Dataset Information

Incorporation of photo-amino acids in HeLa cells

ABSTRACT: In order to assess the efficiency of photo-labeling, total HeLa cell lysates treated with photo-amino acids were subjected to proteome analysis.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Cervix Carcinoma

SUBMITTER: Björn Häupl

LAB HEAD: Andrea Sinz

PROVIDER: PXD005346 | Pride | 2017-04-10

REPOSITORIES: Pride

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Publications

Combining affinity enrichment, cross-linking with photo amino acids, and mass spectrometry for probing protein kinase D2 interactions.

Häupl Björn B Ihling Christian H CH Sinz Andrea A

Proteomics 20170501 10

We present a novel approach that relies on the affinity capture of protein interaction partners from a complex mixture, followed by their covalent fixation via UV-induced activation of incorporated diazirine photoreactive amino acids (photo-methionine and photo-leucine). The captured protein complexes are enzymatically digested and interacting proteins are identified and quantified by label-free LC/MS analysis. Using HeLa cell lysates with photo-methionine and photo-leucine-labeled proteins, we ...[more]

PMID: 28387473

Similar Datasets

Project description:PRIDE > Archive > PXD013420 Project PXD013420 Download Project Files Summary Title A binding site hotspot map of the FKBP12–rapamycin–FRB ternary complex by photo-affinity labeling and mass spectrometry-based proteomics Description Structural characterization of small molecule binding site hotspots within the global proteome is uniquely enabled by photo-affinity labeling (PAL) coupled with chemical enrichment and unbiased analysis by mass spectrometry (MS). MS-based binding site hotspot maps provide structural resolution of interaction sites in conjunction with identification of target proteins. However, binding site hotspot mapping has been confined to relatively simple small molecules to date; extension to more complex compounds would enable the structural definition of new binding modes in the proteome. Here, we extend PAL and MS methods to derive a binding site hotspot map for the immunosuppressant rapamycin, a complex macrocyclic natural product that forms a ternary complex with the proteins FKBP12 and FRB. Photo-rapamycin was developed as a diazirine-based PAL probe for rapamycin, and the FKBP12–photo-rapamycin–FRB ternary complex formed readily in vitro. Photo-irradiation, digestion, and MS analysis of the ternary complex revealed a McLafferty rearrangement product of photo-rapamycin conjugated to specific surfaces on FKBP12 and FRB. Molecular modeling of the ternary complex based on the binding site map revealed a 5.0 Å minimum distance constraint between the conjugated residues and the diazirine carbon. Molecular dynamics further predicted a 9.0 Å labeling radius for the diazirine upon photo-activation that may be useful in the interpretation of binding site measurements from PAL more broadly. Thus, in characterizing the ternary complex of photo-rapamycin by MS, we applied binding site hotspot mapping to a macrocyclic natural product and extracted a precise structural measurement for interpretation of PAL products that may enable the discovery of new ligand space in the “undruggable” proteome.

			Action	DRS
	HeLa_pAA_Rep1.raw	Raw
	HeLa_pAA_Rep1_evidence.txt	Txt
	HeLa_pAA_Rep1_proteinGroups.txt	Txt
	HeLa_pAA_Rep2.raw	Raw
	HeLa_pAA_Rep2_evidence.txt	Txt