Protein Characterization



Activities:

The research activities of protein characterization at the Proteomics Centre cover a variety of areas using unique strategies based on mass spectrometric methods and approaches developed at the Centre, including bottom-up and top-down proteomic approaches. Bottom-up approaches enable the analysis of peptides obtained by proteolysis and are particularly useful for the characterization of specific post-translational modifications (PTMs) including phosphorylation, acetylation, ubiquitination, and glycosylation. Top-down techniques allow for the direct analysis of intact proteins, thus introduce minimal manipulation to the original protein samples. The top-down approach is particularly useful for accurate mass measurements of intact proteins, protein sequencing, combinatorial PTM analysis, and isoform specific PTM characterization.

When combined with hydrogen/deuterium exchange technology, both approaches are used for protein higher-order structure and conformational change characterization including comprehensive biosimilar analysis, with peptide level information from the bottom-up approach and single-residue level information from the top-down. The Centre has developed and applied such approaches to accurately locate protein modification sites, to pinpoint subtle structural differences between therapeutic antibodies, to determine the composition of protein complexes, to accurately reveal the truncation site of proteins, and to elucidate the distribution and types of protein isoforms. Detailed information of the bottom-up and top-down proteomic approaches are described in representative publications listed below, more research articles can be found in Publications of the Centre.


Methods:

MS and LC-MS on 12T FT-ICR, Orbitrap-Velos, and Orbitrap-Fusion
MSMS on 12T FTICR with CID and ECD
LC-MSMS on Orbitrap with CID, HCD, and ETD
Top-down and bottom-up hydrogen/deuterium exchange


Results Highlights:

1. Pan, J., Zhang, S., Parker, CE., Borchers, CH. Subzero temperature chromatography and top-down mass spectrometry for protein higher-order structure characterization: method validation and application to therapeutic antibodies. J Am Chem Soc, 136: 13065-13071. (2014).

2. Pan, J., Borchers, CH. Top-down mass spectrometry and hydrogen/deuterium exchange for comprehensive structural characterization of interferons: Implications for biosimilars. Proteomics, 14, 1249–1258. (2014).

3. Dang, X., Scotcher, J., Wu, S., Chu, RK., Tolic, N., Ntai, I., Thomas, PM., Fellers, RT., Early, BP., Zheng, Y., Durbin, KR., Leduc, RD., Wolff, JJ., Thompson, CJ., Pan, J., Han., J., Shaw, JB., Salisbury, JP., Easterling, M., Borchers, CH., Brodbelt, JS., Agar, JN., Pasa-Tolic, L., Kelleher, NL., Young, NL. The First Pilot Project of the Consortium for Top Down Proteomics: A Status Report. Proteomics, 14(10):1130-40. (2014).

4. Pan, J., Borchers, CH. Top-down structural analysis of posttranslationally modified proteins by Fourier transform ion cyclotron resonance-MS with hydrogen/deuterium exchange and electron capture dissociation. Proteomics, 13(6): 974-81. (2013).

5. Pan, J., Han, J., Borchers, CH. Top-down hydrogen/deuterium exchange and ECD-stitched FTICR-MS for probing structural dynamics of a 29-kDa enzyme. Int J Mass Spectrom, 325, 130-138. (2012).

6. Han, J. and Borchers, C.H. Top-down analysis of recombinant histone H3 and its methylated analogs by ESI/FTICR mass spectrometry. Proteomics, 10(20):3621-30. (2010)

7. Domanski, D., Murphy, L., Borchers, C. Assay Development for the Determination of Phosphorylation Stoichiometry using MRM methods with and without Phosphatase Treatment: Application to Breast Cancer Signaling Pathways. Anal Chem, 82(13): 5610-20. (2010)

8. Pan J., Han, J., Borchers, C.H., Konermann, L. Hydrogen/Deuterium Exchange Mass Spectrometry with Top-Down Electron Capture Dissociation for Characterizing Structural Transitions of a 17 kDa Protein. J Am Chem Soc, 131(35) :12801-8. (2009)

9. Pan, J., Han, J., Borchers, C.H., Konermann, L. Electron Capture Dissociation of Electrosprayed Protein Ions for Spatially-Resolved Hydrogen Exchange Measurements. J Am Chem Soc, 130(35): 11574-5. (2008)

10. Borchers, C.H., Thapar, R., Petrotchenko, E.V., Torres M.P., Speir, S.P., Easterling M., Dominski, Z., Marzluff, W.F., Combined Top-down and Bottom-up Proteomics Identifies a Phosphorylation Site in Stem-Loop Binding Proteins That Contributes to High-affinity RNA Binding. PNAS, 103(9): 3094-3099. (2006)


Group Members:
Jingxi Pan
Suping Zhang