Comparison of alternative analytical techniques for the characterisation of the human serum proteome in HUPO Plasma Proteome Project

Abstract

Based on the same HUPO reference specimen (C1-serum) with the six proteins of highest abundance depleted by immunoaffinity chromatography, we have compared five proteomics approaches, which were (1) intact protein fractionation by anion-exchange chromatography followed by 2-DE-MALDI-TOF-MS/MS for protein identification (2-DE strategy); (2) intact protein fractionation by 2-D HPLC followed by tryptic digestion of each fraction and microcapillary RP-HPLC/microESI-MS/MS identification (protein 2-D HPLC fractionation strategy); (3) protein digestion followed by automated online microcapillary 2-D HPLC (strong cation-exchange chromatography (SCX)-RPC) with IT microESI-MS/MS; (online shotgun strategy); (4) same as (3) with the SCX step performed offline (offline shotgun strategy) and (5) same as (4) with the SCX fractions reanalysed by optimised nanoRP-HPLC-nanoESI-MS/MS (offline shotgun-nanospray strategy). All five approaches yielded complementary sets of protein identifications. The total number of unique proteins identified by each of these five approaches was (1) 78, (2) 179, (3) 131, (4) 224 and (5) 330 respectively. In all, 560 unique proteins were identified. One hundred and sixty-five proteins were identified through two or more peptides, which could be considered a high-confidence identification. Only 37 proteins were identified by all five approaches. The 2-DE approach yielded more information on the pI-altered isoforms of some serum proteins and the relative abundance of identified proteins. The protein prefractionation strategy slightly improved the capacity to detect proteins of lower abundance. Optimising the separation at the peptide level and improving the detection sensitivity of ESI-MS/MS were more effective than fractionation of intact proteins in increasing the total number of proteins identified. Overall, electrophoresis and chromatography, coupled respectively with MALDI-TOF/TOF-MS and ESI-MS/MS, identified complementary sets of serum proteins.

Enhancing Identifications of Lipid-embedded Proteins by Mass Spectrometry for Improved Mapping of Endothelial Plasma Membranes in Vivo

“…Various strategies to enhance MS-based proteomics analysis have been developed, such as different off-line sample fractionations and different mass spectrometers with different ion sources and different search engines (34,35). Recent studies on various isolated cell membranes have used different instruments (36), ionization techniques (11), different temperatures of C 18 column (37), and protein separation methods (11,38) to identify 107 to Ͼ1306 proteins, many based on one peptide identifications.…”

Diversity of human skeletal muscle in health and disease: Contribution of proteomics

“…In earlier studies, the samples were digested by specific proteases (usually trypsin) and the peptides were separated and analyzed by mass spectrometry. The major disadvantage of this method is that nearly all protein isoformspecific information is lost [28]. In 2008, Hojlund et al analyzed human vastus lateralis muscle by combining a fractionation by one-dimensional gel electrophoresis and subsequent HPLC-ESI MS/MS of lysates of 20-24 SDS gel slices digested with trypsin [29].…”

Expression of the Calcium-binding Protein S100A4 Is Markedly Up-regulated by Osmotic Stress and Is Involved in the Renal Osmoadaptive Response

“…In this work we have applied the DIGE approach to study the changes in the cellular proteome in response to chronic adaptation to hypertonic stress in IMCD3 cells. As with any current proteomic technique, DIGE allows for the study of changes to only a fractional subset of the proteome (32,33). In our studies, the small, calcium-binding protein S100A4 was determined to be highly up-regulated by DIGE analysis, Western blot, gene chip analysis, and quantitative PCR.…”