Protein footprinting

Protein footprinting is a term used to refer to a method of biochemical analysis that investigates protein structure, assembly, and interactions within a larger macromolecular assembly. It was originally coined in reference to the use of limited proteolysis to investigate contact sites within a monoclonal antibody - protein antigen complex[1] and a year later to examine the protection from hydroxyl radical cleavage conferred by a protein bound to DNA within a DNA-protein complex.[2] In DNA footprinting the protein is envisioned to make an imprint (or footprint) at a particular point of interaction.[3] This latter method was adapted through the direct treatment of proteins and their complexes with hydroxyl radicals[4][5] and can be generally denoted RP-MS (for Radical Probe - Mass Spectrometry)[6] akin to the designation used for Hydrogen-deuterium exchange Mass Spectrometry (denoted HD-MS or HX-MS).

Hydroxyl radical protein footprinting

Time-resolved hydroxyl radical protein footprinting (HRPF) employing mass spectrometry analysis was originated and developed in the late 1990s in synchrotron radiolysis studies.[7][8] The same year, these authors (Maleknia et al.) reported on the use of an electrical discharge source to effect the oxidation of proteins on millisecond timescales as proteins pass from the electrosprayed solution into the mass spectrometer.[9] Years later in 2005, researchers Hambly and Gross introduced a method for protein oxidation on the microsecond timescale using laser flash photolysis of hydrogen peroxide to generate hydroxyl radicals.[10] This method, fast photochemical oxidation of proteins (FPOP), claimed to footprint proteins faster than they change their fold[11] though this timeframe has been challenged given hydrogen peroxide, not present in the original studies, and secondary radicals, react alone in situ over tens of milliseconds.[12] These approaches have since been used to determine protein structures,[13] protein folding, protein dynamics, and protein–protein interactions.[14]

Unlike nucleic acids, proteins oxidize rather than cleave on these timescales. Analysis of the products by mass spectrometry reveals that proteins are oxidized in a limited manner (some 10–30% of total protein) at a number of amino acid side chains across the proteins. The rate or level of oxidation at the reactive amino acid side chains (Met, Cys, Trp, Tyr, Phe, His, Pro and Leu) provides a measure of their accessibility to the bulk solvent. The mechanisms of side chain oxidation were explored by performing the radiolysis reactions in 18O-labeled water.

Producing OH radicals

A critical feature of these experiments is the need to expose proteins to hydroxyl radicals for limited timescales on the order of 1–50 ms inducing 10-30% oxidation of total protein. A further requirement is to generate hydroxyl radicals from the bulk solvent (i.e. water) (equations 1 and 2) not hydrogen peroxide which can remain to oxidize proteins even without other stimuli.[15]

H2O → H2O+• + e− + H2O*
H2O+• + H2O → H3O+ + OH•

Hydroxyl radicals can be produced in solution by an electrical discharge within a conventional atmospheric pressure electrospray ionization (ESI) source. When a high voltage difference (~8 keV) is held between an electrospray needle and a sampling orifice to the mass analyzer, radicals can be produced in solution at the electrospray needle tip. This method was the first employed to apply protein footprinting to the study of a protein complex.[16]

Method

The exposure of proteins to a "white" X-ray beam of synchrotron light or an electrical discharge for tens of milliseconds provides sufficient oxidative modification to the surface amino acid side chains without damage to the protein structure. These products can be easily detected and quantified by mass spectrometry. By adjusting the time for radiolysis or which protein ions spend in the discharge source, a time-resolved approach is possible which is valuable for the study of protein dynamics.

Analysis

A computer program (PROXIMO) has also been written to help model protein complexes using data from the RP-MS/Protein footprinting approach.[17] RP-MS/Protein footprinting studies of protein complexes can also employ computational approaches to assist with this modeling.[18]

Applications

The application of ion mobility mass spectrometry has conclusively demonstrated that the conditions employed in RP-MS/Protein footprinting experiments do not alter the structure of proteins.[19]

Other studies have extended the method to study early onset protein damage given the radical basis of the method and the significance of oxygen based radicals in the pathogenesis of many diseases including neurological disorders and even blindness.[20]

