High-efficiency nano- and micro-HPLC--high-resolution Orbitrap-MS platform for top-down proteomics

In terms of resolution, mass accuracy, and sensitivity, the Orbitrap represents one of the most potent mass analyzers available today. We here elucidate the potential of interfacing Orbitrap-MS to ion-pair RP HPLC for intact protein analysis. Using gradients of ACN and monolithic columns of 1.0 and 0.10 mm id, peak capacities between 120 and 130 were achievable within 20-25 min separation time. Compared with silica-based stationary phases, protein recovery and carryover from monolithic columns were found clearly superior. Intact proteins were detectable in a mass range covering 5.7-150 kDa with LODs in the low femtomol range. Compared with UV detection, MS detection with a scanning speed of 1.6 s per spectrum on average led to a 26% increase in chromatographic peak widths, whereas chromatographic patterns were mostly preserved in extracted ion chromatograms at an acquisition rate of 0.5 s per spectrum. Isotopic resolution of multiply charged ions was demonstrated for proteins up to 42 kDa. A micro-HPLC-Orbitrap-MS setup employing a 1.0 mm id column was utilized to characterize a 150 kDa recombinant monoclonal antibody. The applicability of nano-HPLC-Orbitrap-MS to the analysis of highly complex protein mixtures is demonstrated for the 70% ethanol extractable subproteome of wheat grains.