High throughput quantification of N-glycans using one-pot sialic acid modification and matrix assisted laser desorption ionization time-of-flight mass spectrometry.

Appropriate glycosylation of recombinant therapeutic glycoproteins has been emphasized in biopharmaceutical industries because the carbohydrate component can affect safety, efficacy, and consistency of the glycoproteins. Reliable quantification methods are essential to ensure consistency of their products with respect to glycosylation, particularly sialylation. Mass spectrometry (MS) has become a popular tool to analyze glycan profiles and structures, showing high resolution and sensitivity with structure identification ability. However, quantification of sialylated glycans using MS is not as reliable because of the different ionization efficiency between neutral and acidic glycans. We report here that amidation in mild acidic conditions can be used to neutralize acidic N-glycans still attached to the protein. The resulting amidated N-glycans can then be released from the protein using PNGase F, and labeled with permanent charges on the reducing end to avoid any modification and the formation of metal adducts during MS analysis. The N-glycan modification, digestion, and desalting steps were performed using a single-pot method that can be done in microcentrifuge tubes or 96-well microfilter plates, enabling high throughput glycan analysis. Using this method we were able to perform quantitative MALDI-TOF MS of a recombinant human glycoprotein to determine changes in fucosylation and changes in sialylation that were in very good agreement with a normal phase HPLC oligosaccharide mapping method.

Combining 2-DE immunoblots and mass spectrometry to identify putative soybean (Glycine max) allergens.

Soybean is recognized as a commonly allergenic food, but the identity of important allergens is not well studied. Recently, some global regulatory agencies started requiring quantitative analysis of individual allergens, including unproven allergens, as part of the risk assessment for genetically engineered (GE) soybeans. We sought to identify soybean proteins that bind IgE from any of 10 individual soybean-sensitized subjects. Soybean IgE binding proteins were identified by 2-DE immunoblots using sera from four soy-allergic and plasma from six soy-sensitized human subjects. Corresponding spots were excised from stained gels, digested, and analyzed using a quadrupole TOF Synapt G2-S tandem mass spectrometer. Results showed the major IgE binding proteins were subunits of either ß-conglycinin (Gly m 5) or glycinin (Gly m 6). Soybean Kunitz trypsin inhibitor (SKTI) was a significant IgE binding protein for four subjects. Soybean agglutinin, seed biotinylated protein (SBP) of 65 kDa, late embryogenesis protein (LEP), and sucrose-binding protein were identified as IgE binding only for soy-sensitized subjects. We conclude that the major soybean allergens are isoforms of Gly m 5, Gly m 6, and possibly SKTI and that requirements for quantitative measurement of proteins that are not clear allergens is not relevant to safety.

Obtaining high sequence coverage in matrix-assisted laser desorption time-of-flight mass spectrometry for studies of protein modification: analysis of human serum albumin as a model.

Several approaches were explored for obtaining high sequence coverage in protein modification studies performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Human serum albumin (HSA, 66.5kDa) was used as a model protein for this work. Experimental factors considered in this study included the type of matrix used for MALDI-TOF MS, the protein digestion method, and the use of fractionation for peptide digests prior to MALDI-TOF MS analysis. A mixture of alpha-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid was employed as the final matrix for HSA. When used with a tryptic digest, this gave unique information on only half of the peptides in the primary structure of HSA. However, the combined use of three enzyme digests based on trypsin, endoproteinase Lys-C, and endoproteinase Glu-C increased this sequence coverage to 72.8%. The use of a ZipTip column to fractionate peptides in these digests prior to analysis increased the sequence coverage to 97.4%. These conditions made it possible to examine unique peptides from nearly all of the structure of HSA and to identify specific modifications to this protein (e.g., glycation sites). For instance, Lys199 was confirmed as a glycation site on normal HSA, whereas Lys536 and Lys389 were identified as additional modification sites on minimally glycated HSA.

Identification and quantitative studies of protein immobilization sites by stable isotope labeling and mass spectrometry.

A method was developed for characterizing immobilization sites on a protein based on stable isotope labeling and MALDI-TOF mass spectrometry. The model for this work was human serum albumin (HSA) immobilized onto silica by the Schiff base method. The immobilized HSA was digested by various proteolytic enzymes in the presence of normal water, while soluble HSA was digested in (18)O-enriched water for use as an internal standard. These two digests were mixed and analyzed, with the (18)O/(16)O ratio for each detected peptide then being measured. Several peptides in the tryptic, Lys-C, and Glu-C digests gave significantly higher (18)O/(16)O ratios than other peptides in the same digests, implying their involvement in immobilization. Analysis of these results led to identification of the N-terminus and several lysines as likely immobilization sites for HSA (e.g., K4, K41, K190, K225, K313, and K317). It was also possible from these results to quantitatively rank these sites in terms of the relative degree to which each might take part in immobilization. This method is not limited to HSA and silica but can be used with other proteins and supports.

Global characterization of porcine intrauterine proteins during early pregnancy.

Total protein secreted in the intrauterine lumen increases between day 10 and 13 post-estrus in both cyclic and pregnant gilts. The objective of this experiment was to identify those intrauterine proteins whose secretion changes during this time period. Sixteen mature gilts were either mated (day 0) or remained cyclic and were slaughtered at either day 10 or day 13 (n = 4 per status by day). At slaughter, each uterine horn was flushed with 20 ml Minimal Essential Medium. Flushings were dialyzed extensively against distilled water. A 0.5 ml aliquot of each was lyophilized, subjected to two-dimensional PAGE, and protein spots were identified following Coomassie staining of each gel. Densitometry was used to compare relative amounts of each spot. After statistical analysis, spots that differed due to either day, status, or day by status interaction were excised and digested in-gel with trypsin. The resulting peptides were analyzed by tandem mass spectrometry (MS/MS). Using MS/MS data, protein identification for each spot was attempted. There were 280 matching spots, of which 132 were significantly (P < 0.05 or 0.01) affected by pregnancy status, day, or the day by status interaction. Most (73%) spots increased from day 10 to day 13 with no effect of pregnancy. Several spots were identified as proteases or their inhibitors. Others potentially modify glycolipids and/or glycoproteins. These results indicate that the concentrations of many proteins within the intrauterine environment during early pregnancy are independent of the conceptus and could play roles in regulating the endometrial or conceptus glycocalyx.