Study of Non-Covalent Complexes by ESI-MS By Quinn Tays
History Overview Background Electrospray Ionization How it is used in study of noncovalent interactions Uses of the Technique Types of molecules Drug Discovery Case Study Hydrophobicity of Proteins
History of ESI-MS The potential of ESI as a soft ionization technique was first reported by Malcolm Dole in 1968 who combined it with MS LC-MS with electrospray as the ionization source was first reported in 1984 Created by Masamichi Yamashita and John Fenn Fenn won the Nobel Prize (2002) for his Analysis of Biological Macromolecules
Background Electrospray Ionization Spray liquid through a capillary tube with an electric field Droplets form and as the solvent evaporates, and the ions will evaporate from the surface of the drops A skimmer only allows ions through ESI is a soft ionization technique Creates primarily in [M+H] + and [M+nH] n+ ions Result: Molecules with non covalent bonds may be ionized yet remain intact Kicman, A.; Parkin, M.; Iles, R. Molecular and Cellular Endocrinology. 2007. 260, 212-27
Background - ESI with Mass Spectrometry Mass spectra are commonly a small range containing [M+nH] n+ ions Analyzers over 4000 m/z are less common Most common is a quadropole mass analyzer Results have been successfully been obtained with most other mass analyzers Detector is commonly a high energy dynode
ESI-MS Pros and Cons Pros Can be combined with other methods of analysis (i.e. MS/MS) Scanning for Inborn Errors of Metabolism is used in clinical labs Ionization is soft enough that even covalent complexes will not fragment Cons Doesn t work for non-polar compounds Spectra of mixtures become very complex
Clinical Applications: Scanning for Inborn Errors of Metabolism (IEM) Rashed, M.; Bucknall, M.; Little, D. Clin Chem. 1997. 43, 1129-41. (https://www.ncbi.nlm.nih.gov/pubmed/9216448/) IEM s are genetic disorders which usually cause a lack of certain protein used in breakdown of food Newborns are often affected by these diseases and must be scanned Some Examples are; Fructose Intolerance, Galactosemia, Maple Sugar Urine Disease (MSUD), and Phenylketonuria Missing some enzyme in the biochemical pathway Blood samples are tested with ESI-MS/MS and abnormal profiles are flagged using a computer algorithm Testing of the algorithm showed it was very good at flagging samples with the disease
Non-Covalent Complexes Primarily proteins are studied as ESI-MS is particularly useful for ionizing large molecules RNA and DNA are also studied Proteins Hydrogen bonding Ionic bonding Hydrophobic effect Nucleotide Complexes Hydrogen bonding between nucleosides
Non-Covalent Complexes Activity relationship studies between two molecules Concentration of bound complex vs. non bound complex reveals affinity for one another Competitive Binding Studies Substrate that binds with the highest affinity will appear with the largest signal in an ESI-MS Spectrum
Uses - Peptide-Peptide Affinities of binding of a protein to a peptide Ex. Ribonuclease cleaves phosphodiester bonds It is composed of a short polypeptide and a protein
Uses - Peptide-Metals Many proteins are bound to metals Important for catalytic function of enzymes Useful for determining Peptide Metal ion stoichiometry Ex. One atom of iron per heme (four heme groups for one hemoglobin) http://www.mn.uio.no/ibv/english/research/sections/bmb/research%20groups/andersson-group/
Uses - Protein Subunits ESI-MS is useful for determining quaternary structure of proteins Homocomplexes or heterocomplexes 33% of proteins are multimeric, 80% of these are dimers or tetramers http://swift.cmbi.ru.nl/gv/students/mtom/qua_1.html
Uses Protein-Nucleic Acid Complexes Proteins commonly serve as regulators of DNA and RNA It useful to know how/where they bind Knowing where on a sequence a protein can bind is useful for targeting tumor cells Competitive binding studies for specific nucleotide sequences
ESI-MS in Drug Discovery Binding affinities of compounds with a target Structure activity relationships of a known drug Vancomycin (blocks cell wall formation of bacteria) was screened for binding to cell wall analogues Screening of libraries of compounds for lead identification Compounds that interact strongly with the protein of interest will remain together for ionization This can be automated can screen large libraries easily
Case Study Probing the Hydrophobic Effect of Noncovalent Complexes by MS (Bich, C.; Baer, S.; Jecklin, M. J Am Soc Mass Spectrom. 2010. 21, 286 289.) Occurs with chains of hydrophobic amino acids The hydrophobic effect occurs when proteins are in solution minimize the number of side chains exposed to water It drives folding of the protein and stabilizes the structure Procedure 4 proteins were purchased A quadropole TOF MS with ESI equipped was used to obtain MS Spectra MALDI TOF MS was also used to measure the proteins Results MALDI was found to effectively give a picture of the non-covalent interactions of the proteins however ESI-MS was not found to be effective at studying compounds exhibiting the hydrophobic effect ESI-MS detected compounds with strong hydrogen bonds and compounds with mixed polar interactions and hydrophobic interactions Compounds with hydrogen bonding survived the transfer to gas
Conclusion ESI-MS works well for non-covalent complexes due to its lack of fragmenation ESI-MS is widely used in the study of proteins A wide variety of biological molecules can be analyzed An essential tool for early steps of drug discovery
References [1] Kicman, A.; Parkin, M.; Iles, R. Molecular and Cellular Endocrinology. 2007. 260, 212-27. [2] Rashed, M.; Bucknall, M.; Little, D. Clin Chem. 1997. 43, 1129-41. [3] Bich, C.; Baer, S.; Jecklin, M. J Am Soc Mass Spectrom. 2010. 21, 286 89. [4] Ho, C.; Lam, C.; Chan, M. Clin Biochem Rev. 2003. 24, 3 12. [5] Loo, J. Mass Spectrometry Reviews. 1997. 16, 1 23.