Multi-lectin Affinity Chromatography (M-LAC) Combined with Abundant Protein Depletion for Analysis of Human Plasma in Clinical Proteomics Applications

  • Marina HincapieEmail author
  • Tatiana Plavina
  • William S. Hancock


In this chapter we describe a robust sample preparation method for proteomic analysis of human plasma and discovery of candidate protein biomarkers. The method consists of depletion of the most abundant plasma proteins and multi-lectin affinity chromatography (M-LAC) to fractionate plasma into flow-through (unbound) and bound fractions, followed by nano-LC-MS/MS analysis of the digested proteins and label-free comparative quantitation. The performance of the method is monitored by several quality control checkpoints to assure reproducibility of the proteomic analysis. This approach reduces the complexity of plasma samples and significantly improves the overall dynamic range of protein detection, enabling detection of tissue leakage proteins present in plasma in ng/mL concentrations. In addition, the lectin-based fractionation of depleted plasma targets the plasma glycoproteome, permitting the identification of glycoproteins with disease-related differences in glycoform populations. The proteomics workflow was applied to the analysis of plasma from diseased patients affected with psoriasis and healthy donors. As an example of the effectiveness of the method we will present data from this biomarker study.


Clinical proteomics Glycoproteins Lectin affinity chromatography Biomarker discovery Sample preparation 



This work was supported by National Institutes of Health/National Cancer Institute (NIH/NCI) grants RO1 CA122591 and U01-CA128427 and by the Korean Research WCU grant R31-2008-000-10086-0. W. S. H and M .H. disclose that they have a financial interest in current efforts by Northeastern University and PeptiFarma to licence the M-LAC technology for biomarker discovery. Contribution Number 960 from the Barnett Institute.


