The UVPD pathways in Mass Matrix were rigorously and statistically optimized using two large data sets with high mass accuracy and high mass resolution for both MS data acquired on an Orbitrap mass spectrometer for complex He La and Halo proteome samples.
Given that ∼50% of peptides/proteins are naturally acidic (6) and that many of the most important post-translational modifications ( phosphorylation, acetylation, sulfonation, etc.) significantly decrease the isoelectric points of peptides (7, 8), there is a compelling need for better analytical methodologies for characterization of the acidic proteome.
A principal reason for the shortage of methods for peptide anion characterization is the lack of MS/MS techniques suitable for the efficient and predictable dissociation of peptide anions.
Despite these advances in instrumentation and methodologies, there are few methods that fully exploit the information available from the acidic proteome or acidic regions of proteins.
Typical high-throughput, bottom-up workflows consist of the chromatographic separation of complex mixtures of digested proteins followed by online mass spectrometry (MS) and MS analysis.
Several widely used or commercial database searching techniques are available for automated “bottom-up” analysis of peptide cations; SEQUEST (21), MASCOT (22), OMSSA (23), X!
Tandem (24), and MASPIC (25) are all popular choices and yield comparable results (26).Because ETD has a significant bias toward identifying highly basic peptides, negative UVPD was used to improve the identification of the more acidic peptides in conjunction with positive ETD for the more basic species.In this case, tryptic peptides from the cytosolic section of He La cells were analyzed by polarity switching nano LC-MS/MS utilizing ETD for cation sequencing and UVPD for anion sequencing.The algorithm also features automated charge determination for low mass accuracy data, precursor filtering (including intact charge-reduced peaks), and the ability to combine both positive and negative MS/MS spectra into a single search, and it is freely open to the public.The accuracy and specificity of the Mass Matrix UVPD search algorithm was also assessed for low resolution, low mass accuracy data on a linear ion trap.The use of ultraviolet photodissociation (UVPD) for the activation and dissociation of peptide anions is evaluated for broader coverage of the proteome.