Analysis of Protein Glycosylation and Phosphorylation Using Liquid Phase Separation, Protein Microarray Technology, and Mass Spectrometry
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.
Key wordsGlycosylation Phosphorylation Posttranslational modification Protein microarrays Liquid phase separation Lectin Mass spectrometry
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.
- 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.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.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.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.CrossRefPubMedGoogle Scholar