The Value of Activated Ion Electron Transfer Dissociation for High-Throughput Top-Down Characterization of Intact Proteins

Abstract

High-throughput top-down proteomic experiments directly identify proteoforms in complex mixtures, making high quality tandem mass spectra necessary to deeply characterize proteins with many sources of variation. Collision-based dissociation methods offer expedient data acquisition but often fail to extensively fragment proteoforms for thorough analysis. Electron-driven dissociation methods are a popular alternative approach, especially for precursor ions with high charge density. Combining infrared photoactivation concurrent with electron transfer dissociation (ETD) reactions, i.e., activated ion ETD (AI-ETD), can significantly improve ETD characterization of intact proteins, but benefits of AI-ETD have yet to be quantified in high-throughput top-down proteomics. Here, we report the first application of AI-ETD to LC-MS/MS characterization of intact proteins (<20 kDa), highlighting improved proteoform identification the method offers over higher energy-collisional dissociation (HCD), standard ETD, and ETD followed by supplemental HCD activation (EThcD). We identified 935 proteoforms from 295 proteins from human colorectal cancer cell line HCT116 using AI-ETD compared to 1014 proteoforms, 915 proteoforms, and 871 proteoforms with HCD, ETD, and EThcD, respectively. Importantly, AI-ETD outperformed each of the three other methods in MS/MS success rates and spectral quality metrics (e.g., sequence coverage achieved and proteoform characterization scores). In all, this four-method analysis offers the most extensive comparisons to date and demonstrates that AI-ETD both increases identifications over other ETD methods and improves proteoform characterization via higher sequence coverage, positioning it as a premier method for high-throughput top-down proteomics.

Capturing site-specific heterogeneity with large-scale N-glycoproteome analysis

“…Activated-ion electron transfer dissociation (AI-ETD) has rapidly developed as a highly effective tandem MS approach for proteomic applications. [33][34][35][36][37][38][39][40][41] The method uses concurrent infrared (IR) photoactivation to improve dissociation efficiencies and increase product ion generation in electron transfer dissociation (ETD) reactions. 11,42 We report here that the combination of simultaneous vibrational activation from IR photon bombardment and electron-driven dissociation via ETD is particularly well-suited for intact glycopeptide fragmentation.…”

“…11,12,[21][22][23][24][25][26][13][14][15][16][17][18][19][20] Even with these methods, large-scale analysis of intact glycopeptides remains largely limited to fewer than ~1,000 unique glycosite identifications from any one system or tissue (approximately an order of magnitude behind other PTMs), [27][28][29][30][31][32] and few studies assess heterogeneity across the glycoproteome with site-specific resolution.Activated-ion electron transfer dissociation (AI-ETD) has rapidly developed as a highly effective tandem MS approach for proteomic applications. [33][34][35][36][37][38][39][40][41] The method uses concurrent infrared (IR) photoactivation to improve dissociation efficiencies and increase product ion generation in electron transfer dissociation (ETD) reactions. 11,42 We report here that the combination of simultaneous vibrational activation from IR photon bombardment and electron-driven dissociation via ETD is particularly well-suited for intact glycopeptide fragmentation.…”

Mapping physiological ADP-ribosylation using Activated Ion Electron Transfer Dissociation (AI-ETD)

“…By contrast, activated ion-ETD (AI-ETD) uses infrared photoactivation during the ETD reaction to overcome ETnoD, and has been shown to benefit glycoproteomics (Riley et al, 2019), top-down proteomics (McCool et al, 2019;Riley et al, 2018a;Riley et al, 2018b), as well as phosphoproteomics (Riley et al, 2017a). We therefore set out to investigate the utility of AI-ETD on an Orbitrap Fusion Lumos mass spectrometer for identification and confident localization of ADPr sites.…”

Top-Down Characterization of Proteins with Intact Disulfide Bonds Using Activated-Ion Electron Transfer Dissociation

“…The ability to effectively fragment highly disulfide linked intact proteins with AI-ETD will likely advance efforts towards the structural characterization of many types of proteins such as intact antibodies, toxins, native proteins, and protein complexes. Additionally, AI-ETD was recently shown to benefit top-down characterization of intact proteins in LC-MS/MS analyses, 60 and the work described here lays the groundwork for future experiments that could be conducted on a chromatographic timescale to screen complex mixtures of proteins with intact disulfide bonds. In all, AI-ETD is a superior fragmentation technique for proteins with intact disulfide bonds and will continue to be explored as a tool for disulfide bond analysis in a variety of applications.…”