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

Abstract

Proteomics has progressed radically in the last 5 years and is now on par with most genomic technologies in throughput and comprehensiveness. Analyzing peptide mixtures by liquid chromatography coupled to high-resolution mass spectrometry (LC-MS) has emerged as the main technology for in-depth proteome analysis whereas two-dimensional gel electrophoresis, low-resolution MALDI, and protein arrays are playing niche roles. MS-based proteomics is rapidly becoming quantitative through both label-free and stable isotope labeling technologies. The latest generation of mass spectrometers combines extremely high resolving power, mass accuracy, and very high sequencing speed in routine proteomic applications. Peptide fragmentation is mostly performed in lowresolution but very sensitive and fast linear ion traps. However, alternative fragmentation methods and high-resolution fragment analysis are becoming much more practical. Recent advances in computational proteomics are removing the data analysis bottleneck. Thus, in a few specialized laboratories, ''precision proteomics'' can now identify and quantify almost all fragmented peptide peaks. Huge challenges and opportunities remain in technology development for proteomics; thus, this is not ''the beginning of the end'' but surely ''the end of the beginning.''

Stepwise evolution of protein native structure with electrospray into the gas phase, 10 −12 to 10 2 s

“…In this ''bottom-up'' approach, these protein fragment sequences are matched against the possible proteins predicted by their precursor DNA; matching peptide sequences can give up to 95% sequence coverage and 99% identification reliability. The ''top-down'' approach has far higher identification reliability and capabilities for locating sequence errors and posttranslational modifications (PTMs), although it requires instrumentation such as Fourier-transform MS of far higher resolving power and expense than instruments used previously for bottom-up proteomics (52)(53)(54). In this, individual molecular ions of a protein in a mixture are separated by MS-I and dissociated (MS-II) to give fragment ions indicative of that protein's sequence and PTM positions.…”

18O Stable Isotope Labeling in MS-based Proteomics

“…Results obtained by global solution-based, multidimensional shotgun investigations suggest that 16 O/ 18 O labeling is a reliable and powerful tool for comparative proteomics and offers significant advantages over the 2D-PAGE-based comparative proteomics by allowing unbiased proteome coverage [33] and high analytical throughput [34]. It is important to stress that relative changes in protein concentrations obtained by shotgun proteomics depict changes in protein abundances only at a given point in time.…”

Top-down high-resolution mass spectrometry of cardiac myosin binding protein C revealed that truncation alters protein phosphorylation state

“…Biomolecular mass spectrometry (MS) (21) is the only technique that can universally provide information about protein posttranslational modifications (PTMs) without a priori knowledge (22)(23)(24)(25)(26). In contrast to the conventional ''bottom-up'' MS approach where proteins of interest are digested with an enzyme before MS analysis providing only partial coverage of the protein sequence with loss of connectivity between modified peptides from disparate regions of the protein (23,27,28), a ''top-down'' MS approach is extremely attractive for characterization of complex PTMs in proteins of 10 to 200 kDa (22,25,(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39).…”