Proteomics

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Labeling preferences of diazirines with protein biomolecules


ABSTRACT: Diazirines are widely used in photoaffinity labeling (PAL) to trap non-covalent interactions with biomolecules. However, design and interpretation of PAL experiments is challenging without a molecular understanding of the reactivity of diazirines with protein biomolecules. Here, we report a systematic evaluation of the labeling preferences of alkyl and aryl diazirines with individual amino acids, single proteins, and in the whole cell proteome. We find that alkyl diazirines exhibit preferential labeling of acidic amino acids in a pH-dependent manner that is characteristic of a reactive alkyl diazo intermediate, while aryl-fluorodiazirines labeling patterns reflect reaction primarily through a carbene intermediate. From a survey of 32 alkyl diazirine probes, we use this reactivity profile to rationalize why alkyl diazirine probes preferentially enrich highly acidic proteins or those embedded in membranes and why probes with a net positive-charge tend to produce higher labeling yields in cells and in vitro. These results indicate that alkyl diazirines are an especially effective chemistry for surveying the membrane proteome, and will facilitate design and interpretation of biomolecular labeling experiments with diazirines.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Brain, Permanent Cell Line Cell, Bone Marrow

DISEASE(S): Neuroblastoma

SUBMITTER: Christina Woo  

LAB HEAD: Christina M. Woo

PROVIDER: PXD025140 | Pride | 2022-07-20

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
181213L_SAM04901_HW201_G1-1_CF.raw Raw
181213L_SAM04902_HW201_G1-2_CF.raw Raw
181213L_SAM04903_HW201_G2-1_CF.raw Raw
181213L_SAM04904_HW201_G2-2_CF.raw Raw
181213L_SAM04905_HW201_G3-1_CF.raw Raw
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