Publications - Published papers

Please find below publications of our group. Currently, we list 508 papers. Some of the publications are in collaboration with the group of Sonja Prohaska and are also listed in the publication list for her individual group. Access to published papers (access) is restricted to our local network and chosen collaborators. If you have problems accessing electronic information, please let us know:

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Optimization of Parameters for Coverage of Low Molecular Weight Proteins

Stephan A. Müller, Tibor Kohajda, Sven Findeiß, Peter F. Stadler, Stefan Washietl, Manolis Kellis, Martin von Bergen and Stefan Kalkhof

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Analytical and Bioanalytical Chemistry

Abstract


Proteins with molecular weights below 25 kDa are involved in major biological processes such as ribosome formation, stress adaption (e.g. temperature reduction) or cell cycle control. Despite their importance, the coverage of smaller proteins in standard proteome studies is rather sparse. Here we investigated biochemical and mass spectrometric parameters that influence coverage and validity of identification. The underrepresentation of low molecular weight (LMW) proteins may be attributed to the low numbers of proteolytic peptides formed by tryptic digestion as well as their tendency to be lost in protein separation and concentration/desalting procedures. In a systematic investigation of the LMW proteome of Escherichia coli, a total of 455 LMW proteins (27 % of the 1672 listed in the SwissProt protein database) were identified which correspond to a coverage of 62% of the known cytosolic LMW proteins. Of these proteins, 93 had not yet been functionally classified and five previously not confirmed on the protein level. In this study, the influence of protein extraction (either urea or TFA), proteolytic digestion (solely and combined usage of trypsin and AspN as endoproteases) and protein separation (gel or non-gel based) were investigated. Compared to the standard procedure based only on urea lysis buffer, in-gel separation and tryptic digestion, the complementary variation of TFA for extraction or endoprotease AspN for proteolysis gains the additional identification of 72 (32 %) and 51 proteins (23 %), respectively. Regarding mass spectrometry analysis with a LTQ Orbitrap mass spectrometer, collision induced fragmentation (CID and HCD) and electron transfer dissociation using the linear ion trap (IT) or the Orbitrap as analyzer were compared. IT-CID was found to yield the best identification rate whereas IT-ETD provided almost comparable results in terms of LMW proteome coverage. The high overlap of the identified proteins found with IT-CID and IT4 ETD allowed the validation of 75 % of the identified proteins with this orthogonal fragmentation technique. Furthermore, with the program “RNAcode” a new approach for evaluation and improvement of the completeness of protein databases was introduced and examined.

Keywords


LTQ Orbitrap, nano-HPLC, nano-ESI-MS/MS, proteomics, low molecular weight proteome, Escherichia coli