Leveraging Ion–Ion and Ion–Photon Activation to Improve the Sequencing of Proteins Carrying Multiple Disulfide Bonds: The Human Serum Albumin Case Study

Gas-phase sequencing of large intact proteins (>30
kDa) via tandem
mass spectrometry is an inherently challenging process that is further
complicated by the extensive overlap of multiply charged product ion
peaks, often characterized by a low signal-to-noise ratio. Disulfide
bonds exacerbate this issue because of the need to cleave both the
S–S and backbone bonds to liberate sequence informative fragments.
Although electron-based ion activation techniques such as electron
transfer dissociation (ETD) have been proven to rupture disulfide
bonds in whole protein ions, they still struggle to produce extensive
sequencing when multiple, concatenated S–S bonds are present
on the same large polypeptide chain. Here, we evaluate the increase
in sequence coverage obtained by combining activated-ion ETD (AI-ETD)
and proton transfer charge reduction (PTCR) in the analysis of 66
kDa human serum albumin, which holds 17 disulfide bridges. We also
describe the combination of AI-ETD with supplemental postactivation
of the ETD reaction products via higher-energy collisional dissociationa
hybrid fragmentation method termed AI-EThcD. AI-EThcD leads to a further
improvement compared to AI-ETD in both the global number of cleaved
backbone bonds and the number of ruptured backbone bonds from disulfide-protected
regions. Our results also demonstrate that the full potential of AI-ETD
and AI-EThcD is unveiled only when combined with PTCR: reduction in
overlap of ion signals leads to a sequence coverage as high as 39%
in a single experiment, highlighting the relevance of spectral simplification
in top-down mass spectrometry of large proteins.