NOVEL TAGS FOR THE STABLE ISOTOPIC LABELING OF CARBOHYDRATES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The increasing interest in glycomics has led to the widespread use of mass spectrometry to determine the carbohydrate components expressed in biological systems. However, the quantification of carbohydrates within different samples remains a daunting task, due to instrument and sample variability. Herein, we show cusyom-synthesized multiplexed stable isotope labeled tags that have broad applicability for the simultaneous quantitation of four samples during the same mass spectrometry experiment. Results exploring the utility of multiplexed stable isotopic labeling from a variety of sources have been demonstrated. A stable isotope-labeled tag in four forms (+0,+4,+8,+12) was synthesized for the purpose of labeling the reducing end of glycans. Glycosaminoglycans (GAGs) from the chondroitin sulfate proteoglycan (CS-PG) class and pharmaceutical low-molecular weight (LMWT) heparins were partially depolymerized using enzymes to form oligosaccharide distributions. These oligosaccharides were labeled with the tetraplex stable isotope-containing tags by reductive amination. The resulting tagged GAGs were combined, separated by high performance size exclusion chromatography (Superdex peptide 3.2/30, Beckman Gold 125 solvent module), and analyzed in the negative electrospray mode using an Applied Biosystems QSTAR Pulsar-I (Q-ToF) mass spectrometer. In addition, the PNGase F released a-1-acid glycans from four species were also multiplex-labeled and analyzed. A series of glycomics tags that incorporate stable isotope modules into the oligosaccharide structure were used to label the glycans released from CS-PGs, LMWT heparins, and N-linked glycans from a-1 acid glycoproteins. The abundances of ions in the MS mode serve to quantify ions produced from a given sample in each of the four samples. These results further demonstrate the principle of quantitation by multiplex analysis of carbohydrate using stable isotope tags. Chondroitinase ABC was used to depolymerize the released GAG chains from aggrecan, biglycan, cartilage extract, and decorin. The CS-proteoglycans were normalized for concentration allowing for a mass spectrometric analysis of relative CS quantities. Additional information on the isomeric content of the CS chains was obtained via tandem mass spectral analysis utilizing the distinctive fragmentation patterns of each glycoform (CSA, CSB, and CSC). Additional experiments were performed to demonstrate greater utility of the tags in examination of GAGs. Low molecular weight heparins (Lovenox and Fragmin) were depolymerized to the greatest extent possible with heparin lyases I, II, and III. A significant fraction of heparin (and LMWT heparin) resistant to the lyase treatment should be composed of a tetrasaccharide containing a trisulfated GlcN residue that derives from the pentasaccharide sequence required for anti-thrombin III binding. The quantities of lyase resistant structures for each LMWT heparin preparation were determined. In addition, using tandem mass spectrometry, the differential fragmentation patterns between porcine heparin, Lovenox, and Fragmin were determined for all component composition. This method provides an alternate method to examine the structural differences between heparin preparations. A final demonstration of the utility of multiplex tagging examined the N-linked glycans released from a-1-acid glycoprotein from four species. Glycoprotein quantities were normalized to quantitate the abundances of glycans present and the tandem MS of each composition was analyzed.