Abstract
Owing to its broad biological significance, the large-scale analysis of protein phosphorylation is more and more getting into the focus of proteomic research. Thousands of phosphopeptides can nowadays be identified using state-of-the-art tandem mass spectrometers in conjunction with sequence database searching, but localizing the phosphate group to a particular amino acid in the peptide sequence is often still difficult. Using 180 individually synthesized phosphopeptides with precisely known phosphorylation sites (p-sites), we have assessed the merits of the Mascot Delta Score (MD score) for the assignment of phosphorylation sites from tandem mass spectra (MS/MS) generated on four different matrix-assisted laser desorption ionization (MALDI) mass spectrometers including tandem time-of-flight (TOF/TOF), quadrupole time-of-flight, and ion trap mass analyzers. The results show that phosphorylation site identification is generally possible with false localization rates of about 10%. However, a comparison to previous work also revealed that phosphorylation site determination by MALDI MS/MS is less accurate than by ESI-MS/MS particularly if several and/or adjacent possible phosphorylation acceptor sites exist in a peptide sequence. We are making the tandem MS spectra and phosphopeptide collection available to the community so that scientists may adapt the MD scores reported here to their analytical environment and so that informatics developers may integrate the MD score into proteomic data analysis pipelines.
Highlights
Owing to their widespread occurrence and broad biological significance, posttranslation modifications (PTMs) of proteins are increasingly studied by proteomic methods [1]
Based on a set of 180 individually synthesized phosphopeptides with precisely known phosphorylation sites and thousands of tandem mass spectra generated by all common types of electrospray ionization (ESI) MS/MS methods, we have recently described and evaluated the Mascot Delta Score (MD score) for phosphorylation site localization [18, 27, 28]
We have evaluated the ability of the MD score to identify phosphorylation sites in matrix-assisted laser desorption ionization (MALDI) tandem MS spectra collected for 180 peptides with precisely known phosphorylation sites on four different instrument platforms
Summary
Owing to their widespread occurrence and broad biological significance, posttranslation modifications (PTMs) of proteins are increasingly studied by proteomic methods [1]. The development of efficient biochemical enrichment procedures [2, 3] and advances in nanoscale chromatography and tandem mass spectrometry allow the analysis of thousands of phosphopeptides from a given biological system [4,5,6,7]. We and others have developed computational approaches that aim to alleviate this problem. Speaking, these methods can be divided into two categories. Well-known representatives of the first category are the Ascore and PTMs score that interpret the tandem mass spectra based on empirical fragmentation rules and score the likelihood that a particular spectrum represents a certain phosphopeptide [5, 9,10,11,12,13,14]. The phosphorylation site localization score uses the results of a sequence database search engine [15,16,17,18,19], and these tools score the likelihood that a particular phosphorylation site isomer of a peptide is best represented by the spectrum that generated the match
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