# On the Utility of Infrared Photoactivation for Native Top-Down and Complex-Down Orbitrap Mass Spectrometry of Soluble Proteoform Complexes

## TL;DR

This paper explores how infrared activation improves mass spectrometry analysis of protein complexes, enhancing the ability to sequence large proteoform subunits.

## Contribution

The study introduces infrared activation as a new and complementary method for improving proteoform sequencing in native and complex-down mass spectrometry.

## Key findings

- Infrared activation (IRMPD) and AI-ETD provided comparable or better sequence coverage than HCD for proteoform subunits.
- For larger subunits like pyruvate kinase, AI-ETD outperformed HCD and IRMPD by approximately 15% in sequence coverage.
- Combining IR activation with electron-based methods enhanced overall sequence coverage and proteoform characterization.

## Abstract

Cellular functions arise from the coordinated action
of proteoforms,
which typically form multiproteoform complexes (MPCs), rather than
functioning as isolated molecular entities. Deciphering the architecture
and composition of MPCs is essential for linking proteoform diversity
to biological function. Native top-down (nTD MS) and complex-down
mass spectrometry (CxD MS) have emerged as powerful strategies to
characterize MPCs, offering intact mass analysis as well as gas-phase
sequencing either at the level of the complete assembly or its constituent
proteoform subunits. Because the attainable sequence coverage is highly
influenced by the ion activation technique, expanding activation strategies
is key to improving proteoform characterization. To this end, we implemented
infrared (IR) activation for the analysis of soluble MPCsalcohol
dehydrogenase (ADH; 147 kDa tetramer), enolase (96 kDa dimer), and
pyruvate kinase (PK; 232 kDa tetramer). IR photons were used to induce
infrared multiphoton dissociation (IRMPD) and to enhance electron-based
fragmentation via activated-ion electron transfer dissociation (AI-ETD),
and performance was benchmarked against higher-energy collisional
dissociation (HCD). For ADH (∼36 kDa subunits), AI-ETD, HCD,
and IRMPD returned similar sequence coverages in nTD MS experiments
(36, 38, and 34%, respectively), with complementary cleavages resulting
in a combined 48% coverage. As subunit mass increased, radical-driven
fragmentation provided a clear advantage: for PK (∼57 kDa subunits),
AI-ETD achieved 28% sequence coverageapproximately 15% higher
than HCD or IRMPD. Together, these results highlight IR irradiationboth
as a standalone dissociation modality and as a complement to electron-based
activationas a versatile strategy to enhance proteoform-level
sequencing in native and complex-down MS workflows.