RT Journal Article
SR Electronic
T1 PhoX – an IMAC-enrichable Crosslinking Reagent
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 556688
DO 10.1101/556688
A1 Steigenberger, Barbara A.
A1 Pieters, Roland J.
A1 Heck, Albert J.R.
A1 Scheltema, Richard A.
YR 2019
UL http://biorxiv.org/content/early/2019/02/21/556688.abstract
AB Chemical crosslinking mass spectrometry is rapidly emerging as a prominent technique to study protein structures. Structural information is obtained by covalently connecting peptides in close proximity by small reagents and identifying the resulting peptide pairs by mass spectrometry. However, sub-stoichiometric reaction efficiencies render routine detection of crosslinked peptides problematic. Here we present a new tri-functional crosslinking reagent, termed PhoX, which is decorated with a stable phosphonic acid handle. This makes the crosslinked peptides amenable to the well-established IMAC enrichment. The handle allows for 300x enrichment efficiency and 97% specificity, dramatically reducing measurement time and improving data quality. We exemplify the approach on various model proteins and protein complexes, e.g. resulting in a structural model of the LRP1/RAP complex. PhoX is also applicable to whole cell lysates. When focusing the database search on ribosomal proteins, our first attempt resulted in 355 crosslinks, out-performing current efforts in less measurement time.