Nano‐high‐performance liquid chromatography in combination with nano‐electrospray ionization Fourier transform ion‐cyclotron resonance mass spectrometry for proteome analysis

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Abstract
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.