Combining metal oxide affinity chromatography (MOAC) and selective mass spectrometry for robust identification of in vivo protein phosphorylation sites

Plant Methods

Wolfram Weckwerth¹  Florian Wolschin¹ 

[1] Max Planck Institute of Molecular Plant Physiology, 14424 Potsdam, Germany

关键词: MS³;    Neutral loss;    MOAC;    IMAC;    Seeds;    Nano-ESI;    Protein phosphorylation;   

Others  :  823154
DOI  :  10.1186/1746-4811-1-9

 received in 2005-08-27, accepted in 2005-11-01,  发布年份 2005

【 摘 要 】

Background

Protein phosphorylation is accepted as a major regulatory pathway in plants. More than 1000 protein kinases are predicted in the Arabidopsis proteome, however, only a few studies look systematically for in vivo protein phosphorylation sites. Owing to the low stoichiometry and low abundance of phosphorylated proteins, phosphorylation site identification using mass spectrometry imposes difficulties. Moreover, the often observed poor quality of mass spectra derived from phosphopeptides results frequently in uncertain database hits. Thus, several lines of evidence have to be combined for a precise phosphorylation site identification strategy.

Results

Here, a strategy is presented that combines enrichment of phosphoproteins using a technique termed metaloxide affinity chromatography (MOAC) and selective ion trap mass spectrometry. The complete approach involves (i) enrichment of proteins with low phosphorylation stoichiometry out of complex mixtures using MOAC, (ii) gel separation and detection of phosphorylation using specific fluorescence staining (confirmation of enrichment), (iii) identification of phosphoprotein candidates out of the SDS-PAGE using liquid chromatography coupled to mass spectrometry, and (iv) identification of phosphorylation sites of these enriched proteins using automatic detection of H₃PO₄ neutral loss peaks and data-dependent MS³-fragmentation of the corresponding MS²-fragment. The utility of this approach is demonstrated by the identification of phosphorylation sites in Arabidopsis thaliana seed proteins. Regulatory importance of the identified sites is indicated by conservation of the detected sites in gene families such as ribosomal proteins and sterol dehydrogenases. To demonstrate further the wide applicability of MOAC, phosphoproteins were enriched from Chlamydomonas reinhardtii cell cultures.

Conclusion

A novel phosphoprotein enrichment procedure MOAC was applied to seed proteins of A. thaliana and to proteins extracted from C. reinhardtii. Thus, the method can easily be adapted to suit the sample of interest since it is inexpensive and the components needed are widely available. Reproducibility of the approach was tested by monitoring phosphorylation sites on specific proteins from seeds and C. reinhardtii in duplicate experiments. The whole process is proposed as a strategy adaptable to other plant tissues providing high confidence in the identification of phosphoproteins and their corresponding phosphorylation sites.

