title: High-speed data reduction, feature detection, and MS/MS spectrum quality assessment of shotgun proteomics data sets using high-resolution mass spectrometry.
abstract: Advances in Fourier transform mass spectrometry have made the acquisition of high-resolution and accurate mass measurements routine on a chromatographic time scale. Here we report an algorithm, Hardklör, for the rapid and robust analysis of high-resolution mass spectra acquired in shotgun proteomics experiments. Our algorithm is demonstrated in the analysis of an Escherichia coli enriched membrane fraction. The mass spectrometry data of the respective peptides are acquired by microcapillary HPLC on an LTQ-orbitrap mass spectrometer with data-dependent acquisition of MS/MS spectra. Hardklör detects 211,272 total peptide isotope distributions over a 2-h analysis (75-min gradient) in only a small fraction of the time required to acquire the data. From these data there are 13,665 distinct, chromatographically persistent peptide isotope distributions. Hardklör is also used to assess the quality of the product ion spectra and finds that more than 11.2% of the MS/MS spectra are composed of fragment ions from multiple different molecular species. Additionally, a method is reported that enzymatically labels N-linked glycosylation sites on proteins, creating a unique isotope signature that can be detected with Hardklör. Using the protein invertase, Hardklör identifies 18O-labeled peptide isotope distributions of four glycosylation sites. The speed and robustness of the algorithm create a versatile tool that can be used in many different areas of mass spectrometry data analysis.
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, Washington 98195, USA.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Hoopmann
authors.forename: Gregory L
authors.initials: GL
authors.lastname: Finney
authors.forename: Michael J
authors.initials: MJ
authors.lastname: MacCoss
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D029968:Escherichia coli Proteins / Q000032:analysis* / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry; D006031:Glycosylation; D007553:Isotope Labeling / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010103:Oxygen Isotopes / Q000737:chemistry; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D029968:Escherichia coli Proteins; D006023:Glycoproteins; D010103:Oxygen Isotopes; D010455:Peptides