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Mass Spectrometry of Proteins and Peptides pp 321–351Cite as

Analysis of Protein Glycosylation and Phosphorylation Using Liquid Phase Separation, Protein Microarray Technology, and Mass Spectrometry

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Part of the book series: Methods In Molecular Biology ((MIMB,volume 492))

Summary

Protein glycosylation and phosphorylation are very common posttranslational modifications. The alteration of these modifications in cancer cells is closely related to the onset and progression of cancer and other disease states. In this protocol, strategies for monitoring the changes in protein glycosylation and phosphorylation in serum or tissue cells on a global scale and specifically characterizing these alterations are included. The technique is based on lectin affinity enrichment for glycoproteins, all liquid-phase two-dimensional fractionation, protein microarray, and mass spectrometry technology. Proteins are separated based on pI in the first dimension using chromatofocusing (CF) or liquid isoelectric focusing (IEF) followed by the second-dimension separation using nonporous silica RP-HPLC. Five lectins with different binding specificities to glycan structures are used for screening glycosylation patterns in human serum through a biotin streptavidin system. Fluorescent phosphodyes and phosphospecific antibodies are employed to detect specific phosphorylated proteins in cell lines or human tissues. The purified proteins of interest are identified by peptide sequencing. Their modifications including glycosylation and phosphorylation could be further characterized by mass-spectrometry-based approaches. These strategies can be used in biological samples for large-scale glycoproteome/phosphoproteome screening as well as for individual protein modification analysis.

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References

  1. Rudd PM, Elliott T, Cresswell P, Wilson IA, Dwek RA. (2001) Glycosylation and the immune system. Science 291 : 2370–2376.

    Article  CAS  PubMed  Google Scholar 

  2. Block TM, Comunale MA, Lowman M, 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.

    Article  CAS  PubMed  Google Scholar 

  3. Zhao J, Simeone DM, Heidt D, Anderson MA, Lubman DM. (2006) Comparative serum glycoproteomics using lectin selected sialic acid glycoproteins with mass spectro-metric analysis: Application to pancreatic cancer serum. J Proteome Res 5 : 1792–1802.

    Article  CAS  PubMed  Google Scholar 

  4. Kobata A, Amano J. (2005) Altered glyco-sylation of proteins produced by malignant cells, and application for the diagnosis and immunotherapy of tumours. Immunol Cell Biol 83 : 429–439.

    Article  CAS  PubMed  Google Scholar 

  5. Robinson DR, Wu YM, Lin SF. (2000) The protein tyrosine kinase family of the human genome. Oncogene 19 : 5548–5557.

    Article  CAS  PubMed  Google Scholar 

  6. Gschwind A, Fischer OM, Ullrich A. (2004) The discovery of receptor tyrosine kinases: Targets for cancer therapy. Nat Rev Cancer 4 : 361–370.

    Article  CAS  PubMed  Google Scholar 

  7. Cohen P. (1982) The role of protein phos-phorylation in neural and hormonal control of cellular activity. Nature 296 : 613–620.

    Article  CAS  PubMed  Google Scholar 

  8. Orchekowski R, Hamelinck D, Li L, et al. (2005) Antibody microarray profiling reveals individual and combined serum proteins associated with pancreatic cancer. Cancer Res 65 : 11193–11202.

    Article  CAS  PubMed  Google Scholar 

  9. Yan F, Sreekumar A, Laxman B, Chinnaiyan AM, Lubman DM, Barder TJ. (2003) Protein microarrays using liquid phase fractiona-tion of cell lysates. Proteomics 3 : 1228–1235.

    Article  CAS  PubMed  Google Scholar 

  10. Patwa TH, Zhao J, Anderson MA, Sime-one DM, Lubman DM. (2006) Screening of glycosylation patterns in serum using natural glycoprotein microarrays and multi-lectin fluorescence detection. Anal Chem 78 : 6411–6421.

    Article  CAS  PubMed  Google Scholar 

  11. Zhu K, Zhao J, Lubman DM, Miller FR, Barder TJ. (2005) Protein pI shifts due to posttranslational modifications in the separation and characterization of proteins. Anal Chem 77 : 2745–2755.

    Article  CAS  PubMed  Google Scholar 

  12. Zhao J, Chang AC, Li C, et al. (2007) Comparative proteomic analysis of Barrett's metaplasia and esophageal adenocarcinomas using 2-D liquid mass mapping. Mol Cell Proteom-ics 6 : 987–999.

    Article  CAS  Google Scholar 

  13. Zhao J, Zhu K, Lubman DM, et al. (2006) Proteomic analysis of estrogen response of premalignant human breast cells using a 2-D liquid separation/mass mapping technique. Proteomics 6 : 3847–3861.

