Proteomic Analysis of Human Blood Serum Using Peptide Library Beads

Abstract

Human serum is thought to contain key information for diagnostics of human disease. However, no single technology is currently nor might ever be available to cope with the complexity and dynamic range of the serum proteome. We here report a large-scale proteomic study of human blood serum using peptide library beads and mass spectrometry. Serum proteins are adsorbed onto polymeric beads coated with a combinatorial library composed of millions of hexameric peptide baits. Analysis of the eluates from this combinatorial library (as obtained with 3 eluants of different strength, able to release 99% of the retentate) via liquid chromatography coupled to high-resolution mass spectrometry resulted in the identification of 1559 proteins or 3869 proteins, respectively, depending on how 95% confidence was estimated. In either case, the analysis showed that ligand beads are able to capture a large number of proteins in a single operation. The ligand bead bound fraction appeared to have a lower dynamic range when compared to the starting material, due to a "normalization" of the protein concentrations in the original mixture. We find that extensive information on the protein composition of complex samples such as serum can be obtained using ligand beads and that these beads enrich the proteomic tool box.

Protein Sample Treatment with Peptide Ligand Library: Coverage and Consistency

“…Manual and scanner examination of the maps after the library treatment did not result in significant difference in spot count, positioning and intensity. The reproducibility of data appeared thus very satisfactory and confirmed what was already reported in the literature for various biological samples and under different conditions for both mass ranges and peptides identification (Sennels et al, 2007;Roux-Dalvai et al, 2008). These data add to reproducibility of masses of intact species proteins by mass spectrometry before trypsination .…”

“…Indications of reproducibility were brought in various circumstances, as for instance in the discovery of novel proteins from human serum (Sennels et al, 2007) The dilemma is generated by the fact that, the library being an assembly of beads each of them carrying a distinct ligand, when a certain volume of beads is taken out of a bulk, the proportions of peptide ligands represented on the beads are not statistically identical. The dilemma becomes even more concerning when one considers that working with very small volumes of biological samples also restricts the volume of beads that can be used and in these situations, the total number of library diversomers largely exceeds the number of beads drawn practically for a given experiment.…”

Quantitative Profiling of Post-translational Modifications by Immunoaffinity Enrichment and LC-MS/MS in Cancer Serum without Immunodepletion

“…The identification of most arginine methylation sites on low abundance proteins such as transcriptional regulators and RNA processing proteins in our study makes it less likely that there was a concentration-dependent, enzyme-independent reaction for methylation in blood. In fact, numerous protein arginine methyltransferases have been identified in plasma in previous studies (44,48). Therefore, arginine methylation could occur either in intracellular compartments followed by release to the bloodstream or be catalyzed in the bloodstream directly.…”

The ProteoMiner and the FortyNiners: Searching for gold nuggets in the proteomic arena

“…Each time the CPLL was applied to protein extracts, the number of proteins detected was significantly larger than in the control, and spanned from a factor of two to a factor of five. Referring to serum proteins, Sennels et al (2007) described an increase of protein spots in 2D map analysis from 115 to 790; for platelet extracts, the number of gene products identified by LC-MS/MS jumped from 197 before treatment to 435. As a last example, relatively old though, in human urines the number of proteins found by FT-ICR-MS before treatment was 134 and 385 after treatment with CPLL (Castagna et al, 2005).…”

“…Its application to proteomics, though, is relatively recent, because the first report appeared only 3 years ago (Thulasiraman et al, 2005). A flurry of applications soon followed; for example, in urine (Castagna et al, 2005), serum Sennels et al, 2007), human platelets (Guerrier et al, 2007a), red blood cell (Roux-Dalvai et al, 2008), bile (Guerrier et al, 2007b), and recombinant DNA product Antonioli et al, 2007) analyses. This technology is presently commercially available under the trade name of ProteoMiner.…”