【 授权许可】

   
2005 Wolschin and PWeckwerth; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

【 参考文献 】

• [1]Agrawal GK, Yonekura M, Iwahashi Y, Iwahashi H, Rakwal R: System, trends and perspectives of proteomics in dicot plants Part II: Proteomes of the complex developmental stages. J Chromatogr B Analyt Technol Biomed Life Sci 2005, 815:125-136.
• [2]Rubin CS, Rosen OM: Protein phosphorylation. Annu Rev Biochem 1975, 44:831-887.
• [3]Ma H: Protein phosphorylation in plants: enzymes, substrates and regulators. Trends Genet 1993, 9:228-230.
• [4]Glinski M, Weckwerth W: The Role of Mass Spectrometry in Plant Biology. Mass Spectrometry Reviews 2005., in press
• [5]Glinski M, Romeis T, Witte CP, Wienkoop S, Weckwerth W: Stable isotope labeling of phosphopeptides for multiparallel kinase target analysis and identification of phosphorylation sites. Rapid Commun Mass Spectrom 2003, 17:1579-1584.
• [6]Glinski M, Weckwerth W: Differential Multisite Phosphorylation of the Trehalose-6-phosphate Synthase Gene Family in Arabidopsis thaliana: A Mass Spectrometry-based Process for Multiparallel Peptide Library Phosphorylation Analysis. Mol Cell Proteomics 2005, 4:1614-1625.
• [7]Martin K, Steinberg TH, Goodman T, Schulenberg B, Kilgore JA, Gee KR, Beechem JM, Patton WF: Strategies and solid-phase formats for the analysis of protein and peptide phosphorylation employing a novel fluorescent phosphorylation sensor dye. Comb Chem High Throughput Screen 2003, 6:331-339.
• [8]Schulenberg B, Goodman TN, Aggeler R, Capaldi RA, Patton WF: Characterization of dynamic and steady-state protein phosphorylation using a fluorescent phosphoprotein gel stain and mass spectrometry. Electrophoresis 2004, 25:2526-2532.
• [9]Schulenberg B, Arnold B, Patton WF: An improved mechanically durable electrophoresis gel matrix that is fully compatible with fluorescence-based protein detection technologies. Proteomics 2003, 3:1196-1205.
• [10]Steinberg TH, Agnew BJ, Gee KR, Leung WY, Goodman T, Schulenberg B, Hendrickson J, Beechem JM, Haugland RP, Patton WF: Global quantitative phosphoprotein analysis using Multiplexed Proteomics technology. Proteomics 2003, 3:1128-1144.
• [11]Goodman T, Schulenberg B, Steinberg TH, Patton WF: Detection of phosphoproteins on electroblot membranes using a small-molecule organic fluorophore. Electrophoresis 2004, 25:2533-2538.
• [12]Salih E: Phosphoproteomics by mass spectrometry and classical protein chemistry approaches. Mass Spectrom Rev 2004, DOI 10.1002/mas.20042.
• [13]Zeller M, Konig S: The impact of chromatography and mass spectrometry on the analysis of protein phosphorylation sites. Anal Bioanal Chem 2004, 378:898-909.
• [14]Laugesen S, Bergoin A, Rossignol M: Deciphering the plant phosphoproteome: tools and strategies for a challenging task. Plant Physiol Biochem 2004, 42:929-936.
• [15]Nuhse TS, Stensballe A, Jensen ON, Peck SC: Phosphoproteomics of the Arabidopsis plasma membrane and a new phosphorylation site database. Plant Cell 2004, 16:2394-2405.
• [16]Aro EM, Rokka A, Vener AV: Determination of phosphoproteins in higher plant thylakoids. Methods Mol Biol 2004, 274:271-285.
• [17]Heintz D, Wurtz V, High AA, Van Dorsselaer A, Reski R, Sarnighausen E: An efficient protocol for the identification of protein phosphorylation in a seedless plant, sensitive enough to detect members of signalling cascades. Electrophoresis 2004, 25:1149-1159.
• [18]Wolschin F, Wienkoop S, Weckwerth W: Enrichment of phosphorylated proteins and peptides from complex mixtures using metal oxide/hydroxide affinity chromatography (MOAC). Proteomics 2005., [Epub ahead of print]
• [19]Weckwerth W, Willmitzer L, Fiehn O: Comparative quantification and identification of phosphoproteins using stable isotope labeling and liquid chromatography/mass spectrometry. Rapid Communications in Mass Spectrometry 2000, 14:1677-1681.
• [20]Jaffe H, Veeranna, Pant HC: Characterization of serine and threonine phosphorylation sites in beta-elimination/ethanethiol addition-modified proteins by electrospray tandem mass spectrometry and database searching. Biochemistry 1998, 37:16211-16224.
• [21]McLachlin DT, Chait BT: Improved beta-elimination-based affinity purification strategy for enrichment of phosphopeptides. Anal Chem 2003, 75:6826-6836.
• [22]Goshe MB, Conrads TP, Panisko EA, Angell NH, Veenstra TD, Smith RD: Phosphoprotein isotope-coded affinity tag approach for isolating and quantitating phosphopeptides in proteome-wide analyses. Anal Chem 2001, 73:2578-2586.
