Optimization and Use of Peptide Mass Measurement Accuracy in Shotgun Proteomics

Abstract

Mass spectrometers that provide high mass accuracy such as FT-ICR instruments are increasingly used in proteomic studies. Although the importance of accurately determined molecular masses for the identification of biomolecules is generally accepted, its role in the analysis of shotgun proteomic data has not been thoroughly studied. To gain insight into this role, we used a hybrid linear quadrupole ion trap/FT-ICR (LTQ FT) mass spectrometer for LC-MS/MS analysis of a highly complex peptide mixture derived from a fraction of the yeast proteome. We applied three data-dependent MS/MS acquisition methods. The FT-ICR part of the hybrid mass spectrometer was either not exploited, used only for survey MS scans, or also used for acquiring selected ion monitoring scans to optimize mass accuracy. MS/MS data were assigned with the SEQUEST algorithm, and peptide identifications were validated by estimating the number of incorrect assignments using the composite target/decoy database search strategy. We developed a simple mass calibration strategy exploiting polydimethylcyclosiloxane background ions as calibrant ions. This strategy allowed us to substantially improve mass accuracy without reducing the number of MS/MS spectra acquired in an LC-MS/MS run. The benefits of high mass accuracy were greatest for assigning MS/MS spectra with low signal-to-noise ratios and for assigning phosphopeptides. Confident peptide identification rates from these data sets could be doubled by the use of mass accuracy information. It was also shown that improving mass accuracy at a cost to the MS/MS acquisition rate substantially lowered the sensitivity of LC-MS/MS analyses. The use of FT-ICR selected ion monitoring scans to maximize mass accuracy reduced the number of protein identifications by 40%. MS has become the method of choice for the characterization of complex protein mixtures from cells or tissues (1). The highest throughput and most comprehensive efforts to catalog protein mixtures have so far been achieved using a strategy known as shotgun proteomics (2). Proteins are proteolytically digested, and the resultant peptide mixture is usually separated by reversed-phase LC coupled on line to a mass spectrometer. Peptides are ionized and subjected to sequencing by MS/MS. Peptide ion sequences are derived based on fragment ions predominately formed through backbone cleavage at amide bonds. Several algorithms such as SEQUEST (3) and Mascot (4) allow automated assignment of MS/MS spectra by matching acquired data with spectra predicted on the basis of protein sequence databases (5). The protein composition of the analyzed sample is ultimately inferred from identified peptides (6).Recent MS technologies facilitate the acquisition of many thousands of MS/MS spectra over the course of one LC-MS/MS analysis. In general, only a portion of these spectra are correctly assigned to peptide ions using current database search algorithms. Low quality MS/MS data, non-peptide ions selected for fragmentation, and peptide ions where the corresponding sequenc...

Phosphoproteome Analysis of Drosophila melanogaster Embryos

“…Figure 2B shows an example of the overlap in phosphopeptide identifications in duplicate samples from fraction 3. As shown previously, 7,23 these data argue that replicate analyses are the most important parameter available for increasing sample sensitivity.…”

“…The LTQ-Orbitrap was operated in the data-dependent mode using the TOP10 strategy. 7 For each cycle, one full MS scan [375-1800 m/z; acquired in the orbitrap at 6 × 10 4 resolution setting and automatic gain control (AGC) target of 10 6 ] was followed by 10 data-dependent MS/MS spectra (AGC target, 5000; threshold 3000) in the linear ion trap from the 10 most abundant ions. Selected ions were dynamically excluded for 30 s. Charge-state screening was used to reject singly charged ions.…”

“…The ability to catalog the precise sites of phosphorylation on a scale of thousands has been accomplished due primarily to the combination of three factors: (i) high mass accuracy precursor ion determination, 7 (ii) optimized enrichment protocols for phosphopeptide isolation, [8][9][10] and (iii) enabling software for false-positive estimation and site localization. 11,12 With these approaches, several large-scale studies have been reported.…”

Comparison of Mascot and X!Tandem Performance for Low and High Accuracy Mass Spectrometry and the Development of an Adjusted Mascot Threshold

“…The recent availability of hybrid-FT mass spectrometers (8,9) enables high mass resolution (30,000 -500,000) together with very high mass accuracy (in the range of a few ppm). On these instruments, throughput and sensitivity are maximized by collecting MS data at a high resolution and accuracy, and MS/MS data are recorded at high speed with low resolution and accuracy (10).…”

Elimination of Systematic Mass Measurement Errors in Liquid Chromatography−Mass Spectrometry Based Proteomics Using Regression Models and a Priori Partial Knowledge of the Sample Content

“…A statistical technique for modeling nonlinear trends in the space of multiple variables is the projection pursuit regression 20 . The model expresses ε systematic in terms of a sum of univariate functions of linear combinations of the explanatory variables, translating the model (1) to (2) The proposed approach reduces systematic MME by estimating model (2) and subtracting off the fitted values from the mass measurements of individual ions, leaving only the random error component.…”