Molecular mass determination of plasma‐derived glycoproteins by ultraviolet matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry with internal calibration
Authors: Belgacem Omar , Buchacher Andrea , Pock Katharina , Josic Djuro , Sutton Chris , Rizzi Andreas , Allmaier Günter 
Published: November 2002

Human plasma‐derived antithrombin III (AT‐III), factor IX (FIX) and vitronectin (VN) were characterized as native glycoproteins and in their de‐N‐glycosylated form by means of MALDI mass spectrometry. The average molecular masses of the three complex glycoproteins were determined applying internal calibration with high‐mass, well‐defined protein calibrants. Internal calibration generated for the 47 kDa yeast protein enolase a mass precision in the continuous and delayed extraction mode of ±0.12 and ±0.022%, respectively. The achievable mass accuracy for such a high‐mass, unmodified protein was in the range of 0.02% in the continuous mode, which turned out to be better than in the delayed extraction mode. Purification of all (glyco) proteins (even the calibration proteins) by means of ZipTip® technology and direct elution with a solvent system containing the appropriate MALDI matrix turned out to be a prerequisite to measure the exact molecular masses with an internal calibration. The average molecular masses of the two different forms of AT‐III, namely AT‐IIIα and AT‐IIIβ, were shown to be 57.26 and 55.04 kDa, respectively. The 2.22 kDa mass difference is attributed to the known difference in carbohydrate content at one specific site (Asn‐135). After exhaustive de‐N‐glycosylation (by means of PNGase F) of the α‐ and β‐form and subsequent MALDI‐MS analysis, average molecular masses of 48.96 and 48.97 kDa, respectively, were obtained. These values are in good agreement (−0.15%) with the calculated molecular mass (49.039 kDa) of the protein part based on SwissProt data. The molecular mass of the heavily post‐translational modified glycoprotein FIX was found to be 53.75 kDa with a peak width at 10% peak height of 4.5 kDa, because of the presence of many different posttranslational modifications (N‐ and O‐glycosylation at multiple sites, sulfation, phosphorylation, hydroxylation and numerous γ‐carboxyglutamic acids). MALDI‐MS molecular mass determination of the native, size‐exclusion chromatography‐purified, VN sample revealed that the glycoprotein was present as dimer with molecular mass of 117.74 kDa, which could be corroborated by non‐reducing SDS‐PAGE. After sample treatment with guanidine hydrochloride and mass spectrometric analysis, a single, new main component was detected. The molecular mass turned out to be 59.45 kDa, representing the monomeric form of VN, known as V75. The determined molecular mass value was shown to be on one hand lower than from SDS‐PAGE and on the other higher than the calculated amino acid sequence molecular mass (52 277 Da), pointing to the well‐known SDS‐PAGE bias and to considerable post‐translational modifications. Further treatment of the sample with a reducing agent and subsequent MALDI‐MS revealed two new components with molecular masses of 49.85 and 9.41 kDa, corresponding to V65 and V10 subunits of VN. PNGase F digest of the V75 and V65 units and MS analysis, exhibiting a molecular mass reduction of 6.37 kDa in both cases, verified the presence of a considerable amount of N‐glycans. Copyright © 2002 John Wiley & Sons, Ltd.