Semiautomated High-Sensitivity Profiling of Human Blood Serum Glycoproteins through Lectin Preconcentration and Multidimensional Chromatography/Tandem Mass Spectrometry

Abstract

We describe an effective analytical approach to identify trace glycoproteins in a small volume of human serum. The system is based on automatable affinity enrichment through silica-based lectin microcolumns and a further separation of the retained glycoproteins on a reversed-phase liquid chromatography with superficially porous packing, operating at high temperature. The fractionated sample is further directed into a 96-well plate for trypsinization and LC-MS/MS analysis. Using a major-component depleted blood serum (16 microg total protein), we were able to identify 271 glycoproteins through this analytical system.

Characterization of glycopeptides by combining collision‐induced dissociation and electron‐transfer dissociation mass spectrometry data

“…To reduce the adverse effects of competitive ionization, a common strategy is to enrich glycopeptides prior to mass spectrometric (MS) analysis. [11][12][13][14] One such approach utilizes lectins, 11,12 which are proteins that are selective toward carbohydrates. Glycoproteins can be enriched by lectins immobilized on a solid-phase support.…”

“…Glycoproteins can be enriched by lectins immobilized on a solid-phase support. 11 The trapped glycoproteins are then released with the proper eluent, enzymatically digested, and subjected to MS analysis. This method can be very effective for determining glycoproteins present in a complex sample, resulting in multiple peptide identifications for a single protein, thus increasing the confidence of a correct identification.…”

“…This method can be very effective for determining glycoproteins present in a complex sample, resulting in multiple peptide identifications for a single protein, thus increasing the confidence of a correct identification. 11 However, due to the competitive ionization, some glycopeptides may still not be identified. To circumvent this problem, glycoproteins can first be digested, while the resulting peptide pool can then be subjected to lectin enrichment.…”

Integrated analytical strategies for the study of phosphorylation and glycosylation in proteins

“…To increase compatibility between the mobile phases used in the two dimensions, the high ionic strength commonly applied to lectin columns was reduced to 10 mM ammonium bicarbonate or pure water without affecting the binding efficiency. The same group used this automated system to profile the glycoproteins in a human sample with an extensive dynamic range (Madera et al, 2006). For that analytical task, SLAC approach was used to pre-concentrate blood serum proteins using lectin-silica microcolumns in series, with immobilized ConA, SNA-I, UEA-I, and PHA-L.…”

“…For that analytical task, SLAC approach was used to pre-concentrate blood serum proteins using lectin-silica microcolumns in series, with immobilized ConA, SNA-I, UEA-I, and PHA-L. The authors demonstrated that the SLAC approach is superior to the multi-lectin affinity approach for the selective enrichment of small volumes of blood serum (Madera et al, 2006(Madera et al, , 2007.…”