Screening for high-affinity ligands to the Src SH2 domain using capillary isoelectric focusing-electrospray ionization ion trap mass spectrometry.
A solution-based microscale approach for determination of high-affinity noncovalent complexes from mixtures of compounds is presented, based on capillary isoelectric focusing coupled on-line with electrospray ionization ion trap mass spectrometry. The studies are performed using the src homology 2 domain and tyrosine-phosphorylated peptide ligands as a model system. Tight complexes are formed in solution, preconcentrated up to 2 orders of magnitude and separated on the basis of their isoelectric points. The complexes are then dissociated in the mass spectrometer and the freed ligands identified. Picomole or less amounts of protein reagent are consumed per experiment. Structural information for the ligands involved in tight complex formation may be obtained using the MSn capabilities of the ion trap. The methodology can potentially be used to screen rapidly combinatorial mixtures of compounds for high-affinity ligands.
Lyubarskaya
Y V
YV
Barnett Institute, Northeastern University, Boston, Massachusetts 02115, USA.
Carr
S A
SA
Dunnington
D
D
Prichett
W P
WP
Fisher
S M
SM
Appelbaum
E R
ER
Jones
C S
CS
Karger
B L
BL
D019075:Electrophoresis, Capillary; D007525:Isoelectric Focusing; D013058:Mass Spectrometry; D010455:Peptides / Q000032:analysis*; D019000:Phosphotyrosine / Q000032:analysis*; D013056:Spectrophotometry, Ultraviolet
D010455:Peptides; D019000:Phosphotyrosine