Quantitative analysis of protein phosphorylation in mouse brain by hypothesis-driven multistage mass spectrometry

被引:27
作者
Jin, M [1 ]
Bateup, H [1 ]
Padovan, JC [1 ]
Greengard, P [1 ]
Nairn, AC [1 ]
Chait, BT [1 ]
机构
[1] Rockefeller Univ, New York, NY 10021 USA
关键词
D O I
10.1021/ac051519m
中图分类号
O65 [分析化学];
学科分类号
070302 ; 081704 ;
摘要
Determination of site-specific changes in the levels of protein phosphorylation in mammals presents a formidable analytical challenge. Here, we demonstrate a strategy for such analyses utilizing a combination of stable isotope chemical labeling and tandem mass spectrometry. Phosphoproteins of interest are isolated from two sets of animals that have undergone differential drug treatments, separated by SDS-PAGE, excised, and subjected to in-gel enzymatic digestion. Using a simple chemical labeling step, we introduce stable, isotopically distinct mass tags into each of the two sets of peptides that originate from the samples under comparison, mix the samples, and subject the resulting mixture to a procedure based on our previously reported hypothesis-driven multistage MS (HMS-MS) method (Chang, E. J.; Archambault, V.; McLachlin, D. T.; Krutchinsky, A. N.; Chait, B. T. Anal. Chem. 2004, 76, 4472-4483). The method takes advantage of the dominant loss of H3PO4 during MS/MS from singly charged phosphopeptide ions produced by matrix-assisted laser desorption/ionization (MALDI) in the ion trap mass spectrometer. In the present work, quantitation is achieved by isolating the range of m/z values that include both isotopic forms of the putative phosphopeptide and measuring the relative intensities of the two resulting -98-Da fragment ion peaks. This MS/MS measurement can be repeated on the same MALDI sample for all potential phosphopeptide ion pairs that we hypothesize might be produced from the protein under study. Use of MS/MS for quantitation greatly increases the sensitivity of the method and allows us to measure relatively low levels of phosphorylation, phosphopeptides, We apply the current method to the determination of changes in the levels of phosphorylation in DARPP-32 from the mouse striatum upon treatment of animals with psychostimulant drugs.
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收藏
页码:7845 / 7851
页数:7
相关论文
共 27 条
[1]   Mass spectrometry-based proteomics [J].
Aebersold, R ;
Mann, M .
NATURE, 2003, 422 (6928) :198-207
[2]   Selective detection of membrane proteins without antibodies - A mass spectrometric version of the Western blot [J].
Arnott, D ;
Kishiyama, A ;
Luis, EA ;
Ludlum, SG ;
Marsters, JC ;
Stults, JT .
MOLECULAR & CELLULAR PROTEOMICS, 2002, 1 (02) :148-156
[3]   High-throughput global peptide proteomic analysis by combining stable isotope amino acid labeling and data-dependent multiplexed-MS/MS [J].
Berger, SJ ;
Lee, SW ;
Anderson, GA ;
Pasa-Tolic, L ;
Tolic, N ;
Shen, YF ;
Zhao, R ;
Smith, RD .
ANALYTICAL CHEMISTRY, 2002, 74 (19) :4994-5000
[4]  
Boyle WJ., 1991, METHOD ENZYMOL, V201, P110
[5]  
Brancia FL, 2000, RAPID COMMUN MASS SP, V14, P2070, DOI 10.1002/1097-0231(20001115)14:21<2070::AID-RCM133>3.0.CO
[6]  
2-G
[7]   Analysis of protein phosphorylation by hypothesis-driven multiple-stage mass spectrometry [J].
Chang, EJ ;
Archambault, V ;
McLachlin, DT ;
Krutchinsky, AN ;
Chait, BT .
ANALYTICAL CHEMISTRY, 2004, 76 (15) :4472-4483
[8]   Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae [J].
Ficarro, SB ;
McCleland, ML ;
Stukenberg, PT ;
Burke, DJ ;
Ross, MM ;
Shabanowitz, J ;
Hunt, DF ;
White, FM .
NATURE BIOTECHNOLOGY, 2002, 20 (03) :301-305
[9]   Analysis of protein phosphorylation by mass spectrometry [J].
Garcia, BA ;
Shabanowitz, J ;
Hunt, DF .
METHODS, 2005, 35 (03) :256-264
[10]   Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway [J].
Gruhler, A ;
Olsen, JV ;
Mohammed, S ;
Mortensen, P ;
Færgeman, NJ ;
Mann, M ;
Jensen, ON .
MOLECULAR & CELLULAR PROTEOMICS, 2005, 4 (03) :310-327