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. Copyright 2003 John Wiley & Sons, Ltd.