Post analysis data acquisition for the iterative MS/MS sampling of proteomics mixtures.
The identification of peptides by microcapillary liquid chromatography-tandem mass spectrometry (microLC-MS/MS) has become routine because of the development of fast scanning mass spectrometers, data-dependent acquisition, and database searching algorithms. However, many peptides within the detection limit of the mass spectrometer remain unidentified because of limitations in MS/MS sampling speed despite the dynamic range and peak capacity of the instrument. We have developed an automated approach that uses the mass spectra from high resolution microLC-MS data to define the molecular species present in the mixture and directs the acquisition of MS/MS spectra to precursors that were missed in prior analyses. This approach increases the coverage of the molecular species sampled by MS/MS and consequently the number of peptides and proteins identified during the acquisition of technical or biological replicates using a simple one-dimensional chromatographic separation. The combination of a unique workflow and custom software contribute to the improved identification of molecular features detected in proteomics experiments of complex protein mixtures.
Hoopmann
Michael R
MR
University of Washington, Department of Genome Sciences, Seattle, Washington 98195, USA.
Merrihew
Gennifer E
GE
von Haller
Priska D
PD
MacCoss
Michael J
MJ
D030562:Databases, Protein; D040901:Proteomics / Q000379:methods*; D012984:Software; D013223:Statistics as Topic / Q000379:methods*; D053719:Tandem Mass Spectrometry