title: Analysis of peptides and protein digests by reversed phase high performance liquid chromatography-electrospray ionisation mass spectrometry using neutral pH elution conditions.
abstract: In this study, the advantages of carrying out the analysis of peptides and tryptic digests of proteins under gradient elution conditions at pH 6.5 by reversed-phase liquid chromatography (RP-HPLC) and in-line electrospray ionisation mass spectrometry (ESI-MS) are documented. For these RP separations, a double endcapped, bidentate anchored n-octadecyl wide pore silica adsorbent was employed in a capillary column format. Compared to the corresponding analysis of the same peptides and protein tryptic digests using low pH elution conditions for their RP-HPLC separation, this alternative approach provides improved selectivity and more efficient separation of these analytes, thus allowing a more sensitive identification of proteins at different abundance levels, i.e. more tryptic peptides from the same protein could be confidently identified, enabling higher sequence coverage of the protein to be obtained. This approach was further evaluated with very complex tryptic digests derived from a human plasma protein sample using an online two-dimensional (2D) strong cation-exchange (SCX)-RP-HPLC-ESI-MS/MS system. Again, at pH 6.5, with mobile phases of different compositions, improved chromatographic selectivities were obtained, concomitant with more sensitive on-line electrospray ionisation tandem mass spectrometric (ESI-MS/MS) analysis. As a consequence, more plasma proteins could be confidently identified, highlighting the potential of these RP-HPLC methods with elution at pH 6.5 to extend further the scope of proteomic investigations.
authors.affiliation: Australian Research Council Special Research Centre for Green Chemistry, School of Chemistry, Monash University, Melbourne, Victoria 3800, Australia.
authors.forename: Yuanzhong
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Australian Research Council Special Research Centre for Green Chemistry, School of Chemistry, Monash University, Melbourne, Victoria 3800, Australia.
authors.forename: Reinhard I
authors.initials: RI
authors.lastname: Boysen
authors.affiliation: Australian Research Council Special Research Centre for Green Chemistry, School of Chemistry, Monash University, Melbourne, Victoria 3800, Australia.
authors.forename: Jamil
authors.initials: J
authors.lastname: Chowdhury
authors.affiliation: Australian Research Council Special Research Centre for Green Chemistry, School of Chemistry, Monash University, Melbourne, Victoria 3800, Australia.
authors.forename: Asif
authors.initials: A
authors.lastname: Alam
authors.affiliation: Australian Research Council Special Research Centre for Green Chemistry, School of Chemistry, Monash University, Melbourne, Victoria 3800, Australia. Electronic address: milton.hearn@monash.edu.
authors.forename: Milton T W
authors.initials: MT
authors.lastname: Hearn
mesh_terms: D000818:Animals; D001798:Blood Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D014357:Trypsin / Q000378:metabolism*
chemical_list: D001798:Blood Proteins; D010446:Peptide Fragments; D012710:Serum Albumin, Bovine; D014357:Trypsin
keywords: Chromatographic efficiency; Electrospray ionisation mass spectrometry; Mobile phase pH; Peptide identification; Reversed-phase liquid chromatography; Selectivity