Oral Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Extending the upper mass range for top-down proteomics with proton-transfer reactions, parallel ion parking, and 21 tesla FT-ICR MS (#153)

Lissa C. Anderson 1 , Chad R. Weisbrod 1 , David Butcher 1 , Zeljka Popovic 2 , Alan G. Marshall 1 2 , Greg T. Blakney 1 , Jeffrey Shabanowitz 3 , Donald F. Hunt 3 4 , Christopher L. Hendrickson 1 2
  1. National High Magnetic Field Laboratory, Tallahassee, FL, United States
  2. Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL, 32306
  3. Department of Chemistry, University of Virginia, Charlottesville, VA, 22904
  4. Department of Pathology, University of Virginina, Charlottesville, VA, USA

Top-down proteomics is routinely complicated by several analytical challenges including the inherent distribution of protein ion signal among multiple charge states, isotopologues, adducts, modification states, and fragment ion channels.  Such challenges are further compounded by poor chromatography, incomplete fragmentation/sequence coverage, slow spectral acquisition rate, and high data complexity.  These effects limit proteoform detection and characterization, and grow exponentially worse as mass increases, making routine analysis of proteins larger than ~30 kDa difficult.  

We will provide performance benchmarks for integration of proton-transfer reactions (PTR), parallel ion parking (PIP), and 21 T FT-ICR MS for analyses of proteins >30 kDa.  Use of PTR coupled with PIP concentrates the majority of ion current from multiple charge states of each precursor proteoform into just a few charge states, which increases sensitivity compared to conventional MS1 spectra.  For example, isotopic distributions of patient-derived human serum albumin proteoforms (66-69 kDa) are resolved without signal averaging in an on-line LC-PTR/PIP-MS experiment.  Signal-to-noise ratio of the most abundant charge state in the PTR/PIP spectra was improved by a factor of 15 compared to corresponding conventional MS1 spectra.  Preliminary results from LC-MS/MS experiments with human whole cell lysate demonstrate significant improvement in the number of proteoforms detected from 1404 (conventional MS1) to 2472 (PTR/PIP).  We will also demonstrate the utility of ETD in tandem with PTR for sequence analysis of large proteins.  ETD/PTR MS/MS reduces fragment ion overlap, enabling more comprehensive sequence analysis compared to ETD alone.  Sequence coverage of recombinant protein AG (50 kDa) was improved from 12% with ETD alone versus 50% with ETD/PTR.  These techniques, combined with the high charge capacity, resolving power, and mass measurement accuracy achieved at 21 T, facilitate an enhanced ability to observe, identify, and characterize higher molecular weight proteins in biological samples.