Combination of Bottom-up 2D-LC-MS and Semi-top-down GelFree-LC-MS Enhances Coverage of Proteome and Low Molecular Weight Short Open Reading Frame Encoded Peptides of the Archaeon Methanosarcina mazei

Cassidy, L., Prasse, D., Linke, D., Schmitz-Streit, Ruth and Tholey, Andreas (2016) Combination of Bottom-up 2D-LC-MS and Semi-top-down GelFree-LC-MS Enhances Coverage of Proteome and Low Molecular Weight Short Open Reading Frame Encoded Peptides of the Archaeon Methanosarcina mazei Journal of Proteome Research, 15 (10). pp. 3773-3783. DOI 10.1021/acs.jproteome.6b00569.

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Abstract

The recent discovery of an increasing number of small open reading frames (sORF) creates the need for suitable analytical technologies for the comprehensive identification of the corresponding gene products. For biological and functional studies the knowledge of the entire set of proteins and sORF gene products is essential. Consequently in the present study we evaluated analytical approaches that will allow for simultaneous analysis of widest parts of the proteome together with the predicted sORF. We performed a full proteome analysis of the methane producing archaeon Methanosarcina mazei strain Gol cytosolic proteome using a high/low pH reversed phase LC-MS bottom-up approach. The second analytical approach was based on semi top-down strategy, encompassing a separation at intact protein level using a GelFree system, followed by digestion and LC-MS analysis. A high overlap in identified proteins was found for both approaches yielding the most comprehensive coverage of the cytosolic proteome of this organism achieved so far. The application of the second approach in combination with an, adjustment of the search criteria for database searches further led to a significant increase of sORF peptide identifications, finally allowing to detect and identify 28 sORF gene products.

Document Type: Article
Additional Information: Times Cited: 0 Cassidy, Liam Prasse, Daniela Linke, Dennis Schmitz, Ruth A. Tholey, Andreas
Research affiliation: Kiel University > Kiel Marine Science
OceanRep > The Future Ocean - Cluster of Excellence
Kiel University
Refereed: Yes
DOI etc.: 10.1021/acs.jproteome.6b00569
ISSN: 1535-3893
Projects: Future Ocean
Date Deposited: 26 Feb 2017 11:09
Last Modified: 06 Feb 2019 10:16
URI: http://eprints.uni-kiel.de/id/eprint/36031

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