Ultratrace liquid chromatography/mass spectrometry analysis of large peptides with post-translational modifications...

This paper describes approaches to optimize the chromatographic performance for our recently developed LC-MS platform, extended range proteomic analysis (ERPA), for comprehensive protein characterization at the ultratrace level. Large digested peptide fragments up to 10 kDa (e.g., from lysyl endopeptidase digestion) with or without modifications were well separated with high resolution using narrow bore (20 and 50 microm I.D.) poly(styrene-divinylbenzene) (PS-DVB) monolithic columns constructed by in situ solution polymerization. Importantly, the macroporous structure of the monolithic columns facilitated mass transport of large peptides with improved recovery relative to small pore size reversed-phase packings. High sequence coverage (>95%), including identification of phosphorylated and glycosylated particles was achieved for beta-casein and epidermal growth factor receptor (EGFR) at the 4 and 20 fmol levels per injection, respectively, using the 20 microm I.D. PS-DVB monolithic column. For peptides with greater ionization efficiency, the detection limit could be lowered to approximately 400 zmol. Typically, the separation system produced a peak capacity of approximately 200 for a 10 cm column. This paper demonstrates that narrow-bore monolithic columns are suitable for high sensitivity and high-resolution separation of large peptide fragments by LC-MS analysis.