Reversible labeling of cysteine‐containing peptides allows their specific chromatographic isolation for non‐gel proteome studies

Abstract

We report upon a novel procedure to specifically isolate cysteine-containing peptides from a complex peptide mixture. Cysteines are converted to hydrophobic residues by mixed disulfide formation with Ellman's reagent. Proteins are subsequently digested with trypsin and the generated peptide mixture is a first time fractionated by reverse-phase high-performance liquid chromatography. Cysteinyl-peptides are isolated out of each primary fraction by a reduction step followed by a secondary peptide separation on the same column, performed under identical conditions as for the primary separation. The reducing agent removes the covalently attached group from the cysteine side chain, making cysteine-peptides more hydrophilic and, thereby, such peptides can be specifically collected during the secondary separation and are finally used to identify their precursor proteins using automated liquid chromatography tandem mass spectrometry. We show that this procedure efficiently isolates cysteine-peptides, making the sample mixture less complex for further analysis. This method was applied for the analysis of the proteomes of human platelets and enriched human plasma. In both proteomes, a significant number of low abundance proteins were identified next to extremely abundant ones. A dynamic range for protein identification spanning 4-5 orders of magnitude is demonstrated.

Morphological and biochemical characterization of a human liver in a uPA‐SCID mouse chimera†‡

“…The presence of these atypical plasma proteins in our chimeric mouse plasma is not unexpected because such proteins are also found in the plasma of healthy blood donors. 17,18 The integrity and spatial organization of the human hepatocytes in the chimeric liver was also examined by histology. Early after engraftment, the human hepatocytes appeared as sharp nodules that expanded and became blurred as time went on and occupied up to 87% of liver parenchyma.…”

Crowdsourcing in proteomics: public resources lead to better experiments

“…Indeed, further processing can dramatically extend the reach of a sequence database and correspondingly increase the number of relevant identifications, by performing serial differential enzymatic digestion (Van Damme et al 2005), amino-or carboxyterminal ragging (Gevaert et al 2003), sequence-based subset selection (Gevaert et al 2004;Gevaert et al 2002), or to create and add decoy sequences ).…”

Current trends in high throughput proteomics in cyanobacteria

“…It most notably led to the introduction of ''shotgun proteomics", a term coined after its seemingly random nature of the analysis. Using shotgun proteomics, proteomes are directly digested and analysed in situ using a combination of rigorous liquid chro- matographic separation and analysis using high speed tandem mass spectrometry [28][29][30]. The rationale of shotgun proteomics was first introduced in 2001 by Yates et al, who applied a combinatorial technique of two-dimensional LC and an electrospray iontrap MS to profile the Saccharomyces cerevisiae proteome [31].…”