Automated metal-free multiple-column nanoLC for improved phosphopeptide analysis sensitivity and throughput

We report on the development and characterization of automated metal-free multiple-column nanoLC instrumentation for sensitive and high-throughput analysis of phosphopeptides with mass spectrometry. The system employs a multiple-column capillary LC fluidic design developed for high-throughput analysis of peptides (Anal. Chem. 2001, 73, 3011-3021), incorporating modifications to achieve broad and sensitive analysis of phosphopeptides. The integrated nanoLC columns (50 mu m i.d. x 30 cm containing 5 mu m C18 particles) and the on-line solid phase extraction Columns (150 mu m i.d. x 4 cm containing 5 mu m C18 particles) were connected to automatic switching valves with non-metal chromatographic accessories. and other modifications to avoid the exposure of the analyte to any metal Surfaces during handling, separation. and electrospray ionization. The nanoLC developed provided a separation peak capacity of similar to 250 for phosphopeptides (and similar to 400 for normal peptides), A detection limit of 0.4 fmol was obtained when a linear ion trap tandem mass spectrometer (Finnegan LTQ) was coupled to a 50-mu m W. column of the nanoLC. The separation power and sensitivity provided by the nanoLC-LTQ enabled identification of similar to 4600 phosphopeptide candidates from similar to 60 mu g COS-7 cell tryptic digest followed by IMAC enrichment and similar to 520 tyrosine phosphopeptides from similar to 2 mg of human T cells digests followed by phosphotyrosine peptide immunoprecipitation. (C) Published by Elsevier B.V.