See also

References

  1. ^ Sheshberadaran H, Payne LG (January 1988). "Protein antigen-monoclonal antibody contact sites investigated by limited proteolysis of monoclonal antibody-bound antigen: protein "footprinting"". Proceedings of the National Academy of Sciences of the United States of America. 85 (1): 1–5. Bibcode:1988PNAS...85....1S. doi:10.1073/pnas.85.1.1. PMC 279469. PMID 2448767.
  2. ^ Shafer GE, Price MA, Tullius TD (1989). "Use of the hydroxyl radical and gel electrophoresis to study DNA structure". Electrophoresis. 10 (5–6): 397–404. doi:10.1002/elps.1150100518. PMID 2504579. S2CID 38355953.
  3. ^ Galas DJ (November 2001). "The invention of footprinting". Trends in Biochemical Sciences. 26 (11): 690–3. doi:10.1016/S0968-0004(01)01979-X. PMID 11701330.
  4. ^ Maleknia SD, Downard KM (May 2014). "Advances in radical probe mass spectrometry for protein footprinting in chemical biology applications". Chemical Society Reviews. 43 (10): 3244–58. doi:10.1039/C3CS60432B. PMID 24590115.
  5. ^ Wang L, Chance MR (October 2011). "Structural mass spectrometry of proteins using hydroxyl radical based protein footprinting". Analytical Chemistry. 83 (19): 7234–41. doi:10.1021/ac200567u. PMC 3184339. PMID 21770468.
  6. ^ Downard KM, Maleknia SD (2019). "Mass spectrometry in structural proteomics: The case for radical probe protein footprinting". Trends in Analytical Chemistry. 110: 293–302. doi:10.1016/j.trac.2018.11.016.
  7. ^ Maleknia SD, Brenowitz M, Chance MR (September 1999). "Millisecond radiolytic modification of peptides by synchrotron X-rays identified by mass spectrometry". Analytical Chemistry. 71 (18): 3965–73. doi:10.1021/ac990500e. PMID 10500483.
  8. ^ Maleknia SD, Ralston CY, Brenowitz MD, Downard KM, Chance MR (February 2001). "Determination of macromolecular folding and structure by synchrotron x-ray radiolysis techniques". Analytical Biochemistry. 289 (2): 103–15. doi:10.1006/abio.2000.4910. PMID 11161303.
  9. ^ Maleknia SD, Chance MR, Downard KM (1999). "Electrospray-assisted modification of proteins: a radical probe of protein structure". Rapid Communications in Mass Spectrometry. 13 (23): 2352–8. Bibcode:1999RCMS...13.2352M. doi:10.1002/(SICI)1097-0231(19991215)13:23<2352::AID-RCM798>3.0.CO;2-X. PMID 10567934.
  10. ^ Hambly DM, Gross ML (December 2005). "Laser flash photolysis of hydrogen peroxide to oxidize protein solvent-accessible residues on the microsecond timescale". Journal of the American Society for Mass Spectrometry. 16 (12): 2057–63. Bibcode:2005JASMS..16.2057H. doi:10.1016/j.jasms.2005.09.008. PMID 16263307. S2CID 24995091.
  11. ^ Gau BC, Sharp JS, Rempel DL, Gross ML (August 2009). "Fast photochemical oxidation of protein footprints faster than protein unfolding". Analytical Chemistry. 81 (16): 6563–71. doi:10.1021/ac901054w. PMC 3164994. PMID 20337372.
  12. ^ Vahidi S, Konermann L (2016). "Probing the time scale of FPOP (fast photochemical oxidation of proteins): radical reactions extend over tens of milliseconds". Journal of the American Society for Mass Spectrometry. 27 (7): 1156–1164. Bibcode:2016JASMS..27.1156V. doi:10.1007/s13361-016-1389-x. PMID 27067899. S2CID 35039048.
  13. ^ Maleknia SD, Kiselar JG, Downard KM (2002). "Hydroxyl radical probe of the surface of lysozyme by synchrotron radiolysis and mass spectrometry". Rapid Communications in Mass Spectrometry. 16 (1): 53–61. Bibcode:2002RCMS...16...53M. doi:10.1002/rcm.543. PMID 11754247.
  14. ^ Maleknia SD, Downard K (2001). "Radical approaches to probe protein structure, folding, and interactions by mass spectrometry". Mass Spectrometry Reviews. 20 (6): 388–401. Bibcode:2001MSRv...20..388M. doi:10.1002/mas.10013. PMID 11997945.
  15. ^ Downard K (24 August 2007). Mass Spectrometry of Protein Interactions. John Wiley & Sons. ISBN 978-0-470-14632-3. Retrieved 14 September 2013.
  16. ^ Wong JW, Maleknia SD, Downard KM (April 2003). "Study of the ribonuclease-S-protein-peptide complex using a radical probe and electrospray ionization mass spectrometry". Analytical Chemistry. 75 (7): 1557–63. doi:10.1021/ac026400h. PMID 12705585.
  17. ^ Gerega SK, Downard KM (July 2006). "PROXIMO--a new docking algorithm to model protein complexes using data from radical probe mass spectrometry (RP-MS)". Bioinformatics. 22 (14): 1702–9. doi:10.1093/bioinformatics/btl178. PMID 16679333.
  18. ^ Downard KM, Kokabu Y, Ikeguchi M, Akashi S (November 2011). "Homology-modelled structure of the βB2B3-crystallin heterodimer studied by ion mobility and radical probe MS". The FEBS Journal. 278 (21): 4044–54. doi:10.1111/j.1742-4658.2011.08309.x. PMID 21848669.
  19. ^ Downard KM, Maleknia SD, Akashi S (February 2012). "Impact of limited oxidation on protein ion mobility and structure of importance to footprinting by radical probe mass spectrometry". Rapid Communications in Mass Spectrometry. 26 (3): 226–30. Bibcode:2012RCMS...26..226D. doi:10.1002/rcm.5320. PMID 22223306.
  20. ^ Shum WK, Maleknia SD, Downard KM (September 2005). "Onset of oxidative damage in alpha-crystallin by radical probe mass spectrometry". Analytical Biochemistry. 344 (2): 247–56. doi:10.1016/j.ab.2005.06.035. PMID 16091281.

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