  1. Anderson, N.L., and Anderson, N.G. (2002). The human plasma proteome: History, character, and diagnostic prospects. Mol Cell Proteomics 1, 845–867.CrossRefGoogle Scholar
  2. Bakry, N., Kamata, Y., and Simpson, L.L. (1991). Lectins from Triticum vulgaris and Limax flavus are universal antagonists of botulinum neurotoxin and tetanus toxin. J Pharmacol Exp Ther 258, 830–836.Google Scholar
  3. Becker, J.W., Reeke, G.N., Jr., Wang, J.L., Cunningham, B.A., and Edelman, G.M. (1975). The covalent and three-dimensional structure of concanavalin A. III. Structure of the monomer and its interactions with metals and saccharides. J Biol Chem 250, 1513–1524.Google Scholar
  4. Block, T.M., Comunale, M.A., Lowman, M., Steel, L.F., Romano, P.R., Fimmel, C., Tennant, B.C., London, W.T., Evans, A.A., Blumberg, B.S., et al. (2005). Use of targeted glycoproteomics to identify serum glycoproteins that correlate with liver cancer in woodchucks and humans. Proc Natl Acad Sci USA 102, 779–784.CrossRefGoogle Scholar
  5. Carlsson, J., Gullstrand, C., Ludvigsson, J., Lundstrom, I., and Winquist, F. (2008). Detection of global glycosylation changes of serum proteins in type 1 diabetes using a lectin panel and multivariate data analysis. Talanta 76, 333–337.CrossRefGoogle Scholar
  6. Cummings, R.D., and Kornfeld, S. (1982). Fractionation of asparagine-linked oligosaccharides by serial lectin-agarose affinity chromatography. A rapid, sensitive, and specific technique. J Biol Chem 257, 11235–11240.Google Scholar
  7. Dai, Z., Liu, Y.K., Cui, J.F., Shen, H.L., Chen, J., Sun, R.X., Zhang, Y., Zhou, X.W., Yang, P.Y., and Tang, Z.Y. (2006). Identification and analysis of altered alpha1,6-fucosylated glycoproteins associated with hepatocellular carcinoma metastasis. Proteomics 6, 5857–5867.CrossRefGoogle Scholar
  8. Dayarathna, M.K., Hancock, W.S., and Hincapie, M. (2008). A two step fractionation approach for plasma proteomics using immunodepletion of abundant proteins and multi-lectin affinity chromatography: Application to the analysis of obesity, diabetes, and hypertension diseases. J Sep Sci 31, 1156–1166.Google Scholar
  9. Dennis, J.W., Laferte, S., Waghorne, C., Breitman, M.L., and Kerbel, R.S. (1987). Beta 1–6 branching of Asn-linked oligosaccharides is directly associated with metastasis. Science 236, 582–585.CrossRefGoogle Scholar
  10. Dwek, M.V., Ross, H.A., Streets, A.J., Brooks, S.A., Adam, E., Titcomb, A., Woodside, J.V., Schumacher, U., and Leathem, A.J. (2001). Helix pomatia agglutinin lectin-binding oligosaccharides of aggressive breast cancer. Int J Cancer 95, 79–85.CrossRefGoogle Scholar
  11. Echan, L.A., Tang, H.Y., Ali-Khan, N., Lee, K., and Speicher, D.W. (2005). Depletion of multiple high-abundance proteins improves protein profiling capacities of human serum and plasma. Proteomics 5, 3292–3303.CrossRefGoogle Scholar
  12. Elias, J.E., and Gygi, S.P. (2007). Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat Methods 4, 207–214.CrossRefGoogle Scholar
  13. Fang, X., and Zhang, W.W. (2008). Affinity separation and enrichment methods in proteomic analysis. J Proteomics 71, 284–303.CrossRefGoogle Scholar
  14. Haab, B.B., Geierstanger, B.H., Michailidis, G., Vitzthum, F., Forrester, S., Okon, R., Saviranta, P., Brinker, A., Sorette, M., Perlee, L., et al. (2005). Immunoassay and antibody microarray analysis of the HUPO Plasma Proteome Project reference specimens: Systematic variation between sample types and calibration of mass spectrometry data. Proteomics 5, 3278–3291.CrossRefGoogle Scholar
  15. Jacobs, J.M., Adkins, J.N., Qian, W.J., Liu, T., Shen, Y., Camp, D.G., 2nd, and Smith, R.D. (2005). Utilizing human blood plasma for proteomic biomarker discovery. J Proteome Res 4, 1073–1085.CrossRefGoogle Scholar
  16. Keller, A., Nesvizhskii, A.I., Kolker, E., and Aebersold, R. (2002). Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal Chem 74, 5383–5392.CrossRefGoogle Scholar
  17. Klammer, A.A., and MacCoss, M.J. (2006). Effects of modified digestion schemes on the identification of proteins from complex mixtures. J Proteome Res 5, 695–700.CrossRefGoogle Scholar
  18. Kreunin, P., Zhao, J., Rosser, C., Urquidi, V., Lubman, D.M., and Goodison, S. (2007). Bladder cancer associated glycoprotein signatures revealed by urinary proteomic profiling. J Proteome Res 6, 2631–2639.CrossRefGoogle Scholar
