Optimization of parameters for coverage of low molecular weight proteins

Abstract

Proteins with molecular weights of <25 kDa are involved in major biological processes such as ribosome formation, stress adaption (e.g., temperature reduction) and 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, corresponding to a coverage of 62% of the known cytosolic LMW proteins. Of these proteins, 93 had not yet been functionally classified, and five had not previously been confirmed at the protein level. In this study, the influences of protein extraction (either urea or TFA), proteolytic digestion (solely, and the combined usage of trypsin and AspN as endoproteases) and protein separation (gel- or non-gel-based) were investigated. Compared to the standard procedure based solely on the use of urea lysis buffer, in-gel separation and tryptic digestion, the complementary use of TFA for extraction or endoprotease AspN for proteolysis permits the identification of an extra 72 (32%) and 51 proteins (23%), respectively. Regarding mass spectrometry analysis with an LTQ Orbitrap mass spectrometer, collision-induced fragmentation (CID and HCD) and electron transfer dissociation using the linear ion trap (IT) or the Orbitrap as the 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 between the proteins identified with IT-CID and IT-ETD allowed the validation of 75% of the identified proteins using this orthogonal fragmentation technique. Furthermore, a new approach to evaluating and improving the completeness of protein databases that utilizes the program RNAcode was introduced and examined.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-010-4093-x) contains supplementary material, which is available to authorized users.

Osteoblast-released Matrix Vesicles, Regulation of Activity and Composition by Sulfated and Non-sulfated Glycosaminoglycans

“…Tryptic peptides gained from cell samples were analyzed using a LTQ Orbitrap XL ETD (Thermo Fisher Scientific) also coupled with a NanoAcquity system as described before (30). Briefly, UPLC parameters were similar to MV sample processing, but peptide elution was conducted using a non-linear 150-min gradient of increasing solvent B (1-40%).…”

RNAcode: Robust discrimination of coding and noncoding regions in comparative sequence data

“…Compared to the overall protein expression level, short peptides often show low abundance, they are easily lost using standard proteomic protocols, and only a limited number of proteolytic peptides can be obtained (Klein et al 2007). To meet these challenges, we developed a protocol that is specifically optimized for small proteins by avoiding sample loss by a simple extraction method and a combined purification and enrichment step using filtration (Müller et al 2010;Materials and Methods). In order to improve the reliability of our results, we applied two different buffer systems for extractions, and for an improved coverage of peptides, we used two different proteases.…”

Stable isotope labeling by amino acids in cell culture based proteomics reveals differences in protein abundances between spiral and coccoid forms of the gastric pathogen Helicobacter pylori

“…In proteomic studies low molecular weight proteins are often underrepresented which can be addressed by a special enrichment, fractionation and the use of multiple proteases [64]. To increase the coverage of low molecular proteins below 50 kDa, an off-gel fractionation was applied.…”