Specific capture of phosphopeptides on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry targets modified by magnetic affinity nanoparticles

被引:24
作者
Tan, Feng
Zhang, Yangjun
Wang, Jinglan
Wei, Junying
Qin, Peibing
Cai, Yun
Qian, Xiaohong
机构
[1] Beijing Proteome Res Ctr, State Key Lab Proteom, Beijing 102206, Peoples R China
[2] Beijing Inst Radiat Med, Beijing 100850, Peoples R China
关键词
D O I
10.1002/rcm.3100
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Specific capture of phosphopeptides from protein digests is a critical step for identification of phosphoproteins by mass spectrometry. In this study, we report a novel phosphopeptide-capture approach based on the specific interaction of phosphopeptides with a stainless steel target modified with magnetic affinity nanoparticles. The modification which was carried out by loading the suspension of nanoparticles into sample wells of the target did not require any pretreatment procedure to the target and did not involve chemical binding reactions. To isolate phosphopeptides, digests were loaded into the wells of the modified target for 10 min incubation, followed by rinsing with washing buffer to remove unbound species; matrix was then added to the captured phosphopeptides prior to analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Capturing the phosphopeptides on the modified target simplified significantly analytical operations and reduced sample loss. This approach has been applied to solution digests of alpha-casein, beta-casein, and a mixture of five proteins; a number of phosphopeptides were confidently detected. Phosphopeptides from digests of 10 fmol beta-casein could be isolated and detected by MALDI-TOFMS with this method. In addition, this approach has been applied successfully to the isolation of phosphopeptides from in-gel digestive products of sub-pmol phosphoproteins after separation by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). Copyright (C) 2007 John Wiley & Sons, Ltd.
引用
收藏
页码:2407 / 2414
页数:8
相关论文
共 31 条
[1]   Mass spectrometry-based proteomics [J].
Aebersold, R ;
Mann, M .
NATURE, 2003, 422 (6928) :198-207
[2]   Rapid enrichment of phosphopeptides and phosphoproteins from complex samples using magnetic particles coated with alumina as the concentrating probes for MALDI MS analysis [J].
Chen, Cheng-Tai ;
Chen, Wei-Yu ;
Tsai, Pei-Jane ;
Chien, Kun-Yi ;
Yu, Jau-Song ;
Chen, Yu-Chie .
JOURNAL OF PROTEOME RESEARCH, 2007, 6 (01) :316-325
[3]   Fe3O4/TiO2 core/shell nanoparticles as affinity probes for the analysis of phosphopeptides using TiO2 surface-assisted laser desorption/ionization mass spectrometry [J].
Chen, CT ;
Chen, YC .
ANALYTICAL CHEMISTRY, 2005, 77 (18) :5912-5919
[4]   The origins of protein phosphorylation [J].
Cohen, P .
NATURE CELL BIOLOGY, 2002, 4 (05) :E127-E130
[5]   Detection of phosphopeptides using Fe(III)-nitrilotriacetate complexes immobilized on a MALDI plate [J].
Dunn, JD ;
Watson, JT ;
Bruening, ML .
ANALYTICAL CHEMISTRY, 2006, 78 (05) :1574-1580
[6]   Systematic analysis of the epidermal growth factor receptor by mass spectrometry reveals stimulation-dependent multisite phosphorylation [J].
Erba, EB ;
Bergatto, E ;
Cabodi, S ;
Silengo, L ;
Tarone, G ;
Defilippi, P ;
Jensen, ON .
MOLECULAR & CELLULAR PROTEOMICS, 2005, 4 (08) :1107-1121
[7]   Identification of novel phosphorylation sites on Xenopus laevis aurora A and analysis of phosphopeptide enrichment by immobilized metal-affinity chromatography [J].
Haydon, CE ;
Eyers, PA ;
Aveline-Wolf, LD ;
Resing, KA ;
Maller, JL ;
Ahn, NG .
MOLECULAR & CELLULAR PROTEOMICS, 2003, 2 (10) :1055-1067
[8]   ON TARGET WITH A NEW MECHANISM FOR THE REGULATION OF PROTEIN-PHOSPHORYLATION [J].
HUBBARD, MJ ;
COHEN, P .
TRENDS IN BIOCHEMICAL SCIENCES, 1993, 18 (05) :172-177
[9]   Signaling - 2000 and beyond [J].
Hunter, T .
CELL, 2000, 100 (01) :113-127
[10]   Weighing in on ubiquitin: the expanding role of mass-spectrometry-based proteomics [J].
Kirkpatrick, DS ;
Denison, C ;
Gygi, SP .
NATURE CELL BIOLOGY, 2005, 7 (08) :750-757