Exploring the fragmentation efficiency of proteins analyzed by MALDI-TOF-TOF tandem mass spectrometry using computational and statistical analyses

Authors: PARK, JIHYUN - Oak Ridge Institute For Science And Education (ORISE); Fagerquist, Clifton - Keith

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/8/2024
Publication Date: 5/3/2024
Citation: Park, J., Fagerquist, C.K. 2024. Exploring the fragmentation efficiency of proteins analyzed by MALDI-TOF-TOF tandem mass spectrometry using computational and statistical analyses. PLOS ONE. 19(5). Article e0299287. https://doi.org/10.1371/journal.pone.0299287.
DOI: https://doi.org/10.1371/journal.pone.0299287

Interpretive Summary: Identification and characterization of proteins is one of the major goals of proteomic science. Mass spectrometry (MS) is the central analytical pillar of proteomics because of its speed, sensitivity, and specificity. Software algorithms, machine learning tools and artificial intelligence (e.g. Alphafold2) are making dramatic advances in providing reliable 3-D structures of proteins for which no crystal structure exists. We have used Alphafold2, residue interaction network analysis and statistical methodologies to extract protein properties of bacterial pathogens. These properties were then compared to the pathways and efficiencies of protein fragmentation as analyzed by matrix-assisted laser desorption/ionization time-of-flight-time-of-flight (MALDI-TOF-TOF) tandem mass spectrometry (MS/MS). In addition to confirmation of the well-known aspartic acid effect pathway, we found that P- and G-residues adjacent to D-residues (C-terminal side) were statistically more likely to result in backbone fragmentation than other residues, and an adjacent P-residue was also found to significantly enhance N-residue backbone cleavage. In addition, backbone cleavage on the N-terminal side of a P-residue was significantly enhanced by adjacent D,E,N residues. In summary, these results demonstrate the power of combining statistical and network analyses with Alphafold2 predictions to rationalize protein fragmentation pathways that are critical to proteomic identification.

Technical Abstract: Matrix-assisted laser desorption/ionization time-of-flight-time-of-flight (MALDI-TOF-TOF) tandem mass spectrometry (MS/MS) is a rapid technique for identifying intact proteins from unfractionated mixtures by top-down proteomic analysis. MS/MS allows isolation of specific intact protein ions prior to fragmentation, allowing fragment ion attribution to a specific precursor ion. However, the fragmentation efficiency of mature, intact protein ions by MS/MS post-source decay (PSD) varies widely, and the biochemical and structural factors of the protein that contribute to it are poorly understood. With the advent of protein structure prediction algorithms such as Alphafold2, we have wider access to protein structures for which no crystal structure exists. In this work, we use a statistical approach to explore the properties of bacterial proteins that can affect their gas phase dissociation via PSD. We extract various protein properties from Alphafold2 predictions and analyze their effect on fragmentation efficiency. Our results show that the fragmentation efficiency from cleavage of the polypeptide backbone on the C-terminal side of glutamic acid (E) and asparagine (N) residues were nearly equal. In addition, we found that the rearrangement and cleavage on the C-terminal side of aspartic acid (D) residues that result from the aspartic acid effect (AAE) were higher than for E- and N-residues. From residue interaction network analysis, we identified several local centrality measures and discussed their implications regarding the AAE. We also confirmed the selective cleavage of the backbone at D-proline bonds in proteins and further extend it to N-proline bonds. Finally, we note an enhancement of the AAE mechanism when the residue on the C-terminal side of D-, E- and N-residues is glycine. To the best of our knowledge, this is the first report of this phenomenon. Our study demonstrates the value of using statistical analyses of protein sequences and their predicted structures to better understand the fragmentation of the intact protein ions in the gas phase.