    Article  CAS  PubMed  Google Scholar 

  14. Yoo C, Zhao J, Pal M, et al. (2006) Automated integration of monolith-based protein separation with on-plate digestion for mass spectrometric analysis of esophageal adeno-carcinoma human epithelial samples. Electro-phoresis 27 : 3643–3651.

    Article  CAS  Google Scholar 

  15. Pal M, Moffa A, Sreekumar A, et al. (2006) Differential phosphoprotein mapping in cancer cells using protein microarrays produced from 2-D liquid fractionation. Anal Chem 78 : 702–710.

    Article  CAS  PubMed  Google Scholar 

  16. Bradford MM. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72 : 248–254.

    Article  CAS  PubMed  Google Scholar 

  17. Rademacher TW, Parekh RB, Dwek RA. (1988) Glycobiology. Annu Rev Biochem 57 : 785–838.

    Article  CAS  PubMed  Google Scholar 

  18. Yang Z, Hancock WS, Chew TR, Bonilla L. (2005) A study of glycoproteins in human serum and plasma reference standards (HUPO) using multilectin affinity chroma-tography coupled with RPLC-MS/MS. Pro-teomics 5 : 3353–3366.

    CAS  Google Scholar 

  19. Banks JF, Gulcicek EE. (1997) Rapid pep-tide mapping by reversed-phase liquid chro-matography on nonporous silica with on-line electrospray time-of-flight mass spectrom-etry. Anal Chem 69 : 3973–3978.

    Article  CAS  PubMed  Google Scholar 

  20. Hanson M, Unger KK, Mant CT, Hodges RS. (1996) Optimization strategies in ultrafast reversed-phase chromatogra-phy of proteins. Trans. Anal. Chem. 15 : 102–110.

    CAS  Google Scholar 

  21. Shibuya N, Goldstein IJ, Broekaert WF, Nsimba-Lubaki M, Peeters B, Peumans WJ. (1987) Fractionation of sialylated oligosac-charides, glycopeptides, and glycoproteins on immobilized elderberry (Sambucus nigra L.) bark lectin. Arch Biochem Biophys 254 : 1–8.

    Article  CAS  PubMed  Google Scholar 

  22. Wang WC, Cummings RD. (1988) The immobilized leukoagglutinin from the seeds of Maackia amurensis binds with high affinity to complex-type Asn-linked oligosaccharides containing terminal sialic acid-linked alpha-2,3 to penultimate galactose residues. J Biol Chem 263 : 4576–4585.

    CAS  PubMed  Google Scholar 

  23. Thomsson KA, Karlsson H, Hansson GC. (2000) Sequencing of sulfated oligosaccha-rides from mucins by liquid chromatogra-phy and electrospray ionization tandem mass spectrometry. Anal Chem 72 : 4543–4549.

    Article  CAS  PubMed  Google Scholar 

  24. Kawasaki N, Itoh S, Ohta M, Hayakawa T. (2003) Microanalysis of N-linked oligosac-charides in a glycoprotein by capillary liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spec-trometry. Anal Biochem 316 : 15–22.

    Article  CAS  PubMed  Google Scholar 

  25. Wuhrer M, Deelder AM, Hokke CH. (2005) Protein glycosylation analysis by liquid chro-matography-mass spectrometry. J Chroma-togr B Analyt Technol Biomed Life Sci 825 : 124–133.

    Article  CAS  Google Scholar 

  26. Harvey DJ, Martin RL, Jackson KA, Sutton CW. (2004) Fragmentation of N-linked gly-cans with a matrix-assisted laser desorption/ ionization ion trap time-of-flight mass spectrometer. Rapid Commun Mass Spectrom 18 : 2997–3007.

    Article  CAS  PubMed  Google Scholar 

  27. Naka R, Kamoda S, Ishizuka A, Kinoshita M, Kakehi K. (2006) Analysis of total N-glycans in cell membrane fractions of cancer cells using a combination of serotonin affinity chromatography and normal phase chroma-tography. J Proteome Res 5 : 88–97.

    Article  CAS  PubMed  Google Scholar 

  28. Merry AH, Neville DC, Royle L, et al. (2002) Recovery of intact 2-aminobenzamide-labeled O-glycans released from glycopro-teins by hydrazinolysis. Anal Biochem 304 : 91–99.

    Article  CAS  PubMed  Google Scholar 

  29. Schulz BL, Packer NH, Karlsson NG. (2002) Small-scale analysis of O-linked oligosaccha-rides from glycoproteins and mucins separated by gel electrophoresis. Anal Chem 74 : 6088–6097.

    Article  CAS  PubMed  Google Scholar 

  30. Hashii N, Kawasaki N, Itoh S, Hyuga M, Kawanishi T, Hayakawa T. (2005) Glycomic/ glycoproteomic analysis by liquid chromatog-raphy/mass spectrometry: Analysis of glycan structural alteration in cells. Proteomics 5 : 4665–4672.

