ABRF-PRG03: phosphorylation site determination

Abstract

A fundamental aspect of proteomics is the analysis of post-translational modifications, of which phosphorylation is an important class. Numerous nonradioactivity-based methods have been described for high-sensitivity phosphorylation site mapping. The ABRF Proteomics Research Group has conducted a study to help determine how many laboratories are equipped to take on such projects, which methods they choose to apply, and how successful the laboratories are in implementing particular methodologies. The ABRF-PRG03 sample was distributed as a tryptic digest of a mixture of two proteins with two synthetic phosphopeptides added. Each sample contained 5 pmol of unphosphorylated protein digest, 1 pmol of each phosphopeptide from the same protein, and 200 fmol of a minor protein component. Study participants were challenged to identify the two proteins and the two phosphorylated peptides, and determine the site of phosphorylation in each peptide. Almost all respondents successfully identified the major protein component, whereas only 10% identified the minor protein component. Phosphorylation site analysis proved surprisingly difficult, with only 3 of the 54 laboratories correctly determining both sites of phosphorylation. Various strategies and instruments were applied to this task with mixed success; chromatographic separation of the peptides was clearly helpful, whereas enrichment by metal affinity chromatography met with surprisingly little success. We conclude that locating sites of phosphorylation remains a significant challenge at this level of sample abundance.

FIGURE 1

Mass spectrometric characterization of the test sample. A: MALDI-TOF analysis of 10% of the unfractionated mixture. Peaks corresponding to PDI (*) and BSA (x) are labeled. Regions containing the phosphopeptides are shown in the insets. B: Ion trap MS/MS of phosphopeptide P1, products of [M+2H]²⁺ = 482.7. The sequence and predicted b and y series fragments are listed; underlined masses were observed in the spectrum. C: MS/MS of phosphopeptide P2, [M+2H]²⁺ = 1013.9, labeled as in B.

FIGURE 2

Success rates for mass spectrometric approaches to protein identification. Five MALDI-TOF as well as all LC-MS and static nanospray analyses used MS/MS.