was determined on 62% (621 of 998) of all phosphopeptides, and ϳ80% of identified phosphorylation sites are novel. Molecular & Cellular Proteomics 5: 914 -922, 2006.

Analytical strategies for phosphoproteomics

“…In addition, highly efficient phosphopeptide fragmentation strategies such as pdMS 3 , MSA, ECD, and ETD enable better identification and sequencing of phosphopeptides for accurate assignment of phosphorylation sites. Combined with the recent development of new and highly specific and sensitive methods for phosphoprotein and phosphopeptide enrichment, MS has for the first time made it feasible to perform large-scale phosphoproteomic studies of highly complex samples [35,43,46,51,86]. However, we believe that further development of phosphoproteomic strategies is still needed in order to be able to characterize signaling pathways employing low abundant protein phosphorylation.…”

“…The trypsin digestion was performed in 1 M guanidine hydrochloride, and the authors suggested that the guanidinium ions could have acted as a competitive inhibitor of trypsin, thereby reducing its activity. This would lead to a high level of missed cleavages, which could explain the higher charge state on the observed peptides [51]. However, recent work by Villén et al in which they fractionated tryptic digests from murine liver using SCX and enriched all 15 fractions for phosphopeptides using IMAC, revealed that phosphopeptides were spread throughout all SCX fractions [35].…”

“…Ficarro and co-workers circumvented this problem by derivatizing the carboxylic groups on acidic amino acid residues in peptides by O-methyl esterification [48] and thereby improving phosphopeptide enrichment considerably [48][49][50]. Further experiments have shown, however, that Omethyl esterification does not derivatize 100% of the available carboxylic acid groups, and that it can increase the complexity of the analysis due to signals originating from peptides with different degrees of O-methylation [51]. Furthermore, the buffer used for O-methyl esterification can result in partial deamidation forming glutamic acid from glutamine as well as aspartic acid from asparagine [52].…”

“…Trinidad and co-workers compared the enrichment efficiency of SCX as a prefractionation method prior to IMAC to both IMAC and SCX alone [51]. They found that the combination of SCX and IMAC led to at least a three-fold increase in the number of phosphopeptides identified relative to either approach alone.…”

New fractionation tools targeting elusive post-translational modifications

“…with IMAC or TiO 2 . 69,75,79, 99, 100 In hydrophilic interaction chromatography (HILIC) the neutral hydrophilic stationary phase is used. 101 Typically the elution is performed in the acetonitrile-water system starting with high organic mobile phase.…”