Abstract
Improvements in ion trap instrumentation have made n-dimensional mass spectrometry more practical. The overall goal of the study was to describe a model for making use of MS(2) and MS(3) information in mass spectrometry experiments. We present a statistical model for adjusting peptide identification probabilities based on the combined information obtained by coupling peptide assignments of consecutive MS(2) and MS(3) spectra. Using two data sets, a mixture of known proteins and a complex phosphopeptide-enriched sample, we demonstrate an increase in discriminating power of the adjusted probabilities compared with models using MS(2) or MS(3) data only. This work also addresses the overall value of generating MS(3) data as compared with an MS(2)-only approach with a focus on the analysis of phosphopeptide data.
Highlights
Improvements in ion trap instrumentation have made ndimensional mass spectrometry more practical
Data generated by the mass spectrometer are processed via the Trans-Proteomic Pipeline (TPP) following normal procedures and using SEQUEST, Mascot, or X! Tandem database search tools for peptide identification up through generation of peptide probabilities from PeptideProphet [32]
Concluding Remarks—The generation of MS3 information is common in directed areas of proteomics such as phosphopeptide identification
Summary
Improvements in ion trap instrumentation have made ndimensional mass spectrometry more practical. One of the most widely used mass analyzers for protein work has historically been the ion trap, and a large proportion of the data from current mass spectrometry-based proteomics experiments are generated on such instruments. A number of researchers are choosing to routinely collect MS3 spectra during LC-MS/MS runs that have the potential to provide additional information useful for peptide identification and characterization This is deemed important in the case of proteins identified by single peptides [10, 11] and for the analysis of phosphopeptides, the spectra of which are frequently dominated by a major fragment ion representing neutral loss of the phosphate group from the precursor peptide. MS3 spectra have proven to be useful in top-down analysis as well both for protein identification and for characterization of specific sites of posttranslational modification [15, 16]
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