N -glycan profiling to identify pancreatic cancer biomarkers
the consensus sequence Asp–Xaa–Ser/ Thr (Xaa is any amino acid but proline). To isolate N-linked glycoproteins from serum, the researchers exploited the glycan-binding properties of conca navalin A (ConA). ConA is a lectin that recognizes -mannose, a constituent of most N-glycans. Lubman and co-workers passed the serum through a ConA column to elute the bound N-linked glycoproteins and to eliminate serum albumin, which comprises 65% of se-
matography. This technique allows efficient separation of glycans with similar Like insectile alien creatures from a scimolecular masses but different strucence fiction novel, metastasizing cantures. Lubman says, “Hydrophilic chrocer cells creep from the primary tumor matography had been used previously to new locations, using bristly, polysacfor analysis of glycans from simple charide-coated “antennae” to contact mixtures, but we showed that it could other cells along the way. Although the be used for screening a large number of precise functions of polysaccharides glycans from a very complex mixture.” (glycans) in cancer progression are The researchers then determined the unknown, aberrant glycosylation of structures of the separated glycans uscell-surface proteins has been linked ing ESI TOFMS and MALDI-quadrupole to cancer-cell adhesion, ion trap-TOFMS/MS. invasion, and metastasis. The researchers deDefining serum glycan tected 92 N-glycosylation K+ profiles for normal, cansites from 51 different K+ cerous, and precancerous proteins, but no obvious + K K+ states, therefore, might aid differences in glycosylNa+ in diagnosis, prognosis, ation sites were observed + K and treatment. among proteins in the 100 K+ In this issue of JPR (pp sera of pancreatic cancer 1126–1138), David M. Lubpatients and controls. + % K man and colleagues at the However, of the 105 University of Michigan and unique carbohydrate 0 the University of Michigan structures detected by the 100 Medical Center introduce group, 44 glycans were a streamlined strategy for distinct in the cancer % N-linked glycosylation serum. Almost all of the profiling by MS, and they overexpressed glycans in 0 use the method in a proofpancreatic cancer serum 960 980 1000 1020 1040 1060 1080 of-principle study to comwere high-mass, fucosylpare the glycan profiles ated, tetra-antennary Peaks aplenty. Differences in MS glycan profiles of pancreatic cancer (top) of control and pancreatic structures with increased and control (bottom) sera. cancer sera. sialic acid content. The Interestingly, most canincreased branching, cers exhibit altered patterns of protein rum protein, and other unwanted profucosylation, and sialylation of glycans glycosylation compared with control teins. Then the researchers digested the in pancreatic cancer serum reflects cells. Glycan characteristics that may glycoproteins with trypsin to generate higher activities of the respective glycobe altered in cancer cells include polyglycopeptides for MS analysis. The trypsyltransferases. saccharide structure, composition, tic digest was applied to a second ConA Lubman says that serum glycan and sites of attachment to proteins. column to extract N-glycopeptides from profiling may be a more accurate tool However, whether these changes are the nonglycosylated peptides. for cancer diagnosis than proteome causes or effects of cancer progression Next, the researchers deglycosylprofiling. “We can see very distinctive is a mystery. Lubman says, “Sometimes ated the purified glycopeptides with changes in glycan profiles between canaltered patterns of glycosylation appear peptide-N-glycosidase F (PNGase F), an cer and normal sera. When you’re lookto reflect global dysregulation in cancer amidase that cleaves the bond between ing at up- and down-regulation of procells, but in other cases it’s a very specifthe asparagine residue of the N-linked teins, sometimes the quantitative levels ic effect. Glycans on proteins often stick glycopeptide and the first monosacoverlap between cancer and normal, so out on cell surfaces and are involved charide of the glycan. This cleavage that’s not always a good diagnostic test,” in cell–cell signaling, so changes in converts the asparagine to aspartic he says. Recently, Lubman and co-workglycosylation may facilitate metastasis, acid, resulting in a 1-Da shift in moers have elucidated a distinct serum which often involves surface–sugar lecular mass. So, a 1-Da shift from the N-glycan profile for pancreatitis, an ininteractions.” expected molecular mass for peptides flammation of the pancreas that is very Lubman and co-workers used double containing asparagine residues in the difficult to distinguish from pancreatic lectin affinity chromatography, capilAsp–Xaa–Ser/Thr consensus sequence cancer with conventional diagnostic lary hydrophilic chromatography, and indicated an N-glycosylation site. tests. The group’s powerful new method MS to compare N-glycans and N-glycoTo profile the serum glycans, the olifor N-glycan profiling, therefore, may sylation sites in proteins from control gosaccharides removed from glycopepfacilitate biomarker discovery for canand pancreatic cancer sera. N-glycans tides by PNGase F were separated by cers as well as for benign disease states. are attached to asparagine residues in capillary hydrophilic interaction chro—Laura Tomky Cassiday
Journal of Proteome Research • Vol. 6, No. 3, 2007 933
