title: Automated, on-line two-dimensional nano liquid chromatography tandem mass spectrometry for rapid analysis of complex protein digests.
abstract: Evaluation of cellular processes and their changes at the level of protein expression and post-translational modifications may allow identification of novel proteins and the mechanisms involved in pathogenic processes. However, the number of proteins and, after tryptic digestion, of peptides from a single cell can be tremendously high. Separation and analysis of such complex peptide mixtures can be performed using multidimensional separation techniques such as two-dimensional gel electrophoresis or two-dimensional-high-performance liquid chromatography (2-D-HPLC). The aim of this work was to establish a fully automated on-line 2-D-HPLC separation method with column switching for the separation of complex tryptic digests. A model mixture of five proteins as well as a nuclear matrix protein sample were digested with trypsin and separated using a strong cation exchange (SCX) column in the first dimension and nano reversed phase in the second dimension. Separated peptides were detected using an ion trap mass spectrometer. The advantages of this new fully automated method are rapid sample loading, the possibility of injecting large volumes and no introduction of salt into the mass spectrometer. Furthermore, column switching allows the independent control and optimization of the two dimensions independently.
authors.affiliation: LC Packings, Abberdaan, Amsterdam, The Netherlands.
authors.forename: Goran
authors.initials: G
authors.lastname: Mitulović
authors.forename: Christoph
authors.initials: C
authors.lastname: Stingl
authors.forename: Marek
authors.initials: M
authors.lastname: Smoluch
authors.forename: Remco
authors.initials: R
authors.lastname: Swart
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Chervet
authors.forename: Ines
authors.initials: I
authors.lastname: Steinmacher
authors.forename: Christopher
authors.initials: C
authors.lastname: Gerner
authors.forename: Karl
authors.initials: K
authors.lastname: Mechtler
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods; D008969:Molecular Sequence Data; D036103:Nanotechnology / Q000295:instrumentation / Q000379:methods; D010446:Peptide Fragments / Q000032:analysis*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D012997:Solvents / Q000737:chemistry
chemical_list: D010446:Peptide Fragments; D011506:Proteins; D012997:Solvents