Maximizing Sequence Coverage in Top-Down Proteomics By Automated Multimodal Gas-Phase Protein Fragmentation

Abstract

Intact protein sequencing by tandem mass spectrometry (MS/MS), known as top-down protein sequencing, relies on efficient gas-phase fragmentation at multiple experimental conditions to achieve extensive amino acid sequence coverage. We developed the "topdownr" R-package for automated construction of multi-modal (i.e. involving CID, HCD, ETD, ETciD, EThcD and UVPD) MS/MS fragmentation methods on an orbitrap instrument platform and systematic analysis of the resultant spectra. We used topdownr to generate and analyze thousands of MS/MS spectra for five intact proteins of 10-30kDa. We achieved 90-100% coverage for the proteins tested and derived guiding principles for efficient sequencing of intact proteins. The data analysis workflow and statistical models of topdownr software and multi-modal MS/MS experiments provide a framework for optimizing MS/MS sequencing for any intact protein. Refined topdownr software will be suited for comprehensive characterization of protein pharmaceuticals and eventually also for de novo sequencing and detailed characterization of intact proteins.

Top-Down Proteomics of Medicinal Cannabis

“…In our study, compiling all HCD fragment masses together in Prosight Lite program yielded a myoglobin sequence coverage of 57%. The fact that the outcome of fragmentation was much less dependent on a particular collisional value for CID than for HCD was also noted by Shliaha and colleagues [25]. Futhermore, they report that, while CID and HCD spectra are very similar, HCD achieves optimal fragmentation at lower energy levels, which we also observed in our study.…”

“…This suggests that parent ions displaying both high m/z (low charge state) and high intensity should be favoured for top-down sequencing experiments. Shliaha and colleagues demonstrate that targeting different charge states provides complementary information; lower charge states respond well to CID and HCD, and higher charge states are more amenable to ETD fragmentation [25]. It is worth mentioning that another type of electron-based fragmentation technology-ECD available on a Q Exactive orbitrap mass spectrometer-produced up to 48% sequence coverage of myoglobin [36].…”

“…The region spanning AAs 100 to 140 of myoglobin was only partially sequenced. Shliaha and colleagues [25] published last year an exhaustive top-down proteomics experiment on infused standards, including myoglobin. The authors indicate that CID and HCD contribute less AA sequence information than ETD, mostly at the protein N and the C-termini, whereas ETD provides coverage throughout the protein sequence.…”

“…For instance, SID, CID, and HCD create b-and y-type ions, while ETD creates c-and z-type ions. Extensive AA sequence coverage of known intact proteins was achieved through efficient multimodal gas-phase fragmentation and sophisticated computational analysis [25]. Top-down proteomic experiments are challenging for the following reasons: low abundance of many proteoforms and low signal-to-noise (S/N) ratios inherent to mass measurement of large molecules, data analysis of complex MS and MS/MS spectra, and co-elution of proteoforms during the initial online separation stage by liquid chromatography (LC) or capillary zone electrophoresis (CZE) [20].…”

Preparing Proteoforms of Therapeutic Proteins for Top-Down Mass Spectrometry

“…For performing a TDMS analysis, a purified individual intact proteoform is transferred into the MS. From the MS spectrum of the intact ions, the molecular weight can be determined. Various techniques are available for fragmentation of the intact proteoform such as HCD, CID, ETD, ETHcD, ECD, UVPD and IRMPD, yielding different types for fragments, which complement each other [5]. After fragmentation, the proteoform can be identified by interpreting the fragment spectrum.…”