Sequential Digestion with Trypsin and Elastase in Cross-Linking Mass Spectrometry.

Analytical Chemistry
Therese DauJuri Rappsilber

Abstract

Cross-linking mass spectrometry has become an important approach for studying protein structures and protein-protein interactions. The amino acid compositions of some protein regions impede the detection of cross-linked residues, although it would yield invaluable information for protein modeling. Here, we report on a sequential-digestion strategy with trypsin and elastase to penetrate regions with a low density of trypsin-cleavage sites. We exploited intrinsic substrate-recognition properties of elastase to specifically target larger tryptic peptides. Our application of this protocol to the TAF4-12 complex allowed us to identify cross-links in previously inaccessible regions.

References

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Citations

Feb 3, 2021·Journal of Proteome Research·Vidur KailashMichael R Hoopmann
Jan 1, 2020·Current Opinion in Biotechnology·Claudio IacobucciAndrea Sinz
May 16, 2019·Journal of Proteome Research·Zheng SerAlex Kentsis
Aug 25, 2020·Journal of Proteome Research·Kathryn R JacobsonSarah Calve
Jan 7, 2020·Analytical Chemistry·Binwen SunFangjun Wang
Nov 21, 2019·Journal of Proteome Research·Petra S J RylJuri Rappsilber

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Methods Mentioned

BETA
ion-exchange chromatography
size-exclusion chromatography

Software Mentioned

Andromeda
Mascot
Xi
xiFDR
ProteoWizard
MaxQuant
CLMS

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