Controlled trypsinolysis of human cancer and non-cancer sera for direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

In order to develop a new approach to MALDI-TOF-mass-spectrometry-based serum proteome profiling without any fractionation, several cancer and non-cancer human sera were treated by different concentrations of trypsin. Serum albumin digestion required much more trypsin in cancer sera than in non-cancer sera, the observation being explained by increased serum level of protease inhibitors in cancer. Moderate concentrations of externally introduced trypsin unexpectedly lead to digestion of proteins different from the serum albumin. Of them, in cancer sera an acute-phase serum amyloid A (SAA) was shown to be digested. Further, using a synthetic SAA peptide with a C-terminal amide as a standard for quantitative MALDI-TOF-MS analysis, it was found that this protein was digested substantially faster in the cancer serum than in the normal serum. Thus, the trypsin being introduced into cancer serum possessed a modified activity. The change in trypsin behavior should be explained by its binding with alpha-2-macroglobulin.

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► Serum digestion with low trypsin quantities yielded selective hydrolysis of proteins other than albumin.
► This process is suggested to be performed by the trypsin-α2M complex.
► Direct quantitative MALDI-TOF analysis in serum.
► The serum amyloid A hydrolysis is substantially faster in cancer serum.