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The application of multiplexed isobaric tandem mass tag (TMT) labeling and an LTQ Orbitrap XL ETD (electron transfer dissociation) hybrid mass spectrometer as a direct approach for qualitative and quantitative characterization of glycoproteins is reported. Bovine fetuin was used as a model glycoprotein in this study. For online liquid chromatography-mass spectrometry (LC-MS) analysis, high-resolution, mass accurate full scan MS spectra were acquired in the Orbitrap mass analyzer followed by data-dependent tandem mass spectrometry (MS/MS) with alternating collision-induced dissociation (CID), ETD, and higher-energy collisional dissociation (HCD) scans. An additional in-source dissociation scan was used as a highly sensitive and selective detection method for eluting glycosylated peptides. By alternatively using three different dissociation methods, 23 glycoforms from all 5 corresponding glycopeptides were identified from a trypsin digest of bovine fetuin. With ETD, labile glycans were retained without any signs of carbohydrate cleavage with concurrent fragmentation of the peptide backbone. Glycosylation sites were clearly localized from the ETD fragmentation data. Glycan structure elucidation was accomplished using CID. The CID experiments generated fragment ions predominantly from cleavage of glycosidic bonds without breaking the peptide bond. Novel to this method, the TMT labeling protocol was modified and adapted for higher labeling efficiency, and a TriVersa NanoMate was used to reinfuse samples to improve ETD and HCD spectra of glycopeptides. Quantification with TMT was verified based on the HCD spectra from multiple nonglycopeptides and glycopeptides. This method can be used as a qualitative and quantitative technique for direct characterization of glycoproteins and has applicability for detection of counterfeit glycoprotein drug products.

During the 2007-2008 heparin crisis, it was found that the United States Pharmacopeia (USP) testing monograph for unfractionated heparin sodium (UFH) did not detect the presence of the contaminant, oversulfated chondroitin sulfate (OSCS) in heparin. In response to this concern, new tests and specifications were developed by the Food and Drug Administration (FDA) and USP and put in place to not only detect the contaminant OSCS but also to improve assurance of quality and purity of the drug product. Additional tests were also developed to monitor the heparin supply chain for other possible economically motivated additives or impurities. In 2009, a new USP monograph was put in place that includes 500 MHz (1)H NMR, SAX-HPLC, %galactosamine in total hexosamine, and anticoagulation time assays with purified factor IIa or factor Xa. These tests represent orthogonal approaches for UFH identification, measurement of bioactivity, and for detection of process impurities or contaminants in UFH. The FDA has applied these analytical approaches to the study of UFH active pharmaceutical ingredients in the marketplace. Here, we describe results from a comprehensive survey of UFH collected from seven different sources after the 2009 monograph revision and compare these data with results obtained on other heparin samples collected during the 2007-2008 crisis.

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