Levin, N. | Search bioRxiv

bioRxiv2023

Characterisation of an Omnitrap-Orbitrap platform equipped with IRMPD, UVPD and ExD for the analysis of peptides and proteins

We describe an instrument configuration based on the Orbitrap Exploris 480 mass spectrometer that has been coupled to an Omnitrap platform. The Omnitrap possesses three distinct ion-activation regions, that can be used to perform resonant based collision induced dissociation, several forms of electron associated fragmentation, and ultraviolet photodissociation. Each section can also be combined with infrared multiphoton dissociation. In this work, we demonstrate all these modes of operation on a range of peptides and proteins. The results show that this instrument configuration produces similar data to previous implementations of each activation technique and at similar efficiency levels. We demonstrate that this unique instrument configuration is extremely versatile for the interrogation of polypeptides.

systems biology↗

bioRxiv2022

Stereoretentive Post-Translational Protein Editing

Chemical post-translational methods now allow convergent side-chain editing of proteins as a form of direct chemical mutagenesis without needing to resort to genetic intervention. Current approaches that allow the creation of constitutionally native side-chains via C-C formation using off-protein carbon-centred C* radicals added to unnatural amino acid radical acceptor SOMOphile tags such as dehydroalanine are benign and wide-ranging. However, they also typically create epimeric mixtures of D-/L-residues. Here we describe a light-mediated desulfurative method that, through the creation and reaction of stereoretained on-protein L-alanyl C{beta}* radicals, allows C{beta}-H{gamma}, C{beta}-O{gamma}, C{beta}-Se{gamma}, C{beta}-B{gamma} and C{beta}-C{gamma} bond formation to flexibly generate site-selectively edited proteins with full retention of native stereochemistry under mild conditions from a natural amino acid. This methodology shows great potential to explore protein side-chain diversity and construct useful bioconjugates. Table of Contents Image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=32 SRC="FIGDIR/small/504816v1_ufig1.gif" ALT="Figure 1"> View larger version (7K): org.highwire.dtl.DTLVardef@1ccd60corg.highwire.dtl.DTLVardef@f8ca72org.highwire.dtl.DTLVardef@1b33835org.highwire.dtl.DTLVardef@12f32e9_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