Detection and structural characterization of ras oncoprotein-inhibitors complexes by electrospray mass spectrometry.
MS based methodology employing electrospray ionization (ESI) is described for the detection of ternary complexes in which SCH 54292 or SCH 54341 and GDP are noncovalently bound to oncogenic ras protein. The observed molecular weights of 19,816 and 19,570 Da confirmed the presence of noncovalent complexes of ras-GDP-SCH 54292 and ras-GDP-SCH 54341, respectively. We have also performed selective chemical modification of lysine residues of the ras protein complex followed by enzymatic digestion and on-line LC-ESI MS peptide mapping to determine protein-drug binding topography. There was a good correlation between nucleotide exchange inhibition as determined by the enzyme assay and evidence of complex formation as determined by MS.
Ganguly
A K
AK
Schering-Plough Research Institute, Kenilworth, NJ 07033, USA.
Pramanik
B N
BN
Huang
E C
EC
Liberles
S
S
Heimark
L
L
Liu
Y H
YH
Tsarbopoulos
A
A
Doll
R J
RJ
Taveras
A G
AG
Remiszewski
S
S
Snow
M E
ME
Wang
Y S
YS
Vibulbhan
B
B
Cesarz
D
D
Brown
J E
JE
del Rosario
J
J
James
L
L
Kirschmeier
P
P
Girijavallabhan
V
V
D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology; D001665:Binding Sites; D004353:Drug Evaluation, Preclinical; D005960:Glucosides / Q000737:chemistry / Q000494:pharmacology; D020662:Guanine Nucleotide Exchange Factors; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D011506:Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry; D013449:Sulfonamides / Q000737:chemistry / Q000494:pharmacology; D020663:ras Guanine Nucleotide Exchange Factors; D018631:ras Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics
D000970:Antineoplastic Agents; D005960:Glucosides; D020662:Guanine Nucleotide Exchange Factors; D046911:Macromolecular Substances; D011506:Proteins; C107861:SCH 54292; C107862:SCH 54341; D013449:Sulfonamides; D020663:ras Guanine Nucleotide Exchange Factors; D018631:ras Proteins