# Enhancing Proteoform Sequence Coverage Using Top-Down Mass Spectrometry with In-Source Fragmentation and Middle-Down Mass Spectrometry

## TL;DR

This paper explores how top-down and middle-down mass spectrometry techniques can improve the sequence coverage of complex proteins.

## Contribution

The study introduces in-source fragmentation and partial digestion to enhance proteoform sequence coverage using top-down and middle-down MS.

## Key findings

- In-source fragmentation improved sequence coverage by generating pseudo-MS3 spectra.
- Short-duration enzymatic digestion in middle-down MS preserved more proteoform sequence information.
- Both methods achieved over 90% sequence coverage for the tested proteins.

## Abstract

The study of complex proteoforms with mutations and post-translational
modifications has gained increasing attention with the advancement
of mass spectrometry (MS)-based techniques. Achieving high proteoform
sequence coverage by MS is essential for accurately characterizing
these complex proteoforms. Extensive efforts have been made to increase
the proteoform sequence coverage using deep bottom-up and top-down
MS strategies. In this study, we evaluated top-down and middle-down
MS approaches for enhancing proteoform sequence coverage using three
proteins: ubiquitin, myoglobin, and carbonic anhydrase II. In the
top-down MS approach, we applied in-source fragmentation (ISF) to
generate pseudo-MS3 spectra, thereby improving sequence
coverage. For the middle-down MS strategy, we performed short-duration
enzymatic digestions to produce longer peptides that preserve more
proteoform sequence information. Our experimental results demonstrated
that ISF and partial digestion significantly increased the sequence
coverage of the proteins, achieving coverage greater than 90%.