Abstract
Glycans are an important class of post-translational modifications that decorate a wide array of protein substrates. These cell-type specific molecules, which are modulated during developmental and disease processes, are attractive biomarker candidates as biology regarding altered glycosylation can be used to guide the experimental design. The mass spectrometry (MS)-based workflow described here incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan motifs. The goal was to design a relatively simple method for the rapid analysis of small plasma volumes (e.g., clinical specimens). As increases in sialylation and fucosylation are prominent among cancer-associated modifications, we focused on Sambucus nigra agglutinin and AAL, which bind sialic acid- and fucose-containing structures, respectively. Positive controls (fucosylated and sialylated human lactoferrin glycopeptides), and negative controls (high-mannose glycopeptides from Saccharomyces cerevisiae invertase) were used to monitor the specificity of lectin capture and optimize the workflow. Multiple Affinity Removal System 14-depleted, trypsin-digested human plasma from healthy donors served as the target analyte. Samples were loaded onto the lectin columns and separated by high performance liquid chromatography (HPLC) into flow through and bound fractions, which were treated with PNGase F, an amidase that removes N-linked glycans and marks the underlying asparagine glycosite by a +1 Da mass shift. The deglycosylated peptide fractions were interrogated by HPLC ESI-MS/MS on a quadrupole time-of-flight mass spectrometer. The method allowed identification of 122 human plasma glycoproteins containing 247 unique glycosites. Notably, glycoproteins that circulate at ng/mL levels (e.g., cadherin-5 at 0.3–4.9 ng/mL, and neutrophil gelatinase-associated lipocalin which is present at ∼2.5 ng/mL) were routinely observed, suggesting that this method enables the detection of low-abundance cancer-specific glycoproteins.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Meezan E, Wu HC, Black PH, Robbins PW (1969) Comparative studies on the carbohydrate-containing membrane components of normal and virus-transformed mouse fibroblasts. II. Separation of glycoproteins and glycopeptides by sephadex chromatography. Biochemistry 8:2518–2524
Barkauskas DA, An HJ, Kronewitter SR, de Leoz ML, Chew HK, de Vere White RW, Leiserowitz GS, Miyamoto S, Lebrilla CB, Rocke DM (2009) Detecting glycan cancer biomarkers in serum samples using MALDI FT-ICR mass spectrometry data. Bioinformatics 25:251–257
Goldberg D, Bern M, Parry S, Sutton-Smith M, Panico M, Morris HR, Dell A (2007) Automated N-glycopeptide identification using a combination of single- and tandem-MS. J Proteome Res 6:3995–4005
Harvey DJ (2005) Structural determination of N-linked glycans by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. Proteomics 5:1774–1786
North SJ, Hitchen PG, Haslam SM, Dell A (2009) Mass spectrometry in the analysis of N-linked and O-linked glycans. Curr Opin Struct Biol 19:498–506
Royle L, Campbell MP, Radcliffe CM, White DM, Harvey DJ, Abrahams JL, Kim YG, Henry GW, Shadick NA, Weinblatt ME, Lee DM, Rudd PM, Dwek RA (2008) HPLC-based analysis of serum N-glycans on a 96-well plate platform with dedicated database software. Anal Biochem 376:1–12
Wong SC, Chan CM, Ma BB, Lam MY, Choi GC, Au TC, Chan AS, Chan AT (2009) Advanced proteomic technologies for cancer biomarker discovery. Expert Rev Proteomics 6:123–134
Barrabes S, Pages-Pons L, Radcliffe CM, Tabares G, Fort E, Royle L, Harvey DJ, Moenner M, Dwek RA, Rudd PM, De Llorens R, Peracaula R (2007) Glycosylation of serum ribonuclease 1 indicates a major endothelial origin and reveals an increase in core fucosylation in pancreatic cancer. Glycobiology 17:388–400
Kuzmanov U, Jiang N, Smith CR, Soosaipillai A, Diamandis EP (2009) Differential N-glycosylation of kallikrein 6 derived from ovarian cancer cells or the central nervous system. Mol Cell Proteomics 8:791–798
Meany DL, Zhang Z, Sokoll LJ, Zhang H, Chan DW (2009) Glycoproteomics for prostate cancer detection: changes in serum PSA glycosylation patterns. J Proteome Res 8:613–619
