Elucidation of Glycoprotein Structures by Unspecific Proteolysis and Direct nanoESI Mass Spectrometric Analysis of ZIC-HILIC-Enriched Glycopeptides

Abstract

Protein glycosylation was explored by direct nanoESI MS and MS/MS analysis of ZIC-HILIC-enriched proteolytic glycopeptides without further separation or purification. In a previous publication, we demonstrated that a direct MS-based analysis of proteolytic glycopeptides is feasible for a number of proteins (Henning , S. J. Mass Spectrom. 2007 , 42 , 1415 - 21). This method has now been refined for two aspects: (1) separation of glycopeptides by use of ZIC-HILIC SPE and (2) the use of unspecific proteases like thermolysin, elastase, or a trypsin/chymotrypsin mixture leading per se to a mass-based separation, that is, small nonglycosylated peptides and almost exclusively glycopeptides at higher m/z values. Furthermore, the glycopeptides produced by the above proteases in general contain short peptide backbones thus improving-probably due to their higher hydrophilicity--the ZIC-HILIC-based separation. The combination of unspecific proteolysis, glycopeptide separation, and their direct MS analysis was successfully accomplished for probing glycoproteins carrying high-mannose type (ribonuclease B), neutral (asialofetuin), and acidic (haptoglobin and α1-acid glycoprotein) complex type glycans as well as for glycopeptides derived from glycoprotein mixtures and, finally, for exploring the glycosylation of a human IgG preparation. Our results show that the presented method is a fast, facile, and inexpensive procedure for the elucidation of protein N-glycosylation.

Immunoglobulin G (IgG) Fab Glycosylation Analysis Using a New Mass Spectrometric High-throughput Profiling Method Reveals Pregnancy-associated Changes

“…An increase in ConA reactivity in vitro has been confirmed by some (27); others observed similar increases in ConA reactivity with low progesterone concentrations, whereas high concentrations resulted in a decrease (51). The increase in ConA reactivity is believed to reflect increased levels of high-mannose glycans only present on the Fab portion, excluding interference from the Fc portion (Asn297), which is known to bear hardly any oligomannosidic glycans (2, 6, 35, 52). However, ConA has also been reported to have affinity for non-bisected glycan structures (41), thereby exhibiting increased binding with decreased levels of bisecting GlcNAc with pregnancy on both Fc and Fab.…”

It is all about the solvent: on the importance of the mobile phase for ZIC-HILIC glycopeptide enrichment

“…Alternatively, the reduction of non-specific enrichment has also been reported by digesting the glycoprotein with non-specific proteases prior HILIC enrichment. This greatly increases glycopeptide hydrophilicity due to the shorter peptide backbone [29]. The drawbacks of this approach are, however, increased sample heterogeneity, impeded accurate site specific glycan structure assignment and lack of accurate relative quantitation of site specific microheterogeneity [30].…”

Comparison of glycopeptide fragmentation by collision induced dissociation and ultraviolet photodissociation

“…Collision-induced dissociation (CID) has been the most popular MS/MS technique for glycopeptide analysis, 11-17 but it typically causes only glycosidic cleavages that are useful for assigning the glycan portions but not for sequencing the peptide portions or pinpointing the glycosylation sites. CID also produces some reporter ions such as m/z 204 ([HexNAc + H] + ), 292 ([NeuNAc + H] + ), and 366 ([Hex-HexNAc + H] + ) which are diagnostic for the presence of glycan modifications.…”