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. Author manuscript; available in PMC: 2021 Oct 20.
Published in final edited form as: Anal Chem. 2020 Oct 6;92(20):14094–14102. doi: 10.1021/acs.analchem.0c03128

Figure 3.

Figure 3.

Experimental overview of iqPIR application to cross-linked protein standards and 17-AAG treated HeLa cells. (A) Cartoon depiction of the RH and SH iqPIR cross-linkers illustrating the primary molecular features including MS-labile bonds, amine reactive groups, location of 13C isotopes (red & blue circles) and biotin affinity tag. (B) Preparation of iqPIR cross-linked standard protein samples consisting of ADH1_YEAST, ALBU_BOVIN, and MYG_HORSE mixed at various RH/SH ratios as indicated, well as an in vivo cross-linked 17-AAG treated vs vehicle (0.1% DMSO) HeLa cell sample. (C) Example total ion chromatogram representing LC–MS/MS analysis of cross-linked peptide pair samples. (D) Data analysis consisted of a Comet database search, XLinkProphet FDR estimation and filtering, quantification of relative abundance of fragment ions in the MS2 spectra and upload of data to XLinkDB.