title: Information-dependent LC-MS/MS acquisition with exclusion lists potentially generated on-the-fly: case study using a whole cell digest of Clostridium thermocellum.
abstract: We have developed a real-time graphic-processor-unit-based search engine capable of high-quality peptide identifications in <500 μs per spectrum. The steps of peptide/protein identification, in-silico prediction of all possible tryptic peptides from these proteins, and the prediction of their expected retention times and m/z values take less than 5 s per cycle over ∼3000 MS/MS spectra. This lays the foundation for information-dependent acquisition with exclusion lists generated on-the-fly, as the instrument continues to acquire data. While a complete evaluation of the dynamic exclusion system requires the participation from instrument vendors, we conducted a series of model experiments using a whole cell tryptic digestion of the bacterium Clostridium thermocellum. We ran a series of five iterative LC-MS/MS runs, adding a new exclusion list at each of four chromatographic "tripping points" - the elution times of the four standard peptides spiked into the sample. Retention times of these standard peptides were also used for real-time "chromatographic calibration." The dynamic exclusion approach gave a ≈ 5% increase in confident protein identification (for typical 2 h LC-MS/MS run), and reduced the average number of identified peptides per protein from 4.7 to 2.9. Its application to a two-times shorter gradient gave a ≈ 17% increase in proteins identified. Further improvements are possible for instruments with better mass accuracy, by employing a more accurate retention prediction algorithm and by developing better understanding of the possible chemical modifications and fragmentations produced during electrospray ionization.
authors.affiliation: Manitoba Centre for Proteomics and Systems Biology, Winnipeg, Canada.
authors.forename: Peter
authors.initials: P
authors.lastname: McQueen
authors.forename: Vic
authors.initials: V
authors.lastname: Spicer
authors.forename: Thomas
authors.initials: T
authors.lastname: Rydzak
authors.forename: Richard
authors.initials: R
authors.lastname: Sparling
authors.forename: David
authors.initials: D
authors.lastname: Levin
authors.forename: John A
authors.initials: JA
authors.lastname: Wilkins
authors.forename: Oleg
authors.initials: O
authors.lastname: Krokhin
mesh_terms: D000465:Algorithms; D002853:Chromatography, Liquid / Q000379:methods*; D048013:Acetivibrio thermocellus / Q000737:chemistry*; D001330:Electronic Data Processing; D010455:Peptides / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D012015:Reference Standards; D015203:Reproducibility of Results; D012984:Software; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry
chemical_list: D010455:Peptides; D014357:Trypsin