• [23]Klemm C, Schroder S, Gluckmann M, Beyermann M, Krause E: Derivatization of phosphorylated peptides with S- and N-nucleophiles for enhanced ionization efficiency in matrix-assisted laser desorption/ionization mass spectrometry. Rapid Communications in Mass Spectrometry 2004, 18:2697-2705.
• [24]Syka JE, Coon JJ, Schroeder MJ, Shabanowitz J, Hunt DF: Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proc Natl Acad Sci U S A 2004, 101:9528-9533.
• [25]DeGnore JP, Qin J: Fragmentation of phosphopeptides in an ion trap mass spectrometer. J Am Soc Mass Spectrom 1998, 9:1175-1188.
• [26]Olsen JV, Mann M: Improved peptide identification in proteomics by two consecutive stages of mass spectrometric fragmentation. Proc Natl Acad Sci U S A 2004, 101:13417-13422.
• [27]Giorgianni F, Beranova-Giorgianni S, Desiderio DM: Identification and characterization of phosphorylated proteins in the human pituitary. Proteomics 2004, 4:587-598.
• [28]Jin WH, Dai J, Zhou H, Xia QC, Zou HF, Zeng R: Phosphoproteome analysis of mouse liver using immobilized metal affinity purification and linear ion trap mass spectrometry. Rapid Commun Mass Spectrom 2004, 18:2169-2176.
• [29]Shu H, Chen S, Bi Q, Mumby M, Brekken DL: Identification of phosphoproteins and their phosphorylation sites in the WEHI-231 B lymphoma cell line. Mol Cell Proteomics 2004, 3:279-286.
• [30]Medzihradszky KF, Darula Z, Perlson E, Fainzilber M, Chalkley RJ, Ball H, Greenbaum D, Bogyo M, Tyson DR, Bradshaw RA, Burlingame AL: O-sulfonation of serine and threonine: mass spectrometric detection and characterization of a new posttranslational modification in diverse proteins throughout the eukaryotes. Mol Cell Proteomics 2004, 3:429-440.
• [31]Lin LJ, Tzen JT: Two distinct steroleosins are present in seed oil bodies. Plant Physiol Biochem 2004, 42:601-608.
• [32]Wolschin F, Lehmann U, Glinski M, Weckwerth. W: An integrated strategy for identification and relative quantification of site-specific protein phosphorylation using liquid chromatography coupled to MS2/MS3. Rapid Commun Mass Spectrom 2005., in press
• [33]Borisjuk L, Rolletschek H, Radchuk R, Weschke W, Wobus U, Weber H: Seed development and differentiation: A role for metabolic regulation. Plant Biology 2004, 6:375-386.
• [34]Walker-Simmons MK: Protein kinases in seeds. Seed Science Research 1998, 8:193-200.
• [35]Wysocki VH, Tsaprailis G, Smith LL, Breci LA: Mobile and localized protons: a framework for understanding peptide dissociation. J Mass Spectrom 2000, 35:1399-1406.
• [36]Salek M, Di Bartolo V, Cittaro D, Borsotti D, Wei J, Acuto O, Rappsilber J, Lehmann WD: Sequence tag scanning: a new explorative strategy for recognition of unexpected protein alterations by nanoelectrospray ionization-tandem mass spectrometry. Proteomics 2005, 5:667-674.
• [37]Samaj J, Baluska F, Hirt H: From signal to cell polarity: mitogen-activated protein kinases as sensors and effectors of cytoskeleton dynamicity. J Exp Bot 2004, 55:189-198.
• [38]Schmid M, Davison TS, Henz SR, Pape UJ, Demar M, Vingron M, Scholkopf B, Weigel D, Lohmann JU: A gene expression map of Arabidopsis thaliana development. Nat Genet 2005, 37:501-506.
• [39]Weckwerth W, Selbig J: Scoring and identifying organism-specific functional patterns and putative phosphorylation sites in protein sequences using mutual information. Biochem Bioph Res Co Biochem Bioph Res Co 2003, 307:516-521.
• [40]Rodriguez-Gabriel MA, Bou G, Briones E, Zambrano R, Remacha M, Ballesta JP: Structure and function of the stalk, a putative regulatory element of the yeast ribosome. Role of stalk protein phosphorylation. Folia Microbiol (Praha) 1999, 44:153-163.
• [41]Nusspaumer G, Remacha M, Ballesta JP: Phosphorylation and N-terminal region of yeast ribosomal protein P1 mediate its degradation, which is prevented by protein P2. Embo J 2000, 19:6075-6084.
• [42]Lin LJ, Tai SS, Peng CC, Tzen JT: Steroleosin, a sterol-binding dehydrogenase in seed oil bodies. Plant Physiol 2002, 128:1200-1211.
• [43]Harris EH: The Chlamydomonas Sourcebook. Academic Press, Inc, San Diego, CA, USA 1989.
• [44]Wessel D, Flugge UI: A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 1984, 138:141-143.
• [45]Bradford MM: Rapid and Sensitive Method for Quantitation of Microgram Quantities of Protein Utilizing Principle of Protein-Dye Binding. Analytical Biochemistry 1976, 72:248-254.
• [46]Otto A, Thiede B, Muller EC, Scheler C, WittmannLiebold B, Jungblut P: Identification of human myocardial proteins separated by two-dimensional electrophoresis using an effective sample preparation for mass spectrometry. Electrophoresis 1996, 17:1643-1650.