  19. Kullolli, M., Hancock, W.S., and Hincapie, M. (2008). Preparation of a high-performance multi-lectin affinity chromatography (HP-M-LAC) adsorbent for the analysis of human plasma glycoproteins. J Sep Sci 31, 2733–2739.CrossRefGoogle Scholar
  20. Kullolli, M., Hancock, W.S., and Hincapie, M. (2010). Automated platform for fractionation of human plasma glycoproteome in clinical proteomics. Anal Chem 82, 115–120.Google Scholar
  21. Liu, T., Qian, W.J., Chen, W.N., Jacobs, J.M., Moore, R.J., Anderson, D.J., Gritsenko, M.A., Monroe, M.E., Thrall, B.D., Camp, D.G., 2nd, et al. (2005). Improved proteome coverage by using high efficiency cysteinyl peptide enrichment: The human mammary epithelial cell proteome. Proteomics 5, 1263–1273.CrossRefGoogle Scholar
  22. Martosella, J., Zolotarjova, N., Liu, H., Nicol, G., and Boyes, B.E. (2005). Reversed-phase high-performance liquid chromatographic prefractionation of immunodepleted human serum proteins to enhance mass spectrometry identification of lower-abundant proteins. J Proteome Res 4, 1522–1537.CrossRefGoogle Scholar
  23. Omenn, G.S., States, D.J., Adamski, M., Blackwell, T.W., Menon, R., Hermjakob, H., Apweiler, R., Haab, B.B., Simpson, R.J., Eddes, J.S., Kapp, E.A., Moritz, R.L., Chan, D.W., Rai, A.J., Admon, A., Aebersold, R., Eng, J., Hancock, W.S., Hefta, S.A., Meyer, H., Paik, Y.K., Yoo, J.S., Ping, P., Pounds, J., Adkins, J., Qian, X., Wang, R., Wasinger, V., Wu, C.Y., Zhao, X., Zeng, R., Archakov, A., Tsugita, A., Beer, I., Pandey, A., Pisano, M., Andrews, P., Tammen, H., Speicher, D.W., and Hanash, S.M. (2005). Overview of the HUPO Plasma Proteome Project: Results from the pilot phase with 35 collaborating laboratories and multiple analytical groups, generating a core dataset of 3020 proteins and a publicly-available database. Proteomics 5, 3226–3245.Google Scholar
  24. Plavina, T., Hincapie, M., Wakshull, E., Subramanyam, M., and Hancock, W.S. (2008). Increased plasma concentrations of cytoskeletal and Ca2+-binding proteins and their peptides in psoriasis patients. Clin Chem 54, 1805–1814.CrossRefGoogle Scholar
  25. Plavina, T., Wakshull, E., Hancock, W.S., and 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.CrossRefGoogle Scholar
  26. Polanski, M.A., and Anderson, N.L (2006). A list of candidate cancer biomarkers for targeted proteomics. Biomarker Insights 2, 1–48.Google Scholar
  27. Purcell, A.W., van Driel, I.R., and Gleeson, P.A. (2008). Impact of glycans on T-cell tolerance to glycosylated self-antigens. Immunol Cell Biol 86, 574–579.CrossRefGoogle Scholar
  28. Qiu, R., and Regnier, F.E. (2005). Comparative glycoproteomics of N-linked complex-type glycoforms containing sialic acid in human serum. Anal Chem 77, 7225–7231.CrossRefGoogle Scholar
  29. Ralin, D.W., Dultz S.C., Silver, J.E., Travis, J.C., Kullolli, M., Hancock, W.S., Hincapie, M. (2008). Kinetic analysis of glycoprotein–lectin interactions by label-free internal reflection ellipsometry. Clin Proteomics 4, 37–46.CrossRefGoogle Scholar
  30. Saulsbury, F.T. (1997). Alterations in the O-linked glycosylation of IgA1 in children with Henoch-Schonlein purpura. J Rheumatol 24, 2246–2249.Google Scholar
  31. Wang, G., Wu, W.W., Zeng, W., Chou, C.L., and Shen, R.F. (2006). Label-free protein quantification using LC-coupled ion trap or FT mass spectrometry: Reproducibility, linearity, and application with complex proteomes. J Proteome Res 5, 1214–1223.CrossRefGoogle Scholar
  32. Yang, Z., and Hancock, W.S. (2004). Approach to the comprehensive analysis of glycoproteins isolated from human serum using a multi-lectin affinity column. J Chromatogr A 1053, 79–88.Google Scholar
  33. Yang, Z., Harris, L.E., Palmer-Toy, D.E., and Hancock, W.S. (2006). Multilectin affinity chromatography for characterization of multiple glycoprotein biomarker candidates in serum from breast cancer patients. Clin Chem 52, 1897–1905.Google Scholar
  34. Yates, J.R., Cociorva, D., Liao, L., and Zabrouskov, V. (2006). Performance of a linear ion trap-orbitrap hybrid for peptide analysis. Anal Chem 78, 493–500.CrossRefGoogle Scholar
  35. Yocum, A.K., Yu, K., Oe, T., and Blair, I.A. (2005). Effect of immunoaffinity depletion of human serum during proteomic investigations. J Proteome Res 4, 1722–1731.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Marina Hincapie
    • 1
    Email author
  • Tatiana Plavina
    • 2
  • William S. Hancock
    • 1
  1. 1.The Barnett Institute of Chemical and Biological SciencesNortheastern UniversityBostonUSA
  2. 2.Biogen Idec, Inc.BostonUSA

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