Phosphorylation-Specific MS/MS Scoring for Rapid and Accurate Phosphoproteome Analysis

Abstract

The promise of mass spectrometry as a tool for probing signal-transduction is predicated on reliable identification of post-translational modifications. Phosphorylations are key mediators of cellular signaling, yet are hard to detect, partly because of unusual fragmentation patterns of phosphopeptides. In addition to being accurate, MS/MS identification software must be robust and efficient to deal with increasingly large spectral data sets. Here, we present a new scoring function for the Inspect software for phosphorylated peptide tandem mass spectra for ion-trap instruments, without the need for manual validation. The scoring function was modeled by learning fragmentation patterns from 7677 validated phosphopeptide spectra. We compare our algorithm against SEQUEST and X! Tandem on testing and training data sets. At a 1% false positive rate, Inspect identified the greatest total number of phosphorylated spectra, 13% more than SEQUEST and 39% more than X!Tandem. Spectra identified by Inspect tended to score better in several spectral quality measures. Furthermore, Inspect runs much faster than either SEQUEST or X!Tandem, making desktop phosphoproteomics feasible. Finally, we used our new models to reanalyze a corpus of 423 000 LTQ spectra acquired for a phosphoproteome analysis of Saccharomyces cerevisiae DNA damage and repair pathways and discovered 43% more phosphopeptides than the previous study.

Large-Scale Arabidopsis Phosphoproteome Profiling Reveals Novel Chloroplast Kinase Substrates and Phosphorylation Networks

“…1A). The resulting data files were analyzed using the MASCOT and INSPECT search algorithms (Tanner et al, 2005;Payne et al, 2008;www.matrixscience. com).…”

“…INSPECT supports Tyr phosphorylation but MASCOT does not, suggesting that the Tyr phosphorylation site is questionable. INSPECT was trained on LTQ data and searches the tandem mass spectrometry (MS/MS) spectrum for sequence tags and the characteristic neutral loss of phosphoric acid (298 atomic mass units [amu]) from the parent ion (Payne et al, 2008). Because phosphotyrosine is more stable than phosphoserine or phosphothreonine, the neutral loss does not readily occur, therefore causing problems for the assignment of Tyr phosphorylation.…”

Phosphoproteomic Profiling Reveals Vasopressin-Regulated Phosphorylation Sites in Collecting Duct

“…The database also contained sequences for common MS contaminants, such as human keratin and porcine trypsin. Data from the phosphoproteome profiling experiment were searched with SE-QUEST, 44 InsPecT, 45,46 and OMSSA. 47 InsPecT and OMSSA searches used the high-performance computational capabilities of the Biowulf Linux cluster at the National Institutes of Health, Bethesda, MD (http://biowulf.nih.gov).…”

Linking the proteins—Elucidation of proteome‐scale networks using mass spectrometry

“…Detection of phosphate neutral losses in tandem mass spectra supports the phosphorylated nature of the sequences matched to the experimental spectra (Bodenmiller et al, 2007c;Lu et al, 2007). Programs were developed to specifically test the validity of phosphosite assignments (Beausoleil et al, 2006;Albuquerque et al, 2008;Payne et al, 2008), and to compile MS 2 and MS 3 search results Hoffert et al, 2007;Ulintz et al, 2008).…”