Peptide Sequencing of 2-DE Gel-Isolated Proteins by Nanoelectrospray Tandem Mass Spectrometry

Shortly after the introduction of electrospray as a viable ionization technique for large molecules (1 ), electrospray tandem mass spectrometry (ES MS/MS) techniques, such as HPLC-ES MS/MS, were used successfully for peptide sequencing at picomole and subpicomole levels (2 –4 ). In LC-MS/MS, peptide sequence information is generated during the short time, 10–30 s, that a peptide elutes from the HPLC column run at a flow rate of 0.5–5 μL/min. This time frame rarely allows optimization of experimental parameters for MS/MS sequencing of individual peptides unless several LC-MS/MS experiments can be performed on a sample. With the introduction of the nanoelectrospray ion source (5 –7 ), the time constraint of tandem mass spectrometry has been removed, and peptide sequencing has been reliably extended to the femtomole level of gel-isolated protein.