Adoption of targeted mass spectrometry (MS) approaches such as multiple reaction monitoring (MRM) to study biological and biomedical questions is well underway in the proteomics community. Successful application depends on the ability to generate reliable assays that uniquely and confidently identify target peptides in a sample. Unfortunately, there is a wide range of criteria being applied to say that an assay has been successfully developed. There is no consensus on what criteria are acceptable and little understanding of the impact of variable criteria on the quality of the results generated. Publications describing targeted MS assays for peptides frequently do not contain sufficient information for readers to establish confidence that the tests work as intended or to be able to apply the tests described in their own labs. Guidance must be developed so that targeted MS assays with established performance can be made widely distributed and applied by many labs worldwide. To begin to address the problems and their solutions, a workshop was held at the National Institutes of Health with representatives from the multiple communities developing and employing targeted MS assays. Participants discussed the analytical goals of their experiments From the ‡Broad Institute of MIT and Harvard, Cambridge, Massachusetts; §Eli
Combinatorial biosynthesis of type I polyketide synthases is a promising approach for the generation of new structural derivatives of polyketide-containing natural products. A target of this approach has been to change the extender units incorporated into a polyketide backbone to alter the structure and activity of the natural product. One limitation to these efforts is that only four extender units were known: malonyl-CoA, methylmalonyl-CoA, ethylmalonyl-CoA, and methoxymalonyl-acyl carrier protein (ACP). The chemical attributes of these extender units are quite similar, with the exception of the potential hydrogen bonding interactions by the oxygen of the methoxy moiety. Furthermore, the incorporated extender units are not easily modified by using simple chemical approaches when combinatorial biosynthesis is coupled to semisynthetic chemistry. We recently proposed the existence of two additional extender units, hydroxymalonyl-ACP and aminomalonyl-ACP, involved in the biosynthesis of zwittermicin A. These extender units offer unique possibilities for combinatorial biosynthesis and semisynthetic chemistry because of the introduction of free hydroxyl and amino moieties into a polyketide structure. Here, we present the biochemical and mass spectral evidence for the formation of these extender units. This evidence shows the formation of ACP-linked extender units for polyketide synthesis. Interestingly, aminomalonyl-ACP formation involves enzymology typically found in nonribosomal peptide synthesis.antibiotics ͉ combinatorial biosynthesis
Top Down analysis revealed that at least fourteen genes encoding histone H2A are coexpressed in HeLa cells. Characterization of these species revealed that all except H2A.Z and H2A.F/Z were alpha-N-acetylated, H2A.O and H2A.C,D,I,N,P were the most abundant, and those exceeding approximately 10% abundance lacked post-translational modifications. This unequivocal identification of H2A forms illustrates the advantages of Top Down Mass Spectrometry and provides a global perspective of H2A regulation through the cell cycle.
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
Proteomics has grown significantly with the aid of new technologies that consistently are becoming more streamlined. While processing of proteins from a whole cell lysate is typically done in a bottom-up fashion utilizing MS/MS of peptides from enzymatically digested proteins, top-down proteomics is becoming a viable alternative that until recently has been limited largely to offline analysis by tandem mass spectrometry. Here we describe a method for high-resolution tandem mass spectrometery of intact proteins on a chromatographic time scale. In a single liquid chromatography-tandem mass spectrometry (LC-MS/MS) run, we have identified 22 yeast proteins with molecular weights from 14 to 35 kDa. Using anion exchange chromatography to fractionate a whole cell lysate before online LC-MS/MS, we have detected 231 metabolically labeled (14N/15N) protein pairs from Saccharomyces cerevisiae. Thirty-nine additional proteins were identified and characterized from LC-MS/MS of selected anion exchange fractions. Automated localization of multiple acetylations on Histone H4 was also accomplished on an LC time scale from a complex protein mixture. To our knowledge, this is the first demonstration of top-down proteomics (i.e., many identifications) on linear ion trap Fourier transform (LTQ FT) systems using high-resolution MS/MS data obtained on a chromatographic time scale.
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