    Article  PubMed  Google Scholar 

  31. Deguchi K, Takegawa Y, Ito H, et al. (2006) Structural assignment of isomeric 2-ami-nopyridine-derivatized monosialylated bian-tennary N-linked oligosaccharides using negative-ion multistage tandem mass spectral matching. Rapid Commun Mass Spectrom 20 : 1480–1481.

    Article  CAS  Google Scholar 

  32. Deguchi K, Ito H, Takegawa Y, Shinji N, Nakagawa H, Nishimura S. (2006) Complementary structural information of positive-and negative-ion MSn spectra of glycopeptides with neutral and sialylated N-glycans. Rapid Commun Mass Spectrom 20 : 741–746.

    Article  CAS  PubMed  Google Scholar 

  33. Harvey DJ. (1999) Matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates. Mass Spectrom Rev 18 : 349–450.

    Article  CAS  PubMed  Google Scholar 

  34. Fountain ST, Lee H, Lubman DM. (1994) Ion fragmentation activated by matrix-assisted laser desorption/ionization in an ion-trap/ reflectron time-of-flight device. Rapid Com-mun Mass Spectrom 8 : 407–416.

    Article  CAS  Google Scholar 

  35. Ding L, Kawatoh E, Koichi T, Smith AJ, Kumashiro S. (1999) High efficiency MALDI-QIT-ToF mass spectrometer. Proc Int Soc Optical Eng 3777 : 144.

    CAS  Google Scholar 

  36. Chien BM, Michael SM, Lubman DM. (1993) Enhancement of resolution in matrix-assisted laser-desorption using an ion-trap stroage reflectron time-of-flight mass spectrometer. Rapid Commun Mass Spectrom 7 : 837–843.

    Article  CAS  Google Scholar 

  37. Doroshenko VM, Cotter RJ. (1998) A quad-rupole ion trap/time-of-flight mass spectrometer with a parabolic reflectron. J Mass Spectrom 33 : 305–318.

    Article  CAS  PubMed  Google Scholar 

  38. Demelbauer UM, Zehl M, Plematl A, All-maier G, Rizzi A. (2004) Determination of glycopeptide structures by multistage mass spectrometry with low-energy collision-induced dissociation: Comparison of elec-trospray ionization quadrupole ion trap and matrix-assisted laser desorption/ionization quadrupole ion trap reflectron time-of-flight approaches. Rapid Commun Mass Spectrom 18 : 1575–1582.

    Article  CAS  PubMed  Google Scholar 

  39. Ojima N, Masuda K, Tanaka K, Nishimura O. (2005) Analysis of neutral oligosac-charides for structural characterization by matrix-assisted laser desorption/ioniza-tion quadrupole ion trap time-of-flight mass spectrometry. J Mass Spectrom 40 : 380–388.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported in part by the National Cancer Institute under grant R01CA106402 (D.M.L.), the National Institute of Health under grant R01GM49500 (D.M.L.), and a Michigan Economic Development Grant MEDC03–622 (D.M.S.). Support was also generously provided by Eprogen, Inc. and Beck-man-Coulter. We thank Bio-Rad for the gift of the micro-Rotofor device.

Author information

Authors and Affiliations

  1. Department of Chemistry, University of Michigan Medical Center, Ann Arbor, MI, USA

    Jia Zhao, Tasneem H. Patwa, Manoj Pal & Weilian Qiu

  2. Department of Chemistry, Comprehensive Cancer Center, Department of Surgery, University of Michigan Medical Center, Ann Arbor, MI, USA

    David M. Lubman

Authors
  1. Jia Zhao
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  2. Tasneem H. Patwa
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  3. Manoj Pal
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  4. Weilian Qiu
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  5. David M. Lubman
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Editor information

Editors and Affiliations

  1. Fundamental & Computational Sciences Directorate, Pacific Northwest National Laboratory, Richland, WA, USA

    Mary S. Lipton

  2. Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA, USA

    Ljiljana Paša-Tolic

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© 2009 Humana Press, a part of Springer Science+Business Media, LLC

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Zhao, J., Patwa, T.H., Pal, M., Qiu, W., Lubman, D.M. (2009). Analysis of Protein Glycosylation and Phosphorylation Using Liquid Phase Separation, Protein Microarray Technology, and Mass Spectrometry. In: Lipton, M.S., Paša-Tolic, L. (eds) Mass Spectrometry of Proteins and Peptides. Methods In Molecular Biology, vol 492. Humana Press. https://doi.org/10.1007/978-1-59745-493-3_20

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  • DOI: https://doi.org/10.1007/978-1-59745-493-3_20

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-934115-48-0

  • Online ISBN: 978-1-59745-493-3

  • eBook Packages: Springer Protocols

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