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

Abstract

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 approximately 3 days, as in most of the traditional off-line methods, to approximately 1 h.

Glycoscience aids in biomarker discovery

“…These may be easily assigned according to accurate mass using software algorithms designed to simulate non-specific cleavage of glycoproteins, such as GlycoX (81) or its in-house descendants in labs around the world. In cases where accurate mass is ambiguous or further verification is desired, tandem MS experiments can further elucidate glycopeptide composition and structure (22,(82)(83)(84)(85).…”

N-glycoproteomics: mass spectrometry-based glycosylation site annotation

“…For cleavage, most studies use trypsin cleaving with high-specifi city C-terminal of arginine and lysine residues. Alternatively, Lys-C (Nielsen et al , 2005 ), Arg-C (Muller et al , 1997 ), Glu-C, Asp-N, chymotrypsin, or unspecifi c enzymes such as pronase Temporini et al , 2007 ) or proteinase K (Zauner et al , 2010 ) may be used, as complementary digestion methods can increase coverage of glycosylation sites otherwise not accessible to tryptic cleavage (Parker et al , 2011 ). However, missing enzyme specifi city and missed cleavage sites commonly lead to reduced reliability of peptide identifi cations due to the necessary increase in search space during data evaluation.…”

On‐line multi‐enzymatic approach for improved sequence coverage in protein analysis

“…Even though the unspecific cleavage can be a valuable method to maximize the digestion of isolated proteins with post-translational modifications [11,20,21], it cannot be used for complex samples because it would lead to false positive results when automatic search is carried out for protein identification using large databases [15].…”

“…Based on our experience on the development and application of bioreactors based on enzymes (penicillin G acylase [8,9], trypsin [10], pronase [11]) immobilized on epoxy-modified monolithic silica supports and on the recently described chymotrypsin bioreactor [12], we here report the preparation and characterization of a mixed bioreactor based on trypsin and chymotrypsin.…”