Pilsoo Kang

Breast cancer diagnosis and prognosis through quantitative measurements of serum glycan profiles.

BACKGROUND Glycosylated proteins play important roles in cell-to-cell interactions, immunosurveillance, and a variety of receptor-mediated and specific protein functions through a highly complex repertoire of glycan structures. Aberrant glycosylation has been implicated in cancer for many years. METHODS We performed specific MALDI mass spectrometry (MS)-based glycomic profile analyses of permethylated glycans in sera from breast cancer patients (12, stage I; 11, stage II; 9, stage III; and 50, stage IV) along with sera from 27 disease-free women. The serum glycoproteins were enzymatically deglycosylated, and the released glycans were purified and quantitatively permethylated before their MALDI-MS analyses. We applied various statistical analysis tools, including ANOVA and principal component analysis, to evaluate the MS profiles. RESULTS Two statistical procedures implicated several sialylated and fucosylated N-glycan structures as highly probable biomarkers. Quantitative changes according to a cancer stage resulted when we categorized the glycans according to molecular size, number of oligomer branches, and abundance of sugar residues. Increases in sialylation and fucosylation of glycan structures appeared to be indicative of cancer progression. Different statistical evaluations confirmed independently that changes in the relative intensities of 8 N-glycans are characteristic of breast cancer (P < 0.001), whereas other glycan structures might contribute additionally to distinctions in the statistically recognizable patterns (different stages). CONCLUSIONS MS-based N-glycomic profiling of serum-derived constituents appears promising as a highly sensitive and informative approach for staging the progression of cancer.

High-throughput solid-phase permethylation of glycans prior to mass spectrometry.

Permethylation of glycans prior to their mass spectrometric determination has now become a time-honored methodology in glycoconjugate analysis due to the advantage of a simultaneous analysis of neutral and acidic glycans as well as enhanced sensitivity and easier tandem mass spectrometry interpretation. While the different solvent extraction-based versions of this method often suffice in different structural studies, they are generally less satisfactory in the quantitative determinations aiming at minor quantities of the analyzed materials. To overcome these difficulties, we recently introduced a solid-phase capillary permethylation technique (Kang et al., Rapid Commun. Mass Spectrom. 2005; 19: 3421) for microscale determination. Here, we describe a very useful high-throughput extension of the solid-phase methodology utilizing spin columns packed with sodium hydroxide beads. This procedure has been thoroughly optimized to match the analytical performance parameters of the previously used capillary technique. As demonstrated with a high-precision glycomic profiling analysis of human blood serum, this methodological improvement offers simplicity and reproducibility, allowing the complete permethylation of 12-18 samples in less than 20 min.

Solid-Phase Permethylation for Glycomic Analysis

This chapter discusses in detail a miniaturized version of the widely used permethylation technique which permits quantitative derivatization of oligosaccharides derived from minute quantities of glycoprotein. The approach involves packing of sodium hydroxide powder or beads in a microcolumn format, including spin columns, fused silica capillaries (500 microm i.d.) and plastic tubes (1 mm i.d.). The derivatization proceeds effectively in less than a minute time scale and it is applicable to glycans derived from femtomole quantities of glycoproteins. Prior to mass spectrometry (MS), methyl iodide is added to analytes suspended in dimethyl sulfoxide solution containing traces of water. The reaction mixture is then immediately infused through the microreactor. The packed sodium hydroxide powder or beads inside the microcolumns minimize oxidative degradation and peeling reactions which are otherwise commonly associated with the conventional permethylation technique. In addition, this solid-phase permethylation approach eliminates the need for excessive sample clean-up. As demonstrated below, picomole amounts of various types of glycans derived from model glycoproteins as well as real samples, including linear and branched, sialylated and neutral glycans were shown to become rapidly and efficiently permethylated through this approach.

Analysis of site-specific glycosylation of renal and hepatic γ-glutamyl transpeptidase from normal human tissue

The cell surface glycoprotein γ-glutamyl transpeptidase (GGT) was isolated from healthy human kidney and liver to characterize its glycosylation in normal human tissue in vivo. GGT is expressed by a single cell type in the kidney. The spectrum of N-glycans released from kidney GGT constituted a subset of the N-glycans identified from renal membrane glycoproteins. Recent advances in mass spectrometry enabled us to identify the microheterogeneity and relative abundance of glycans on specific glycopeptides and revealed a broader spectrum of glycans than was observed among glycans enzymatically released from isolated GGT. A total of 36 glycan compositions, with 40 unique structures, were identified by site-specific glycan analysis. Up to 15 different glycans were observed at a single site, with site-specific variation in glycan composition. N-Glycans released from liver membrane glycoproteins included many glycans also identified in the kidney. However, analysis of hepatic GGT glycopeptides revealed 11 glycan compositions, with 12 unique structures, none of which were observed on kidney GGT. No variation in glycosylation was observed among multiple kidney and liver donors. Two glycosylation sites on renal GGT were modified exclusively by neutral glycans. In silico modeling of GGT predicts that these two glycans are located in clefts on the surface of the protein facing the cell membrane, and their synthesis may be subject to steric constraints. This is the first analysis at the level of individual glycopeptides of a human glycoprotein produced by two different tissues in vivo and provides novel insights into tissue-specific and site-specific glycosylation in normal human tissues.

Use of a stable‐isotope‐labeled reporter peptide and antioxidants for reliable quantification of methionine oxidation in a monoclonal antibody by liquid chromatography/mass spectrometry

RATIONALE Accurate quantification of methionine oxidation in therapeutic proteins by liquid chromatography/mass spectrometry (LC/MS) is challenging due to the potential artifacts introduced during sample preparation and analysis in the peptide mapping workflow. In this study, a systematic approach for optimization of the peptide mapping procedure to achieve reliable quantification of endogenous methionine oxidation in monoclonal antibodies was developed. METHODS The approach is based on usage of a stable-isotope-labeled reporter peptide, identical in sequence to the tryptic peptide of an IgG1 monoclonal antibody containing the methionine residue most prone to oxidation. This approach was applied to evaluating various desalting procedures, and tested on nanoLC/MS, microLC/MS and UPLC/MS for the peptide mapping analysis of a model monoclonal antibody IgG1 sensitive to oxidation. RESULTS Several steps in the peptide mapping procedure with LC/MS detection at which protein oxidation occurred were identified and optimized using the reference stable-isotope-labeled peptide. Thus, reliable quantification of methionine oxidation in the target monoclonal antibody was validated. CONCLUSIONS The methodology which utilizes the reference stable-isotope-labeled reporter peptide is applicable to monoclonal antibody oxidation analysis and could be extended to other biotherapeutics once oxidation-prone methionine(s) in the protein sequence are identified. Copyright