Extensive backbone cleavage coverage of intact proteoforms in a mass range of 10-70 kDa by integrating electron, collision, and photon-based fragmentation techniques during an electrophoretic timescale

ABSTRACT: Capillary zone electrophoresis (CZE) –-tandem mass spectrometry (MS/MS) has been documented as a useful tool for top-down proteomics (TDP). However, CZE-MS/MS-based TDP typically has limited backbone cleavage coverage for identified proteoforms due to the use of traditional collision-based fragmentation methods (i.e., higher-energy collisional dissociation, HCD). Here, for the first time, we coupled CZE to a high-end Orbitrap Ascend mass spectrometer to investigate the performance of collision, electron, and photon-based fragmentation methods and their combinations for boosting the backbone cleavage coverage of proteoforms during the electrophoretic timescale using a standard protein mixture covering a mass range of about 10-70 kDa. CZE-MS achieved reproducible measurement of six proteins, including three insulin-like growth factor (IGF) proteoforms with different modifications. Systematic investigations of HCD, electron-transfer dissociation (ETD), electron-transfer/HCD (EThcD), and ultraviolet photodissociation (UVPD) during CZE-MS/MS analysis revealed distinct yet complementary fragmentation characteristics. ETD, EThcD, and UVPD, in general, provided higher backbone cleavage coverage than HCD. The integration of HCD, ETD, EThcD, and UVPD data offered a 74% and 98% sequence coverage for carbonic anhydrase (a 30-kDa protein) and thioredoxin (a 12-kDa protein), which is 185% and 100% higher than that produced by HCD alone. Adding internal fragments further boosted the backbone cleavage coverage substantially, for example, from 74% to 94% for 30-kDa carbonic anhydrase, and from 35% to 94% for 50-kDa Protein AG. The results demonstrate the capability of CZE-MS/MS with the integration of various fragmentation techniques for comprehensive characterization of proteoforms in a wide mass range.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Qianjie Wang  

LAB HEAD: Qianjie Wang

PROVIDER: PXD070486 | Pride | 2026-02-16

REPOSITORIES: Pride