Speaker
Description
Top-down mass spectrometry (TD MS) is a powerful approach for the in-depth analysis of protein modifications, offering detailed, and sometimes unique, insights into intact proteoform structures. As the demand for proteoform-level information grows in biomarker discovery, drug development, and structural biology, there is increasing need for specialized fee-for-service (core) facilities equipped with TD MS instrumentation, advanced data analysis tools, and the expertise required to execute these complex experiments reliably, rapidly, and efficiently.
We present the TD MS workflow implemented in our MS service facility for proteoform-level characterization. As a representative example, we analyzed a set of proteins (18 - 40 kDa) chemically modified on lysine residues with a retinal group, aiming to localize the modification sites. All experiments were performed on an Orbitrap Exploris™ 240 instrument equipped with the Intact Protein Mode and HCD fragmentation.
After intact mass determination, precursor ions across multiple charge states were subjected to multiplexed fragmentation using various normalized collision energies (NCEs). Data were processed using the Peak-by-Peak software (Spectroswiss), enabling comprehensive analysis from RAW files to annotated sequence maps with putative modification localization. The workflow includes isotopically resolved deconvolution (Hardklör algorithm with ±1 Da correction), charge state grouping, spectral recalibration, peak similarity scoring, user-defined modification input, multiplexed result fusion, and final interactive visualization of sequence maps.
We will share our experience and highlight selected user-submitted projects successfully completed using this workflow, demonstrating its value for proteoform analysis in a fee-for-service context. Despite its promise, broader adoption of TD MS in Switzerland remains limited, primarily due to restricted access to TD MS-grade platforms combining high-resolution mass spectrometers with suitable LC configurations, particularly in core facilities focused on peptides or small molecules.
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