Precise and Parallel Characterization of Coding Polymorphisms, Alternative Splicing, and Modifications in Human Proteins by Mass Spectrometry

Abstract

The human proteome is a highly complex extension of the genome wherein a single gene often produces distinct protein forms due to alternative splicing, RNA editing, polymorphisms, and posttranslational modifications. Due to the presence of polymorphisms, alternative splicing, and posttranslational modifications (PTMs) 1 the human proteome is highly complex, often encoding multiple protein forms for a given gene (1). This biological complexity poses a significant analytical and bioinformatic challenge to the detailed analysis of mammalian proteomes by MS and is exacerbated by the presence of gene families sharing high sequence identity (2, 3). Protein modifications are often indicative of changes in cellular or tissue dynamics and therefore play central roles in regulation of the cell cycle or development of disease. Whether for new diagnostics or understanding molecular mechanisms in cell biology, protein identification using tryptic peptides has revolutionized the analysis of complex mixtures by mass spectrometry (1, 4). High throughput platforms based on MALDI (5) and ESI use MS/MS engines capable of spectral acquisition at a rate of Ͼ10 4 /week (6, 7). Recent studies indicate significant inefficiencies associated with such large scale "bottom up" analyses in mammalian systems including imperfect enzymatic cleavage (8, 9) and some MS/MS spectra requiring manual interpretation/validation for identification. Despite the lingering difficulties with peptide analysis, it provides the best and most general method for large scale protein identification today with information on nonsynonymous coding single nucleotide polymorphisms (cSNPs), alternative splicing (10), and PTMs challenging to obtain (2).Recent developments by MacCoss et al. (11), Wu et al. (12), and Zhu et al. (13) use three proteases and multidimensional protein identification technology ("MudPIT") or isoelectric focusing, reversed-phase chromatography, and three mass spectrometers (13), respectively, to obtain mass information on ϳ70 -99% of the primary protein structure. Combining intact protein measurement with near exhaustive peptide analysis of five proteins from human cells allowed detection of N-terminal modifications and one alternatively spliced transcript (13). Although cSNP analysis of abundant blood proteins is possible (14), a general informatic strategy has yet to systematically integrate DNA and RNA level data with the MS-based interrogation of the human proteome. This is accomplished here using a data base of human proteins tailored

Precision proteomics: The case for high resolution and high mass accuracy

“…5, bottom). High mass accuracy allows much more variability of proteins (vide supra) to be ''locked down'' during database searches (56). Current-generation FTMS instruments can record fragmentation data with better than 5 ppm mass accuracy routinely.…”

Detection of Alternative Splice Variants at the Proteome Level in Aspergillus flavus

“…1 Probable explanations include quality of spectrum, search engine algorithm, sequence polymorphism, post-translational modification, and transcriptional variation by RNA splicing and editing. 1,2 Alternative pre-mRNA splicing is a mechanism that removes the intervening introns (noncoding sequences) and joins the flanking exons (coding regions) in different arrangements. This process allows a single gene to generate various mRNAs, then multiple protein variants, which might have diverse and even antagonistic functions.…”

Parallel Reaction Monitoring for High Resolution and High Mass Accuracy Quantitative, Targeted Proteomics

“…Namely, developments in time-of-flight (TOF) technology (45), and the advent of the Orbitrap mass analyzer in 2005 (46 -49), have made fast and sensitive MS/MS scanning (50 -53) with Ͻ10 parts-per-million (ppm) mass measurement error routine (54). For discovery experiments, the ability to acquire MS/MS scans with high resolution and low-ppm mass errors offers several advantages, including higher confidence sequence identification (44,55), post-translational modification site localization (56), and improved quantitative accuracy. Coming from this perspective, we wondered whether the highly accurate mass measurement capabilities of today's new-generation MS instrumentation could be leveraged to provide benefits within the targeted proteomics domain.…”