# 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 Time Scale

## TL;DR

This study shows how combining different fragmentation methods in mass spectrometry improves the analysis of large proteins and their modified forms.

## Contribution

First integration of multiple fragmentation techniques with CZE-MS/MS to enhance proteoform analysis in a wide mass range.

## Key findings

- Combining HCD, ETD, EThcD, and UVPD increased sequence coverage for carbonic anhydrase and thioredoxin.
- Internal fragments significantly boosted cleavage coverage for proteins like carbonic anhydrase and protein AG.

## 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 an
Orbitrap Ascend Tribrid 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 time scale 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 67 and 98% sequence coverage for carbonic
anhydrase (a 30 kDa protein) and thioredoxin (a 12 kDa protein), which
is 158 and 100% higher than that produced by HCD alone. Adding internal
fragments further boosted the backbone cleavage coverage substantially,
for example, from 67 to 94% for 30 kDa carbonic anhydrase and from
21 to 82% 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.