Speaker
Description
Although the characterization of intact proteins remains a demanding endeavour – particularly as protein size increases - ongoing technological/bioinformatic advancements are steadily enhancing the capabilities of top-down mass spectrometry (TDMS) for deep sequencing. Different ion activation techniques – such as electron transfer dissociation (ETD), electron transfer higher-energy collisional dissociation (EThcD), and ultraviolet photodissociation (UVPD) available on the Thermo Scientific Orbitrap Tribrid MS platform - can provide complementary fragmentation patterns, and when combined, offer elevated sequence coverage. Despite the advancements in fragmentation techniques, data interpretation remains challenging due to extensive overlapping of ion signals, which compromises product ion matching. Herein, we present strategies for resolving spectral ambiguities, implemented on the Orbitrap Ascend BioPharma MS.
Proteins in the 8-46 kDa Mw range were interrogated with higher-energy collisional dissociation (HCD), ETD, EThcD, and UVPD. Data collection was carried out using a range of resolving powers (RP). A gradual increase in coverage was observed moving from an RP of 60,000 towards 480,000 in all cases - for enolase reaching 53% from the initial 14% yielded by lower resolution. However, high RP alone could not effectively resolve spectral congestion due to fragment ions signals being restricted to m/z < 2000. Additional increase in coverage (~8-15%) could only be achieved with proton transfer charge reduction (PTCR), due to its ability to distribute the product ion population over a broader m/z range (up to m/z 8000). The PTCR MS3 workflow yielded a coverage of 98% for carbonic anhydrase (~29 kDa) when results from all tested fragmentation techniques were combined.
To evaluate the approach under chromatographic time constraints, we applied the workflow for the middle-down analysis of mAb and antibody-drug conjugate (ADC) ~25 kDa subunits. PTCR MS3 enabled not only increased coverage (87% for Fd’ subunit) but also the unambiguous localization of payload conjugation sites for the ADC.
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