Pronase-Immobilized Enzyme Reactor: a New Approach for Automation in Glycoprotein Analysis by LC/LC-ESI/MSn

An automated analytical approach is proposed for simultaneous
characterization of glycan and peptide moieties
in pronase-generated glycopeptides. The proposed method
is based on the use of a new pronase-immobilized enzyme
reactor for the on-line rapid digestion of the target glycoprotein.
By coupling the bioreactor to a Hypercarb chromatographic
trap column, on-line selective glycopeptide
enrichment prior to normal-phase liquid chromatography-
mass spectrometry was obtained. A detailed study
was carried out for integration and automation of each
phase of the proposed analytical procedure. On-line
digestion allowed extensive cleavage of the model protein
(ribonuclease B), yielding to glycopeptides with peptide
moieties up to eight amino acids, carrying the Man5-Man9
N-glycans each, selectively resolved on an Amide-80
column. The use of a linear ion trap instrument resulted
in efficient ion capture and led to MS3 acquisition times
and spectra quality similar to those for MS2, allowing the
unambiguous identification of glycan (MS2) and peptide
(MS3) sequences. The proposed procedure reduces the
glycoprotein analysis time from 3 days, as in most of
the traditional off-line methods, to 1 h.