Direct monitoring of protein-chemical reactions utilising nanoelectrospray mass spectrometry

被引:32
作者
Fligge, TA [1 ]
Kast, J [1 ]
Bruns, K [1 ]
Przybylski, M [1 ]
机构
[1] Univ Konstanz, Fak Chem, D-78457 Constance, Germany
关键词
D O I
10.1016/S1044-0305(98)00131-7
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
The feasibility of nanoelectrospray mass spectrometry (nanoESI) for the direct analysis of protein chemical reactions and structural changes of proteins has been evaluated. Taking advantage of the long spraying time and the capability of nanoESI for employing a wide range of solvent conditions such as buffers and detergents, applications of monitoring reaction pathways, and dynamics have been carried out with several peptides and proteins. The time course of proteolytic digestions with trypsin and pepsin was investigated for several model polypeptides, and nanoESI showed to provide an efficient tool for optimising digestion conditions for the mass spectrometric peptide mapping analysis. Examples of specific protein chemical modification reactions at arginine and tyrosine residues illustrate the feasibility of nanoESI to monitoring reaction yields and modification sites for more than 180 min. Furthermore, changes of the pattern of protonated molecules caused by temperature effects and by protein unfolding due to disulfide bond reduction have been studied with the model proteins cytochrome c and hen eggwhite lysozyme. The results indicate that nanoESI is an efficient technique for the direct, molecular characterisation of protein-chemical reactions in solution. (J Am Soc Mass Spectrom 1999, 10, 112-118) (C) 1999 American Society for Mass Spectrometry.
引用
收藏
页码:112 / 118
页数:7
相关论文
共 35 条
[1]   Characterization of the structural difference between active and inactive forms of the Ras protein by chemical modification followed by mass spectrometric peptide mapping [J].
Akashi, S ;
Shirouzu, M ;
Terada, T ;
Ito, Y ;
Yokoyama, S ;
Takio, K .
ANALYTICAL BIOCHEMISTRY, 1997, 248 (01) :15-25
[2]   DESIGN AND PERFORMANCE OF A NOVEL ELECTROSPRAY INTERFACE [J].
ALLEN, MH ;
VESTAL, ML .
JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY, 1992, 3 (01) :18-26
[3]  
Bosshard H R, 1979, Methods Biochem Anal, V25, P273, DOI 10.1002/9780470110454.ch4
[4]   Disulfide bonds in protein folding studies: friends or foes? [J].
Dadlez, M .
ACTA BIOCHIMICA POLONICA, 1997, 44 (03) :433-452
[5]  
DOBRYSZYCKA W, 1975, ACTA BIOCHIM POL, V22, P143
[6]   ELECTROSPRAY IONIZATION FOR MASS-SPECTROMETRY OF LARGE BIOMOLECULES [J].
FENN, JB ;
MANN, M ;
MENG, CK ;
WONG, SF ;
WHITEHOUSE, CM .
SCIENCE, 1989, 246 (4926) :64-71
[7]   Molecular characterization of a conformational epitope of hen egg white lysozyme by differential chemical modification of immune complexes and mass spectrometric peptide mapping [J].
Fiedler, W ;
Borchers, C ;
Macht, M ;
Deininger, SO ;
Przybylski, M .
BIOCONJUGATE CHEMISTRY, 1998, 9 (02) :236-241
[8]   Analytical development of electrospray and nanoelectrospray mass spectrometry in combination with liquid chromatography for the characterization of proteins [J].
Fligge, TA ;
Bruns, K ;
Przybylski, M .
JOURNAL OF CHROMATOGRAPHY B, 1998, 706 (01) :91-100
[9]  
FLIGGE TA, 1997, P 14 INT MASS SPECTR
[10]   MOLECULAR CHARACTERIZATION OF SURFACE-TOPOLOGY IN PROTEIN TERTIARY STRUCTURES BY AMINO-ACYLATION AND MASS-SPECTROMETRIC PEPTIDE-MAPPING [J].
GLOCKER, MO ;
BORCHERS, C ;
FIEDLER, W ;
SUCKAU, D ;
PRZYBYLSKI, M .
BIOCONJUGATE CHEMISTRY, 1994, 5 (06) :583-590