Intramolecular cross-linking experiments on cytochrome c and ribonuclease A using an isotope multiplet method

被引:90
作者
Pearson, KM
Pannell, LK
Fales, HM [1 ]
机构
[1] NHLBI, NIH, Bethesda, MD 20892 USA
[2] NIDDKD, NIH, Bethesda, MD 20892 USA
关键词
D O I
10.1002/rcm.554
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Mass spectral analysis of tryptic digests of cross-linked proteins offers considerable promise as a simple technique to probe protein structure and study protein-protein interactions. We describe the use of a 1:1 mixture of isotopically labeled and unlabeled cross-linkers, disuccinimidyladipate (DSA) and dimethyladipimidate (DMA), to enhance visualization of cross-linked peptides in a tryptic digest. Optimized intramolecular reactions of cytochrome c and ribonuclease A (RNase A) with DSA yielded an average of two cross-links per protein molecule. After digestion of the crosslinked cytocbrome c with trypsin and analysis by liquid chromatography/mass spectrometry (LC/MS) and matrix-assisted laser desorption/ionization (MALDI), eight modified peptides, five crosslinked and two end-capped, were detected by virtue of their doublet character. An eighth modified peptide's identity remained ambiguous because of its inability to fragment. The lysine-lysine distance constraints obtained are discussed in the context of the known NMR and X-ray structures of cytochrome c. Analysis of cross-linked RNase A by LC/MS and MALDI yielded nine modified peptides, four of which were modified twice, as indicated by the isotopic triplets. Although seven of these peptides contained cross-links, few distance constraints were gained due to the fact that the cross-linked products were variations of modification of the same three lysine residues. Published in 2001 by John Wiley Sons, Ltd.
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页码:149 / 159
页数:11
相关论文
共 15 条
[1]   Solution structure of oxidized horse heart cytochrome c [J].
Banci, L ;
Bertini, I ;
Gray, HB ;
Luchinat, C ;
Reddig, T ;
Rosato, A ;
Turano, P .
BIOCHEMISTRY, 1997, 36 (32) :9867-9877
[2]   Chemical cross-linking with thiol-cleavable reagents combined with differential mass spectrometric peptide mapping -: A novel approach to assess intermolecular protein contacts [J].
Bennett, KL ;
Kussmann, M ;
Björk, P ;
Godzwon, M ;
Mikkelsen, M ;
Sorensen, P ;
Roepstorff, P .
PROTEIN SCIENCE, 2000, 9 (08) :1503-1518
[3]   CHEMICAL CROSS-LINKING OF ALPHA-SUBUNITS IN THE F1 ADENOSINE-TRIPHOSPHATASE OF ESCHERICHIA-COLI [J].
BRAGG, PD ;
HOU, C .
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 1986, 244 (01) :361-372
[4]   HIGH-RESOLUTION 3-DIMENSIONAL STRUCTURE OF HORSE HEART CYTOCHROME-C [J].
BUSHNELL, GW ;
LOUIE, GV ;
BRAYER, GD .
JOURNAL OF MOLECULAR BIOLOGY, 1990, 214 (02) :585-595
[5]   Protein cross-links: Universal isolation and characterization by isotopic derivatization and electrospray ionization mass spectrometry [J].
Chen, XH ;
Chen, YH ;
Anderson, VE .
ANALYTICAL BIOCHEMISTRY, 1999, 273 (02) :192-203
[6]   CROSS-LINKS BETWEEN RIBOSOMAL-PROTEINS OF 30S SUBUNITS IN 70S TIGHT COUPLES AND IN 30S SUBUNITS [J].
LAMBERT, JM ;
BOILEAU, G ;
COVER, JA ;
TRAUT, RR .
BIOCHEMISTRY, 1983, 22 (16) :3913-3920
[7]   SPATIAL ARRANGEMENT OF GENE-PRODUCTS OF THE P1 OPERON IN THE MEMBRANE OF MYCOPLASMA-PNEUMONIAE [J].
LAYHSCHMITT, G ;
HERRMANN, R .
INFECTION AND IMMUNITY, 1994, 62 (03) :974-979
[8]   Isotope tagged cross linking reagents.: A new tool in mass spectrometric protein interaction analysis [J].
Müller, DR ;
Schindler, P ;
Towbin, H ;
Wirth, U ;
Voshol, H ;
Hoving, S ;
Steinmetz, MO .
ANALYTICAL CHEMISTRY, 2001, 73 (09) :1927-1934
[9]   Structure, subunit topology, and actin-binding activity of the Arp2/3 complex from Acanthamoeba [J].
Mullins, RD ;
Stafford, WF ;
Pollard, TD .
JOURNAL OF CELL BIOLOGY, 1997, 136 (02) :331-343
[10]   A generic strategy to analyze the spatial organization of multi-protein complexes by cross-linking and mass spectrometry [J].
Rappsilber, J ;
Siniossoglou, S ;
Hurt, EC ;
Mann, M .
ANALYTICAL CHEMISTRY, 2000, 72 (02) :267-275