Speaker
Description
Introduction
Protein-centric proteomics is an emerging mass spectrometric field where to answer a biological question related to cells or tissues, one analyzes proteins directly, without relying on their digestion into smaller peptides. The unique ability of protein-centric proteomics to disentangle isoforms and proteoforms is directly beneficial to the complex functional analysis usually needed to answer a given biological question.
Methods
We cleaved the fragment antigen-binding (Fabs) regions from immunoglobulins using a hinge-cleaving protease. We then used reverse phase liquid chromatography (LC) to separate Fabs according to their hydrophobicity. MS analysis was performed on the new timsOmniTM instrument (Bruker) combining the capabilities of a timsTOF with the fragmentation techniques and flexibility of an OmnitrapTM platform. Practically, we analyzed the ~50 kDa Fabs inline relying on MS1 scans to identify precursors belonging to the charge state distribution of a given clone or proteoform. A given charge state was then automatically selected, accumulated, and fragmented using reagent-free electron-based fragmentation techniques. The resulting data was processed using OmniScape.
Preliminary data
We demonstrate that the timsOmni can overcome the limitations of current LC-MS methods in which many species coelute, potentially resulting into congested spectra unsuitable to the analysis of complex samples. Specifically, we use cDDA-ExD method to identify the charge states of a given-mass species characterized, select relevant precursors, and selectively fragment them. The parameters used by the cDDA-ExD method were adapted to sample complexity and chromatographic separation. Under these conditions, we show accurate determination of the masses of the light and heavy chains and good sequence coverage of the hypervariable CDR3 regions of therapeutic recombinant Fabs.
We demonstrate that, focusing on the CDR3 regions, we can generate easy-to-read sequence ladders that provide a unique insight into the complexity of the serum antibody repertoires.
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