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      Mapping Intact Protein Isoforms in Discovery Mode Using Top Down Proteomics

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          Abstract

          A full description of the human proteome relies on the challenging task of detecting mature and changing forms of protein molecules in the body. Large scale proteome analysis 1 has routinely involved digesting intact proteins followed by inferred protein identification using mass spectrometry (MS) 2 . This “bottom up” process affords a high number of identifications (not always unique to a single gene). However, complications arise from incomplete or ambiguous 2 characterization of alternative splice forms, diverse modifications ( e.g., acetylation and methylation), and endogenous protein cleavages, especially when combinations of these create complex patterns of intact protein isoforms and species 3 . “Top down” interrogation of whole proteins can overcome these problems for individual proteins 4, 5 , but has not been achieved on a proteome scale due to the lack of intact protein fractionation methods that are well integrated with tandem MS. Here we show, using a new four dimensional (4D) separation system, identification of 1,043 gene products from human cells that are dispersed into >3,000 protein species created by post-translational modification, RNA splicing, and proteolysis. The overall system produced >20-fold increases in both separation power and proteome coverage, enabling the identification of proteins up to 105 kilodaltons and those with up to 11 transmembrane helices. Many previously undetected isoforms of endogenous human proteins were mapped, including changes in multiply-modified species in response to accelerated cellular aging (senescence) induced by DNA damage. Integrated with the latest version of the Swiss-Prot database 6 , the data provide precise correlations to individual genes and proof-of-concept for large scale interrogation of whole protein molecules. The technology promises to improve the link between proteomics data and complex phenotypes in basic biology and disease research 7 .

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          Most cited references24

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          Escape from therapy-induced accelerated cellular senescence in p53-null lung cancer cells and in human lung cancers.

          Accelerated cellular senescence (ACS) has been described for tumor cells treated with chemotherapy and radiation. Following exposure to genotoxins, tumor cells undergo terminal growth arrest and adopt morphologic and marker features suggestive of cellular senescence. ACS is elicited by a variety of chemotherapeutic agents in the p53-null, p16-deficient human non-small cell H1299 carcinoma cells. After 10 to 21 days, infrequent ACS cells (1 in 10(6)) can bypass replicative arrest and reenter cell cycle. These cells express senescence markers and resemble the parental cells in their transcription profile. We show that these escaped H1299 cells overexpress the cyclin-dependent kinase Cdc2/Cdk1. The escape from ACS can be disrupted by Cdc2/Cdk1 kinase inhibitors or by knockdown of Cdc2/Cdk1 with small interfering RNA and can be promoted by expression of exogenous Cdc2/Cdk1. We also present evidence that ACS occurs in vivo in human lung cancer following induction chemotherapy. Viable tumors following chemotherapy also overexpress Cdc2/Cdk1. We propose that ACS is a mechanism of in vivo tumor response and that mechanisms aberrantly up-regulate Cdc2/Cdk1 promotes escape from the senescence pathway may be involved in a subset of tumors and likely accounts for tumor recurrence/progression.
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            Quantitative analysis of intact apolipoproteins in human HDL by top-down differential mass spectrometry.

            Top-down mass spectrometry holds tremendous potential for the characterization and quantification of intact proteins, including individual protein isoforms and specific posttranslationally modified forms. This technique does not require antibody reagents and thus offers a rapid path for assay development with increased specificity based on the amino acid sequence. Top-down MS is efficient whereby intact protein mass measurement, purification by mass separation, dissociation, and measurement of product ions with ppm mass accuracy occurs on the seconds to minutes time scale. Moreover, as the analysis is based on the accurate measurement of an intact protein, top-down mass spectrometry opens a research paradigm to perform quantitative analysis of "unknown" proteins that differ in accurate mass. As a proof of concept, we have applied differential mass spectrometry (dMS) to the top-down analysis of apolipoproteins isolated from human HDL(3). The protein species at 9415.45 Da demonstrates an average fold change of 4.7 (p-value 0.017) and was identified as an O-glycosylated form of apolipoprotein C-III [NANA-(2 --> 3)-Gal-beta(1 --> 3)-GalNAc, +656.2037 Da], a protein associated with coronary artery disease. This work demonstrates the utility of top-down dMS for quantitative analysis of intact protein mixtures and holds potential for facilitating a better understanding of HDL biology and complex biological systems at the protein level.
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              The pros and cons of peptide-centric proteomics.

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                Author and article information

                Journal
                0410462
                6011
                Nature
                Nature
                Nature
                0028-0836
                1476-4687
                16 September 2011
                30 October 2011
                08 June 2012
                : 480
                : 7376
                : 254-258
                Affiliations
                [1 ]University of Illinois at Urbana-Champaign, Urbana, IL, 61801
                [2 ]Northwestern University, Evanston, IL, 60208
                [3 ]Institute for Genomic Biology, Urbana, IL, 61801
                [7 ]University of Wisconsin, Madison WI, 53706
                Author notes
                [* ]Corresponding author: n-kelleher@ 123456northwestern.edu , Department of Chemistry, Department of Molecular Biosciences, and The Chemistry of Life, Processes Institute, Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Evanston, IL 60208
                [4]

                Current address: Korea Institute of Science and Technology, Seoul, South Korea

                [5]

                Current address: The Methodist Hospital Research Institute, Houston, TX, 77030

                [6]

                Current address: Harvard Medical School, Boston, MA, 02115

                Article
                nihpa325382
                10.1038/nature10575
                3237778
                22037311
                070d3e2d-395b-472b-8d32-19933cace646

                Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

                History
                Funding
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM067193-08 || GM
                Funded by: National Institute on Drug Abuse : NIDA
                Award ID: P30 DA018310-06 || DA
                Funded by: National Institute on Drug Abuse : NIDA
                Award ID: F30 DA026672-03 || DA
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