Towards a reliable molecular mass determination of intact glycoproteins by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Abstract

Different matrices and sample-matrix preparation procedures have been tested in order to study their influence on the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra of intact glycoproteins, which present different degrees of glycosylation (human transferrin; bovine fetuin; bovine alpha(1)-acid-glycoprotein; recombinant human erythropoietin; and the novel erythropoiesis stimulating protein). Using sinapinic acid (SA) and the fast evaporation method, the studied glycoproteins became susceptible to fragmentation at any laser intensity, suggesting that this 'hot' matrix is unsuitable for a reliable molecular mass determination of glycosylated compounds. In contrast, 2,5-dihydroxybenzoic acid (DHB) and 6-aza-2-thiothymine (ATT), with an adequate sample-matrix preparation, provided improved results. Samples containing DHB after crystallization by vacuum drying demonstrated the best performance because the labile functional groups from the glycoforms were apparently fragmented to a lower extent. The average molecular masses obtained using this methodology were in all cases a better estimation than those values reported in the literature. The results were reproducible, and sensitivity was similar to that obtained with SA and the fast evaporation method. These excellent results suggest that this MALDI-TOF-MS methodology could be useful for an improved determination of the average molecular mass values of microheterogeneous compounds such as glycoproteins, glycosylated compounds or, in general, molecular mass values of molecules with similar labile functional groups.

Mapping the glycation sites in the neoglycoconjugate from hexasaccharide antigen of Vibrio cholerae, serotype Ogawa and the recombinant tetanus toxin C‐fragment carrier

“…As for the previously discussed glycated peptides, we also observed product ions corresponding to the loss of the entire carbohydrate portion on peptidic fragment ions: [y 14 −B 6 ] + at m/z 1727.8164 and [y 10 −B 6 ] + at m/z 1348.6460. The glycated peptide only contains one lysine residue (Lys 61), and as we already reported that the squaric acid chemistry allows the conjugation on exclusively lysine residues [15,17–19] we can conclude that the glycation site corresponds to the Lys 61.…”

“…Matrix-assisted laser desorption/ionization (MALDI) and surface-enhanced laser desorption/ionization coupled to a time-of-flight (TOF) mass analyzer are the methods of choice for the determination of the molecular weight of glycoproteins [16,19] and carbohydrate hapten-to-carrier protein ratios. [12,20–22] Recently, we reported on the determination of the glycation sites of several different vaccine models prepared by dialkyl squarate chemistry: a synthetic tetrasaccharide hapten of Bacillus anthracis –BSA conjugate vaccine, a synthetic lactose-BSA conjugate and neoglycoconjugates from the terminal monosaccharide hapten of the O-specific polysaccharide of Vibrio cholerae O1, serotype Ogawa and BSA.…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2007–2008

“…Several matrices and sample preparation techniques have been examined with respect to their influence on the measured mass of intact glycoproteins with different degrees of glycosylation (Giménez et al, 2007). Glycoproteins examined included human transferrin, bovine fetuin, bovine a1-acidglycoprotein and recombinant human erythropoietin.…”

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

“…The on-line coupling of MALDI-TOF-MS to high-performance separation techniques, is still in an early development, and for the moment it is difficult to evaluate its potential to separate and characterize the elevated number of glycoforms found in complex glycoproteins. Recently, Gimenez et al (2007) proposed a promising methodology for the analysis of intact glycoproteins by MALDI-TOF-MS that used different matrices and sample-matrix preparation procedures.…”