Covalent labeling in combination with mass spectrometry is a powerful approach used in structural biology to study protein structures, interactions, and dynamics. Recently, the toolbox of covalent labeling techniques has been expanded with fast fluoroalkylation of proteins (FFAP). FFAP is a novel radical labeling method that utilizes fluoroalkyl radicals generated from hypervalent Togni reagents for targeting aromatic residues. This report further demonstrates the benefits of FFAP as a new method for structural characterization of therapeutic antibodies and interaction interfaces of antigen-antibody complexes. The results obtained from human trastuzumab and its complex with human epidermal growth factor receptor 2 (HER2) correlate well with previously published structural data and demonstrate the potential of FFAP in structural biology.
- MeSH
- alkylace MeSH
- footprinting proteinů metody MeSH
- halogenace MeSH
- imunokomplex chemie MeSH
- lidé MeSH
- mapování epitopu * metody MeSH
- monoklonální protilátky chemie imunologie MeSH
- receptor erbB-2 * chemie imunologie MeSH
- trastuzumab * chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Fast photochemical oxidation of proteins (FPOP) footprinting is a structural mass spectrometry method that maps proteins by fast and irreversible chemical reactions. The position of oxidative modification reflects solvent accessibility and site reactivity and thus provides information about protein conformation, structural dynamics, and interactions. Bottom-up mass spectrometry is an established standard method to analyze FPOP samples. In the bottom-up approach, all forms of the protein are digested together by a protease of choice, which results in a mixture of peptides from various subpopulations of proteins with varying degrees of photochemical oxidation. Here, we investigate the possibility to analyze a specifically selected population of only singly oxidized proteins. This requires utilization of more specific top-down mass spectrometry approaches. The key element of any top-down experiment is the selection of a suitable method of ion isolation, excitation, and fragmentation. Here, we employ and compare collision-induced dissociation, electron-transfer dissociation, and electron-capture dissociation combined with multi-continuous accumulation of selected ions. A singly oxidized subpopulation of FPOP-labeled ubiquitin was used to optimize the method. The top-down approach in FPOP is limited to smaller proteins, but its usefulness was demonstrated by using it to visualize structural changes induced by co-factor removal from the holo/apo myoglobin system. The top-down data were compared with the literature and with the bottom-up data set obtained on the same samples. The top-down results were found to be in good agreement, which indicates that monitoring a singly oxidized FPOP ion population by the top-down approach is a functional workflow for oxidative protein footprinting.
- MeSH
- elektrony * MeSH
- footprinting proteinů * metody MeSH
- konformace proteinů MeSH
- myoglobin chemie MeSH
- oxidační stres MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- myoglobin MeSH
Methods of structural mass spectrometry have become more popular to study protein structure and dynamics. Among them, fast photochemical oxidation of proteins (FPOP) has several advantages such as irreversibility of modifications and more facile determination of the site of modification with single residue resolution. In the present study, FPOP analysis was applied to study the hemoglobin (Hb) - haptoglobin (Hp) complex allowing identification of respective regions altered upon the complex formation. FPOP footprinting using a timsTOF Pro mass spectrometer revealed structural information for 84 and 76 residues in Hp and Hb, respectively, including statistically significant differences in the modification extent below 0.3%. The most affected residues upon complex formation were Met76 and Tyr140 in Hbα, and Tyr280 and Trp284 in Hpβ. The data allowed determination of amino acids directly involved in Hb - Hp interactions and those located outside of the interaction interface yet affected by the complex formation. Also, previously modeled interaction between Hb βTrp37 and Hp βPhe292 was not confirmed by our data. Data are available via ProteomeXchange with identifier PXD021621.
- Klíčová slova
- FPOP, Footprinting, Haptoglobin, Hemoglobin, timsTOF pro,
- MeSH
- aminokyseliny chemie metabolismus MeSH
- footprinting proteinů metody MeSH
- haptoglobiny chemie metabolismus MeSH
- hemoglobiny chemie metabolismus MeSH
- hmotnostní spektrometrie metody MeSH
- hydroxylový radikál chemie MeSH
- lidé MeSH
- molekulární modely MeSH
- molekulární struktura MeSH
- oxidace-redukce MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- aminokyseliny MeSH
- haptoglobin-hemoglobin complex MeSH Prohlížeč
- haptoglobiny MeSH
- hemoglobiny MeSH
- hydroxylový radikál MeSH
Several proteins that specifically bind to DNA modified by cisplatin, including those containing HMG-domains, mediate antitumor activity of this drug. Oligodeoxyribonucleotide duplexes containing a single, site-specific interstrand cross-link of cisplatin were probed for recognition by the rat chromosomal protein HMGB1 and its domains A and B using the electrophoretic mobility-shift assay. It has been found that the full-length HMGB1 protein and its domain B to which the lysine-rich region (seven amino acid residues) of the A/B linker is attached at the N-terminus (the domain HMGB1b7) specifically recognize DNA interstrand cross-linked by cisplatin. The affinity of these proteins to the interstrand cross-link of cisplatin is not very different from that to the major 1,2-GG intrastrand cross-link of this drug. In contrast, no recognition of the interstrand cross-link by the domain B lacking this region or by the domain A with or without this lysine-rich region attached to its C-terminus is noticed under conditions when these proteins readily bind to 1,2-GG intrastrand adduct. A structural model for the complex formed between the interstrand cross-linked DNA and the domain HMGB1b7 was constructed and refined using molecular mechanics and molecular dynamics techniques. The calculated accessible areas around the deoxyribose protons correlate well with the experimental hydroxyl radical footprint. The model suggests that the only major adaptation necessary for obtaining excellent surface complementarity is extra DNA unwinding (approximately 40 degrees ) at the site of the cross-link. The model structure is consistent with the hypothesis that the enhancement of binding affinity afforded by the basic lysine-rich A/B linker is a consequence of its tight binding to the sugar-phosphate backbone of both DNA strands.
- MeSH
- adukty DNA chemie MeSH
- aminokyselinové motivy MeSH
- aminokyseliny chemie MeSH
- antitumorózní látky chemie MeSH
- cisplatina chemie MeSH
- deoxyribosa chemie MeSH
- footprinting proteinů MeSH
- heteroduplexy nukleové kyseliny chemie MeSH
- hydroxylový radikál chemie MeSH
- krysa rodu Rattus MeSH
- lysin chemie MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- párování bází MeSH
- peptidové fragmenty chemie MeSH
- povrchové vlastnosti MeSH
- protein HMGB1 chemie izolace a purifikace MeSH
- protony MeSH
- reagencia zkříženě vázaná chemie MeSH
- rozpouštědla MeSH
- sekvence aminokyselin MeSH
- terciární struktura proteinů MeSH
- vazba proteinů MeSH
- vodíková vazba MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- adukty DNA MeSH
- aminokyseliny MeSH
- antitumorózní látky MeSH
- cisplatina MeSH
- deoxyribosa MeSH
- heteroduplexy nukleové kyseliny MeSH
- hydroxylový radikál MeSH
- lysin MeSH
- peptidové fragmenty MeSH
- protein HMGB1 MeSH
- protony MeSH
- reagencia zkříženě vázaná MeSH
- rozpouštědla MeSH
