Poster Presentation 27th Annual Lorne Proteomics Symposium 2022

Internal fragment assignments enhance top-down protein sequencing experiments which is beneficial for interrogating protein structure and localising protein-ligand binding (#186)

Benqian Wei 1 , Carter Lantz 1 , Wonhyuek Jung 1 , Muhammad A Zenaidee 1 2 , Rachel R Ogorzalek Loo 1 , Joseph A Loo 1
  1. School of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California, United States
  2. Australian Proteome Analysis Facility, Macquarie University, Sydney, NSW, Australia

Top-down mass spectrometry (TD-MS) analysis of intact protein ions can be utilised to return information on the primary amino acid sequence of proteins and localise protein modifications and/or protein-ligand binding sites. In TD-MS experiments, intact protein ions are generated by electrospray ionisation, isolated, and subsequently fragmented in a mass spectrometer to generate fragment ions, that can be matched to the primary protein sequence. Traditionally, TD-MS experiments only utilise terminal fragments ions (containing the N- or C- terminus) for protein sequencing which leads to many mass spectral signals going unassigned, limiting protein sequence information.

 

In TD-MS, internal fragments (fragment ions containing neither the N- nor C- terminus) can also be generated during the fragmentation process. Internal fragments often go unassigned due to the computational complexity associated with assigning internal fragments.1,2 However, assignment of internal fragments can enhance TD-MS experiments and extend the information that can be gleaned from these experiments.

 

Here, we describe Clips-MS, a program written in Python that can precisely assign internal fragments from a TD-MS experiment.3 By including internal fragments, sequence information was increased from ~30% to >80%.4 Internal fragments were found to explain portions of the sequence within the centre of the protein, complementing terminal fragments which show limited coverage of the interior of the protein sequence. This suggests that modifications located within the centre of the protein can be interrogated using internal fragments. We demonstrate that for disulfide intact proteins, internal fragments can be utilised to extend the characterisation of disulphide intact proteins and localise disulfide connectivity. Further, internal fragments can aid in the localisation of ligand binding on membrane proteins. Combining terminal and internal fragment assignments in TD-MS should allow for more efficient TD-MS experiments which could further enhance localisation of protein modifications and/or protein-ligand binding sites.

  1. 1. Durbin, K. R., Skinner, O. S., Fellers, R. T., & Kelleher, N. L. (2015). Analyzing internal fragmentation of electrosprayed ubiquitin ions during beam-type collisional dissociation. Journal of the American Society for Mass Spectrometry, 26(5), 782-787.
  2. 2. Zenaidee, M. A., Lantz, C., Perkins, T., Jung, W., Loo, R. R. O., & Loo, J. A. (2020). Internal Fragments Generated by Electron Ionization Dissociation Enhance Protein Top-Down Mass Spectrometry. Journal of the American Society for Mass Spectrometry, 31(9), 1896-1902.
  3. 3. Lantz, C., Zenaidee, M. A., Wei, B., Hemminger, Z., Ogorzalek Loo, R. R., & Loo, J. A. (2021). ClipsMS: An Algorithm for Analyzing Internal Fragments Resulting from Top-Down Mass Spectrometry. Journal of Proteome Research, 20(4), 1928-1935.
  4. 4. Zenaidee, Muhammad A., Benqian Wei, Carter Lantz, Hoi Ting Wu, Tyler R. Lambeth, Jolene K. Diedrich, Rachel R. Ogorzalek Loo, Ryan R. Julian, and Joseph A. Loo. (2021). Internal Fragments Generated from Different Top-Down Mass Spectrometry Fragmentation Methods Extend Protein Sequence Coverage. Journal of the American Society for Mass Spectrometry, 32(7), 1752-1758