On-chip proteolytic digestion and analysis using "wrong-way-round" electrospray time-of-flight mass spectrometry

Rapid protein digestion and analysis using a hybrid microchip nanoelectrosp ray device and time-of-flight mass spectrometry detection are reported. The device consists of a planar glass chip with microfabricated channels coupl ed to a disposable nanospray emitter. Reactions between substrate and enzym e (trypsin), mixed off-chip and then immediately loaded into a sample reser voir on the device, are monitored in real time following the onset of elect rospray, Protein cleavage products are determined at the optimum pH for gen erating tryptic fragments, directly from the digestion buffer using "wrong- way-round" electrospray, i.e., monitoring (MH)(+) ions from basic solutions . Intense tryptic peptide ions are observed within a few minutes following sample loading on the microchip. Proteins were identified from low femtomol e or even attomole quantities of analyte/spectrum using peptide mass finger printing, loading 0.1-2 pmol/muL of sample on the chip. The sequence covera ge for analyzed proteins ranged from 70 to 95%. The rapid analysis of human hemoglobin is demonstrated using the technique.