title: Two-dimensional reversed-phase x ion-pair reversed-phase HPLC: an alternative approach to high-resolution peptide separation for shotgun proteome analysis.
abstract: A two-dimensional separation scheme for shotgun proteome analysis employing high-pH reversed-phase HPLC in the first and low-pH ion-pair reversed-phase HPLC in the second dimension (RP x IP-RP-HPLC) has been developed and evaluated. Compared to the classical strong cation exchange x ion-pair reversed-phase (SCX x IP-RP-HPLC) approach, the RP x IP-RP-HPLC system was characterized by a lower degree of orthogonality, which was, however, more than counterbalanced by higher separation efficiency, more homogeneous distribution of peptide elution, and easier experimental handling. Peptide fragment fingerprinting by electrospray-ionization tandem mass spectrometry (ESI-MS/MS) was employed for peptide detection and identification. About 13% more peptides and 7% more proteins could be identified with the alternative approach in 30% less analysis time, enabling the analysis of the proteome of Corynebacterium glutamicum with a coverage of 24.9% (745 proteins). Combining the identification results both of the SCX- x IP-RP-HPLC-ESI-MS/MS and RP- x IP-RP-HPLC-ESI-MS/MS methods, a total of 871 proteins were identified in a cytosolic protein preparation, which represented 29.1% of all proteins annotated in the genome of C. glutamicum.
authors.affiliation: Department of Chemistry, Instrumental Analysis and Bioanalysis, and Biochemical Engineering, Saarland University, 66123 Saarbrücken, Germany.
authors.forename: Nathanaël
authors.initials: N
authors.lastname: Delmotte
authors.forename: Maria
authors.initials: M
authors.lastname: Lasaosa
authors.forename: Andreas
authors.initials: A
authors.lastname: Tholey
authors.forename: Elmar
authors.initials: E
authors.lastname: Heinzle
authors.forename: Christian G
authors.initials: CG
authors.lastname: Huber
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods; D048230:Corynebacterium glutamicum / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007477:Ions; D013058:Mass Spectrometry / Q000379:methods; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D020543:Proteome; D040901:Proteomics / Q000295:instrumentation* / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry
chemical_list: D007477:Ions; D010455:Peptides; D011506:Proteins; D020543:Proteome; D014357:Trypsin