The Utility of Accurate Mass and LC Elution Time Information in the Analysis of Complex Proteomes

Abstract

The combination of mass and normalized elution time (NET) of a peptide identified by liquid chromatography-mass spectrometry (LC-MS) measurements can serve as a unique signature for that peptide. However, the specificity of an LC-MS measurement depends upon the complexity of the proteome (i.e., the number of possible peptides) and the accuracy of the LC-MS measurements. In this work, theoretical tryptic digests of all predicted proteins from the genomes of three organisms of varying complexity were evaluated for specificity. Accuracy of the LC-MS measurement of mass-NET pairs (on a 0 to 1.0 NET scale) was described by bivariate normal sampling distributions centered on the peptide signatures. Measurement accuracy (i.e., mass and NET standard deviations of Ϯ0.1, 1, 5, and 10 ppm, and Ϯ0.01 and 0.05, respectively) was varied to evaluate improvements in process quality. The spatially localized confidence score, a conditional probability of peptide uniqueness, formed the basis for the peptide identification. Application of this approach to organisms with comparatively small proteomes, such as Deinococcus radiodurans, shows that modest mass and elution time accuracies are generally adequate for confidently identifying most peptides. For more complex proteomes, more accurate measurements are required. However, the study suggests that the majority of proteins for even the human proteome should be identifiable with reasonable confidence by using LC-MS measurements with mass accuracies within Ϯ1 ppm and high efficiency separations having elution time measurements within Ϯ0.01

New Model for Electron Flow for Sulfate Reduction in Desulfovibrio alaskensis G20

“…Proteomic analyses of cell pellets were performed by the Environmental Molecular Science Laboratory of the Pacific Northwest National Laboratory (38) according to the following protocol. Whole-cell lysis of quick-frozen cell pellets from D. alaskensis G20 was achieved by bead beating as previously described (39).…”

“…The peptide identifications were made with the spatially localized confidence scoring (SLiC) algorithm (38,40), which incorporates a number of constraints and estimates the confidence of each peptide identification by yielding a score of 0 to 1. In this work, a minimum SLiC score of 0.3 (default) or higher was required for confident identification (38,40).…”

“…In this work, a minimum SLiC score of 0.3 (default) or higher was required for confident identification (38,40). Further, at least one high-confidence unique peptide (i.e., a peptide mapping to only one possible parent protein) and a total of two peptides were required for protein identification in each technical replicate.…”

Advances in proteomics data analysis and display using an accurate mass and time tag approach

“…The extent to which this is true depends on 1) the complexity of the system, 2) the complexity of the measurable components of the system being analyzed, 3) the overall separation efficiency (i.e., peak capacity) of the 2D analysis, and 4) the separation time and mass accuracy of the measurements. Thus, the approach uses the distinctiveness of mass and LC separation time information to associate detected "features" with either their matching peptides or to other species previously identified , Norbeck, et al, 2005. By using LC-FTICRMS instrumentation, the AMT tag strategy provides increased sensitivity, coverage, and throughput, and facilitates quantitative studies that involve many analyses of different perturbations or time points.…”

“…When a detected UMC can potentially be assigned to two or more PMT tags (as is illustrated here), the best match can be determined on the basis of the mass and NET deviations. For this purpose, an algorithm has been developed that utilizes the standardized squared distance between a given UMC's mass and NET and each PMT tag's mass and NET to estimate the uniqueness of the match ,Norbeck, et al, 2005. The distances are weighted by the variance in mass and time for each PMT tag and are used to compute a conditional probability of the likelihood that the given PMT tag is a unique match to a given UMC.…”