title: 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.
authors.affiliation: Department of Genomics and Proteomics, Beijing Institute of Radiation Medicine, Beijing, China.
authors.forename: Xiaohai
authors.initials: X
authors.lastname: Li
authors.forename: Yan
authors.initials: Y
authors.lastname: Gong
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.forename: Songfeng
authors.initials: S
authors.lastname: Wu
authors.forename: Yun
authors.initials: Y
authors.lastname: Cai
authors.forename: Ping
authors.initials: P
authors.lastname: He
authors.forename: Zhuang
authors.initials: Z
authors.lastname: Lu
authors.forename: Wantao
authors.initials: W
authors.lastname: Ying
authors.forename: Yangjun
authors.initials: Y
authors.lastname: Zhang
authors.forename: Liyan
authors.initials: L
authors.lastname: Jiao
authors.forename: Hongzhi
authors.initials: H
authors.lastname: He
authors.forename: Zisen
authors.initials: Z
authors.lastname: Zhang
authors.forename: Fuchu
authors.initials: F
authors.lastname: He
authors.forename: Xiaohang
authors.initials: X
authors.lastname: Zhao
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Qian
mesh_terms: D001798:Blood Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D002845:Chromatography / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D010455:Peptides / Q000737:chemistry; D020033:Protein Isoforms; D011506:Proteins / Q000737:chemistry; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013347:Subcellular Fractions; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D014462:Ultrafiltration
chemical_list: D001798:Blood Proteins; D010455:Peptides; D020033:Protein Isoforms; D011506:Proteins; D020543:Proteome; D014357:Trypsin