Fast trypsin digestion of proteins on a cross-linked [Os(dmebpy)_2Cl]^(+/2+)-derivatized copolymer of acrylamide and vinylimidazole column

Abstract

Fast digestion of proteins was observed when they were loaded together with trypsin onto the crosslinked [Os(dmebpy)_2Cl]^(+/2+)-derivatized copolymer of acrylamide and vinylimidazole column. The insoluble Os-complexed polymer particles were packed into an electrospray tip to monitor peptides eluted during loading, washing and elution periods with a mass spectrometer. The proteolytic cleavage of proteins was observed immediately when the mixture of trypsin and substrates in 0.2mM ammonium bicarbonate 50:50 H_2O/acetonitrile reached the column tip, and continued through the loading period. Some tryptic peptides were released from the column during the loading and following washing periods. The others still stayed on the column until the low pH elution buffer reached the column. If a protein was first loaded onto the column, no tryptic peptides of the protein were observed when trypsin was loaded later for the on-column digestion. Only the autolysis peptides of trypsin were observed. On-column digestion of 100 fmol myoglobin was successfully detected with a low sensitivity quadrupole mass spectrometer. A hybrid Os-polymer/C_(18) column tip was constructed for the online trypsin digestion of proteins in the aqueous buffers and the following trapping and elution of peptides from the C_(18) column. The digestion of reduced and alkylated bovine serum albumin and human transferrin in 2.5mM ammonium bicarbonate and 0.2M urea buffer was observed on the column, with more peptide coverage than conventional 4 h in-solution digestion at 37°C. Control experiments without the Os-polymer in the column tip excluded the spontaneous insolution digestion of proteins in the short time window of buffer delivery onto the column, indirectly confirming the contribution of Os-polymer on the fast trypsin digestion.

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