Research and Teaching Interests1. Analysis of Protein and Lipid Glycosylation For many years cancer biologists have recognized that, as a general rule, tumor cells display aberrant glycan (sugar polymer) structures. This means that if glycans can be analyzed in the right way(s), they should serve as excellent markers for the presence and progression of cancer. Altered activity of the enzymes that build glycans (glycotransferases, GTs) is the immediate upstream cause of abnormal glycan production. GTs build glycans in an opportunistic non-template driven, first-come-first-build way, with each type of GT adding a specific sugar residue to a specific position of a growing glycan "tree". Thus increased expression of a particular GT results in an increased number of specific glycan polymer branch points (or linear linkages) and a highly diverse set of final glycan structures. We have invented a new gas chromatography-mass spectrometry (GC-MS) based technique to quantify N-, O-, and lipid-linked glycans in whole biofluids and tissues on the basis of these specific glycan polymer branch points and linkages rather than on the basis of intact glycan structure. This provides a direct surrogate readout for GT activity and, as such, a promising new angle by which to leverage glycans as cancer biomarkers. The technique is illustrated below (Figure 1). | | Figure 1: Conceptual overview of our approach to the analysis of glycans as cancer markers. As illustrated, an upregulated GT (e.g., GnT-V) causes an increase in the quantity of specific uniquely linked glycan monosaccharide residues (glycan "nodes")-which can lead to formation of a mixture of heterogeneous whole-glycan structures at low copy number each-all of which, together, can be difficult to detect and quantify consistently. But when the glycan "nodes" are pooled together analytically from amongst all the aberrant glycan structures their combined numbers add up to produce larger-than-normal GC-MS signals (actual extracted ion chromatograms from 10-μl blood plasma samples shown). [Numbers adjacent to monosaccharide residues in glycan structures indicate the position at which the higher residue is linked to the lower residue. If no linkage positions are indicated in the chromatogram annotation the residue is either in the terminal position or free in solution (e.g. glucose). All residues link downward via their 1-position. Split in chromatogram indicates change in extracted ion chromatograms.] |
2. Protein Posttranslational Modifications (PTMs) Many if not most proteins are molecularly modified after translation. Abnormal protein modification may exist as either a cause or an effect of disease; it therefore warrants exploration as a potentially rich source of disease markers. In combination with optimized sample preparation techniques, modern bioanalytical tools such as mass spectrometry are now capable of routinely characterizing and quantifying the relative abundance of most protein PTMs (Figure 2). | | Figure 2: Charge deconvoluted electrospray ionization (ESI)-mass spectrum of osteocalcin extracted from human blood plasma. Numerous truncated forms of the protein with variable numbers of Vitamin K dependent gamma-carboxylated glutamic acid residues are evidenced and readily quantified in terms of their relative fractional abundance. |
Yet caution is warranted as certain protein modifications-oxidative PTMs in particular-may arise as artifacts of less-than-optimal sample handling(Figure 3), potentially confounding their use as disease markers and interfering with conventional clinical laboratory tests based on binding interactions (e.g., ELISAs where protein quantification is based on interaction of the target protein with an antibody). We are interested in tracking protein modifications due to artifactual oxidation, determining how they affect conventional clinical laboratory tests, and figuring out how to prevent them from occurring. | | Figure 3: Charge deconvoluted electrospray ionization (ESI)-mass spectra of human albumin and apolipoprotein A-I, illustrating how improper sample storage may lead to artifactual protein oxidation. For albumin, ex vivo oxidation takes the form of S-cysteinylation and in apolipoprotein A-I it takes the form of methionine sulfoxidation. |
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Selected Publications"Gamma-Carboxylation and Fragmentation of Osteocalcin in Human Serum Defined by Mass Spectrometry," Rehder, D.S., Gundberg, C.M., Booth, S.L., and Borges, C.R. , Molecular and Cellular Proteomics 14 1546-1555 (2015) "Techniques for the Analysis of Cysteine Sulfhydryls and Oxidative Protein Folding," C.R. Borges and N.D. Sherma, Antioxid Redox Signal 21 511-531 (2014) "Elevated Plasma Albumin and Apolipoprotein A-I Oxidation under Suboptimal Specimen Storage Conditions.," C.R. Borges, D.S. Rehder, S. Jensen, M.R. Schaab, N.D. Sherma, H. Yassine, B. Nikolova,C. Breburda , Molecular and Cellular Proteomics 13 1890-1899 (2014) "Multiplexed surrogate analysis of glycotransferase activity in whole biospecimens," Borges, C.R., Rehder, D.S., Boffetta, P., Anal Chem 85 2927-2936 (2013) "Mass spectrometric immunoassay revisited," Nelson, R.W., Borges, C.R., J Am Soc Mass Spectrom 22 960-968 (2011) "Cysteine Sulfenic Acid as an Intermediate in Disulfide Bond Formation and Nonenzymatic Protein Folding," Rehder, D.S., Borges, C.R. ,Biochemistry 49 7748-7755 (2010) |
