Non-Covalent Interaction Analysis

of Macromolecules using

Electrospray Ionization-Mass

Spectrometry

LUKIAN MAXIMIUK

7749218

FEBRUARY 28TH,2020

Outline

Introduction

Principles/Layout

Why NCB?

Current Developments

Advantages and Disadvantages

Introduction Non-covalent interactions are essential for the structural

organization biomacromolecules.

Non-covalent forces play important roles in molecular recognition

processes

This includes interactions with proteins, glycans, lipids, DNA and RNA.

The use of electrospray ionization coupled with mass spectrometry

aids in the understanding/quantification of structural architecture of

macromolecules.

Used in fields of pharmacology, medical research (chemical

therapeutics), proteomics, toxicology and biochemistry.

Frieden, E. Non-Covalent Interactions: Key to Biological Flexibility and Specificity. Journal of Chemical Education 1975, 52 (12), 754.

Ho CS, Lam CW, Chan MH, et al. Electrospray ionisation mass spectrometry: principles and clinical applications. Clin Biochem Rev. 2003, 24 (1), 3–12.

Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000. Section 2.2, Noncovalent Bonds.

Przybylski, M.; Glocker, M. O. Electrospray Mass Spectrometry of Biomacromolecular Complexes with Noncovalent Interactions—New Analytical Perspectives for Supramolecular Chemistry and Molecular

Recognition Processes. Angewandte Chemie International Edition in English 1996, 35 (8), 806–826.

Veenstra, T. D. Electrospray Ionization Mass Spectrometry in the Study of Biomolecular Non-Covalent Interactions. Biophysical Chemistry 1999, 79 (2), 63–79.

ESI-MS Principles

and layout

Analyte prepared in solution.

Pumped through a stainless steel or Quartz silica capillary under high voltage.

Parent droplet is formed.

Solvent evaporation occurs, decreasing the droplet size.

Droplet will decrease to where the parent molecule breaks into smaller daughter molecules.

Daughter molecules now have high charge to mass ratio.

Ionized particles enter heated capillary where its subjected to a drying agent (Eg. Heated nitrogen gas).

Sent into mass spectrometer.

Banerjee, S.; Mazumdar, S. Electrospray Ionization Mass Spectrometry: A Technique to Access the

Information beyond the Molecular Weight of the Analyte. International Journal of Analytical Chemistry 2012,

2012, 1–40.

Electrospray. https://www.sciencedirect.com/topics/immunology-and-microbiology/electrospray

(accessed Feb 27, 2020).

What is electrospray? http://www.newobjective.com/electrospray/whatisESI.shtml (accessed

Feb 27, 2020).

Electrospray ionization - Mass Spectrometer (ESI – MS): Central Mechanical Engineering

Research Institute. https://www.cmeri.res.in/electrospray-ionization-mass-spectrometer-esi-–-ms

(accessed Feb 27, 2020).

Mass Spectrometry Analysis Triple-quadrupole analysis

Allows the analysis of proteins within the 4000 m/z (less than 100 kDa) range.

Limitation: Has low resolving power at higher m/z outputs.

Time-of-Flight analysis

Allows the analysis of proteins above 5000 m/z (above 100 kDa) range.

Limitation: ????

Hybrid Q-ToF analysis

Combined the m/z range of ToF with the selection abilities of the triple-quadrupole.

Currently the tandem-MS on macromolecules aids in the reveal of their compositions.

Heck, A. J. R.; Robert H. H. Van Den Heuvel. Investigation of Intact Protein Complexes by Mass Spectrometry. Mass Spectrometry Reviews 2004, 23 (5), 368–389.

Banerjee, S.; Mazumdar, S. Electrospray Ionization Mass Spectrometry: A Technique to Access the Information beyond the Molecular Weight of the Analyte. International Journal of

Analytical Chemistry 2012, 2012, 1–40.

Why Noncovalent bonds? Noncovalent bonds are crucial for the three-dimensional structures

of macromolecules and supramolecular structures.

Includes Hydrogen, Ionic, Van der Waals and Hydrophobic bonds.

Average energy released from NCB is approximately 1-5kcal/moI.

During electrospray ionization daughter droplets containing the macromolecule undergoes thermal re-equilibration (loss of hydrophobic bonding and electrostatic interactions)

The unfolded macromolecules form conformers with new noncovalent bonds.

Preserves the non-covalent bonds and interactions.

Data from noncovalent interactions can be placed into specialized software to build a visual representation.

Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000. Section 2.2, Noncovalent Bonds.

Przybylski, M.; Glocker, M. O. Electrospray Mass Spectrometry of Biomacromolecular Complexes with Noncovalent Interactions—New Analytical Perspectives for Supramolecular Chemistry and Molecular Recognition

Processes. Angewandte Chemie International Edition in English 1996, 35 (8), 806–826.

Veenstra, T. D. Electrospray Ionization Mass Spectrometry in the Study of Biomolecular Non-Covalent Interactions. Biophysical Chemistry 1999, 79 (2), 63–79.

Breuker, K.; Mclafferty, F. W. Stepwise Evolution of Protein Native Structure with Electrospray into the Gas Phase, 10-12 to 102 s. Proceedings of the National Academy of Sciences

2008, 105 (47), 18145–18152.

Results

Siuzdak, G. An Introduction to Mass Spectrometry Ionization: An Excerpt from The Expanding

Role of Mass Spectrometry in Biotechnology, 2nd Ed.; MCC Press: San Diego, 2005. Journal of

the Association for Laboratory Automation 2004, 9 (2), 50–63.

Banerjee, S.; Mazumdar, S. Electrospray Ionization Mass Spectrometry: A Technique to Access

the Information beyond the Molecular Weight of the Analyte. International Journal of Analytical

Chemistry 2012, 2012, 1–40.