Misonou Y, Shida K, Korekane H, Seki Y, Noura S, Ohue M, Miyamoto Y (2009) Comprehensive clinico-glycomic study of 16 colorectal cancer specimens: elucidation of aberrant glycosylation and its mechanistic causes in colorectal cancer cells. J Proteome Res 8:2990–3005
Mizuochi T, Nishimura R, Derappe C, Taniguchi T, Hamamoto T, Mochizuki M, Kobata A (1983) Structures of the asparagine-linked sugar chains of human chorionic gonadotropin produced in choriocarcinoma. Appearance of triantennary sugar chains and unique biantennary sugar chains. J Biol Chem 258:14126–14129
Ohyama C, Hosono M, Nitta K, Oh-eda M, Yoshikawa K, Habuchi T, Arai Y, Fukuda M (2004) Carbohydrate structure and differential binding of prostate specific antigen to Maackia amurensis lectin between prostate cancer and benign prostate hypertrophy. Glycobiology 14:671–679
Taylor AD, Hancock WS, Hincapie M, Taniguchi N, Hanash SM (2009) Towards an integrated proteomic and glycomic approach to finding cancer biomarkers. Genome Med 1:57
Cho W, Jung K, Regnier FE (2008) Use of glycan targeting antibodies to identify cancer-associated glycoproteins in plasma of breast cancer patients. Anal Chem 80:5286–5292
Heo SH, Lee SJ, Ryoo HM, Park JY, Cho JY (2007) Identification of putative serum glycoprotein biomarkers for human lung adenocarcinoma by multilectin affinity chromatography and LC-MS/MS. Proteomics 7:4292–4302
Jung K, Cho W, Regnier FE (2009) Glycoproteomics of plasma based on narrow selectivity lectin affinity chromatography. J Proteome Res 8:643–650
Kaji H, Saito H, Yamauchi Y, Shinkawa T, Taoka M, Hirabayashi J, Kasai K, Takahashi N, Isobe T (2003) Lectin affinity capture, isotope-coded tagging and mass spectrometry to identify N-linked glycoproteins. Nat Biotechnol 21:667–672
Plavina T, Wakshull E, Hancock WS, Hincapie M (2007) Combination of abundant protein depletion and multi-lectin affinity chromatography (M-LAC) for plasma protein biomarker discovery. J Proteome Res 6:662–671
Zhang H, Li XJ, Martin DB, Aebersold R (2003) Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Nat Biotechnol 21:660–666
Drake PM, Schilling B, Niles RK, Braten M, Johansen E, Liu H, Lerch M, Sorensen DJ, Li B, Allen S, Hall SC, Witkowska HE, Regnier FE, Gibson BW, Fisher SJ (2011) A lectin affinity workflow targeting glycosite-specific, cancer-related carbohydrate structures in trypsin-digested human plasma. Anal Biochem 408:71–85
Keshishian H, Addona T, Burgess M, Kuhn E, Carr SA (2007) Quantitative, multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution. Mol Cell Proteomics 6:2212–2229
Soeki T, Tamura Y, Shinohara H, Sakabe K, Onose Y, Fukuda N (2004) Elevated concentration of soluble vascular endothelial cadherin is associated with coronary atherosclerosis. Circ J 68:1–5
Krokhin OV, Antonovici M, Ens W, Wilkins JA, Standing KG (2006) Deamidation of -Asn-Gly- sequences during sample preparation for proteomics: consequences for MALDI and HPLC-MALDI analysis. Anal Chem 78:6645–6650
Matsumoto A, Yoshima H, Takasaki S, Kobata A (1982) Structural study of the sugar chains of human lactoferrin: finding of four novel complex-type asparagine-linked sugar chains. J Biochem 91:143–155
Spik G, Strecker G, Fournet B, Bouquelet S, Montreuil J, Dorland L, van Halbeek H, Vliegenthart JF (1982) Primary structure of the glycans from human lactotransferrin. Eur J Biochem 121:413–419
Trimble RB, Atkinson PH (1986) Structure of yeast external invertase Man8-14GlcNAc processing intermediates by 500-megahertz 1H NMR spectroscopy. J Biol Chem 261:9815–9824
Acknowledgement
This work was supported by the Clinical Proteomic Technologies for Cancer initiative, 5U24CA126477-04.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Drake, P., Schilling, B., Gibson, B., Fisher, S. (2013). Elucidation of N-Glycosites Within Human Plasma Glycoproteins for Cancer Biomarker Discovery. In: Kohler, J., Patrie, S. (eds) Mass Spectrometry of Glycoproteins. Methods in Molecular Biology, vol 951. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-146-2_21
Download citation
DOI: https://doi.org/10.1007/978-1-62703-146-2_21
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-145-5
Online ISBN: 978-1-62703-146-2
eBook Packages: Springer Protocols