Greisch, J.-F. | Search bioRxiv

bioRxiv2025

Activation of oligonucleotide polyanions using collisions, electrons and photons in a timsOmni platform

We describe here various ion activation experiments realized in the Omnitrap platform integrated on the timsOmniTM mass spectrometer for the analysis of oligonucleotides in the negative ion mode. The activation methods include resonance collision-induced dissociation (RCID), electron detachment dissociation (EDD), infrared laser multiple-photon activation (IRMPD) and UV laser photodissociation (UVPD). Special emphasis is given to EDD, either as a standalone technique or in conjunction with vibrational re-activation of the ion radicals. We describe EDD on standard 6-mer DNA sequences that have been extensively characterized on other instruments, followed by a comparison of several activation approaches for the phosphorothioate-based oligonucleotide therapeutics Fomivirsen, and concluding with the fragmentation analysis of 46-mer DNA and RNA. EDD alone already provides excellent sequence information on Fomivirsen, but MS3 combinations such as EDD-RCID or EDD-IRMPD proved even more effective, including for the 46-mer DNA (less prone to fragmentation than RNA) at a relatively low charge state. The diversity of ion activation combinations available on the Omnitrap platform is demonstrated by an MS4 experiment investigating the fate of a* and z* radical fragments produced by EDD. TOC graphics O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/687689v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@534c32org.highwire.dtl.DTLVardef@af8ab0org.highwire.dtl.DTLVardef@ba2c3dorg.highwire.dtl.DTLVardef@ae9196_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

Benefits of Collisional Cross Section Assisted Precursor Selection (caps-PASEF) for Cross-linking Mass Spectrometry

Ion mobility separates molecules in the gas-phase on their physico-chemical properties, providing information about their size as collisional cross-sections. The timsTOF Pro combines trapped ion mobility with a quadrupole, collision cell and a time-of-flight mass analyzer, to probe ions at high speeds with on-the-fly fragmentation. Here, we show that on this platform ion mobility is beneficial for cross-linking mass spectrometry (XL-MS). Cross-linking reagents covalently link amino acids in close proximity, resulting in peptide pairs after proteolytic digestion. These cross-linked peptides are typically present at low abundance in the background of normal peptides, which can partially be resolved by using enrichable cross-linking reagents. Even with a very efficient enrichable cross-linking reagent, like PhoX, the analysis of cross-linked peptides is still hampered by the co-enrichment of peptides connected to a partially hydrolyzed reagent - termed mono-linked peptides. For experiments aiming to uncover protein-protein interactions these are unwanted byproducts. Here, we demonstrate that gas-phase separation by ion mobility enables the separation of mono-linked peptides from cross-linked peptide pairs. A clear partition between these two classes is observed at a CCS of 500 [A]2 and a monoisotopic mass of 2 kDa, which can be used for targeted precursor selection. A total of 50 - 70% of the mono-linked peptides are prevented from sequencing, allowing the analysis to focus on sequencing the relevant cross-linked peptide pairs. In applications to both simple proteins and protein mixtures and a complete highly complex lysate this approach provides a substantial increase in detected cross-linked peptides.

biochemistry↗