Leveraging Ion–Ion\nand Ion–Photon Activation\nto Improve the Sequencing of Proteins Carrying Multiple Disulfide Bonds: The Human Serum Albumin\nCase Study

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