Nano-high-performance liquid chromatography in combination with nano-electrospray ionization Fourier transform ion-cyclotron resonance mass spectrometry for proteome analysis.
Fourier transform ion-cyclotron resonance (FTICR) mass spectrometry offers several advantages for the analysis of biological samples, including excellent mass resolution, ultra-high mass measurement accuracy, high sensitivity, and wide mass range. We report the application of a nano-HPLC system coupled to an FTICR mass spectrometer equipped with nanoelectrospray source (nano-HPLC/nano-ESI-FTICRMS) for proteome analysis. Protein identification in proteomics is usually conducted by accurately determining peptide masses resulting from enzymatic protein digests and comparing them with theoretically digested protein sequences from databases. A tryptic in-solution digest of bovine serum albumin was used to optimize experimental conditions and data processing. Spots from Coomassie Blue and silver-stained two-dimensional (2D) gels of human thyroid tissue were excised, in-gel digested with trypsin, and subsequently analyzed by nano-HPLC/nano-ESI-FTICRMS. Additionally, we analyzed 1D-gel bands of membrane preparations of COS-6 cells from African green monkey kidney as an example of more complex protein mixtures. Nano-HPLC was performed using 1-mm reverse-phase C-18 columns for pre-concentration of the samples and reverse-phase C-18 capillary columns for separation, applying water/acetonitrile gradient elution conditions at flow rates of 200 nL/min. Mass measurement accuracies smaller than 3 ppm were routinely obtained. Different methods for processing the raw data were compared in order to identify a maximum number of peptides with the highest possible degree of automation. Parallel identification of proteins from complex mixtures down to low-femtomole levels makes nano-HPLC/nano-ESI-FTICRMS an attractive approach for proteome analysis.
Ihling
Christian
C
Biotechnological-Biomedical Center, Institute of Analytical Chemistry, University of Leipzig, 04103 Leipzig, Germany.
Berger
Kerstin
K
Medical Department III, University of Leipzig, 04103 Leipzig, Germany.
Höfliger
Martin M
MM
Institute of Biochemistry, University of Leipzig, 04103 Leipzig, Germany.
Führer
Dagmar
D
Medical Department III, University of Leipzig, 04103 Leipzig, Germany.
Beck-Sickinger
Annette G
AG
Institute of Biochemistry, University of Leipzig, 04103 Leipzig, Germany.
Sinz
Andrea
A
Biotechnological-Biomedical Center, Institute of Analytical Chemistry, University of Leipzig, 04103 Leipzig, Germany.
D000818:Animals; D019556:COS Cells; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D017357:Cyclotrons; D004583:Electrons; D015180:Electrophoresis, Gel, Two-Dimensional; D005583:Fourier Analysis; D006801:Humans; D008832:Microchemistry / Q000379:methods*; D036103:Nanotechnology / Q000379:methods*; D011506:Proteins / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D011506:Proteins