• Flavonoids as polyphenolic secondary metabolites are ubiquitously found in many plants.

• Due to their high reactivity with reactive oxygen species such as hydroxyl, alkoxyl, or peroxyl

radicals and efficient inhibition of lipid peroxidationin micelle systems, flavonoids are thought to be

associated with antiaging, antifungal, anti-inflammatory, and especially anticancer activities.

• Transport and disposition of flavonoids in the human body are regulated by the noncovalent

interaction between human serum albumin (HSA) and flavonoids.

Wang, B.; Qin, Q.; Chang, M.; Li, S.; Shi, X.; Xu, G. Molecular Interaction Study of Flavonoids with Human Serum Albumin

Using Native Mass Spectrometry and Molecular Modeling. Analytical and Bioanalytical Chemistry 2017, 410 (3), 827–837.

Wang, B.; Qin, Q.; Chang, M.; Li, S.; Shi, X.; Xu, G. Molecular Interaction Study of Flavonoids with Human Serum Albumin Using Native Mass Spectrometry and

Molecular Modeling. Analytical and Bioanalytical Chemistry 2017, 410 (3), 827–837.

Summary

Qualitative binding stoichiometry and binding constants

determined.

Showed characterization and quantification of the structure–affinity

relationship.

Determined HSA binding site markers and flavonoids.

Observed competition between apigenin and warfarin at the

subdomain IIA of HSA (hydrogen bonds).

Quantified binding constants for HSA–flavonoid interactions.

Determined the relative Ka values for binding affinities.

Wang, B.; Qin, Q.; Chang, M.; Li, S.; Shi, X.; Xu, G. Molecular Interaction Study of Flavonoids

with Human Serum Albumin Using Native Mass Spectrometry and Molecular Modeling.

Analytical and Bioanalytical Chemistry 2017, 410 (3), 827–837.

• Endogenous mechanisms of cardioprotection under hypoxia and ischemia of

cardiac hystiocytes is related to activation and opening of ATP sensitive potassium

channels (KATP channels) of sarcolemma and mitochondrial membranes.

• Flokalin, a fluorine containing analogue of pinacidil, has shown high effectiveness in

the treatment of myocardial infarction and ischemia in in vitro and in vivo

experiments with mammals.

• Investigate the biologically relevant interactions of flokalin with selected amino acids

involved in KATP channel-activator binding sites using the electrospray ionization mass

spectrometry.

• Lysine and Threonine are the amino acids selected for the investigation, as they are

involved in the regulatory subunits of KATP channel proteins and are relevant in the

channel activation by pinacidil. Pashynska, V.; Stepanian, S.; Gömöry, Á.; Vékey, K.; Adamowicz, L. New Cardioprotective Agent

Flokalin and Its Supramolecular Complexes with Target Amino Acids: An Integrated Mass-

Spectrometry and Quantum-Chemical Study. Journal of Molecular Structure 2017, 1146, 441–

449.

Pashynska, V.; Stepanian, S.; Gömöry, Á.; Vékey, K.; Adamowicz, L. New Cardioprotective Agent

Flokalin and Its Supramolecular Complexes with Target Amino Acids: An Integrated Mass-

Spectrometry and Quantum-Chemical Study. Journal of Molecular Structure 2017, 1146, 441–

449.

Summary

Electrospray ionization mass spectrometry is found to be an effective method to study flokalin in polar solvents, as well as to study its noncovalent interactions with possible biological targets involved in the flokalin biological activity.

B3LYP/aug-cc-pVDZ calculations provide the molecular structure of the most stable tautomer of flokalin in vacuum.

Most stable flokalin-lysine protonated complex is stabilized by a strong electrostatic interaction between the protonated side-chain amino group of the most stable conformer of protonated lysine and the nitrile nitrogen of the neutral flokalin.

Formation of the stable noncovalent complexes of flokalin with Lys and Thr gives support to the idea of a strong possibility of interaction of flokalin with the K+-channel domains.

Pashynska, V.; Stepanian, S.; Gömöry, Á.; Vékey, K.; Adamowicz, L. New Cardioprotective Agent

Flokalin and Its Supramolecular Complexes with Target Amino Acids: An Integrated Mass-

Spectrometry and Quantum-Chemical Study. Journal of Molecular Structure 2017, 1146, 441–

449.

Advantages

Measure a mass range of approximately 70,000Da.

Excellent sensitivity.

Soft ionization method, capable of generating/preserving

noncovalent complexes in gaseous phase.

Can be coupled with LC or HPLC.

Detectors can be modified with tandem mass analyzers (such as ion

traps and triple quadrupole instruments).

Multiple charging allows for high mass ion analysis with relatively low

m/z ranged instruments.

No matrix interference.

Siuzdak, G. An Introduction to Mass Spectrometry Ionization: An Excerpt from The Expanding

Role of Mass Spectrometry in Biotechnology, 2nd Ed.; MCC Press: San Diego, 2005. Journal

of the Association for Laboratory Automation 2004, 9 (2), 50–63

Disadvantages

Must be absent of a salt environment.

Complex mixtures can reduce sensitivity.

Simultaneous mixture analysis can be poor in quality.

Multiple charging can obscure results (especially in mixture analysis).

Sample purity is VERY IMPORTANT!!!

Siuzdak, G. An Introduction to Mass Spectrometry Ionization: An Excerpt from The Expanding

Role of Mass Spectrometry in Biotechnology, 2nd Ed.; MCC Press: San Diego, 2005. Journal of

the Association for Laboratory Automation 2004, 9 (2), 50–63

Thank-you for

listening.