Synthesis of S-adenosyl-L-homocysteine capture compounds for selective photoinduced isolation of methyltransferases.
Understanding the interplay of different cellular proteins and their substrates is of major interest in the postgenomic era. For this purpose, selective isolation and identification of proteins from complex biological samples is necessary and targeted isolation of enzyme families is a challenging task. Over the last years, methods like activity-based protein profiling (ABPP) and capture compound mass spectrometry (CCMS) have been developed to reduce the complexity of the proteome by means of protein function in contrast to standard approaches, which utilize differences in physical properties for protein separation. To isolate and identify the subproteome consisting of S-adenosyl-L-methionine (SAM or AdoMet)-dependent methyltransferases (methylome), we developed and synthesized trifunctional capture compounds containing the chemically stable cofactor product S-adenosyl-L-homocysteine (SAH or AdoHcy) as selectivity function. SAH analogues with amino linkers at the N6 or C8 positions were synthesized and attached to scaffolds containing different photocrosslinking groups for covalent protein modification and biotin for affinity isolation. The utility of these SAH capture compounds for selective photoinduced protein isolation is demonstrated for various methyltransferases (MTases) acting on DNA, RNA and proteins as well as with Escherichia coli cell lysate. In addition, they can be used to determine dissociation constants for MTase-cofactor complexes.
Dalhoff
Christian
C
Caprotec Bioanalytics, GmbH, Volmerstrasse 5, 12489 Berlin (Germany), Fax: (+49) 30-6392-3989.
Hüben
Michael
M
Lenz
Thomas
T
Poot
Peter
P
Nordhoff
Eckhard
E
Köster
Hubert
H
Weinhold
Elmar
E
D003432:Cross-Linking Reagents / Q000737:chemistry; D007700:Kinetics; D008280:Magnetics; D008780:Methyltransferases / Q000302:isolation & purification*; D055668:Photochemical Processes; D012435:S-Adenosylhomocysteine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019809:Streptavidin / Q000737:chemistry / Q000378:metabolism; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D019809:Streptavidin; D012435:S-Adenosylhomocysteine; D008780:Methyltransferases