Application of Mass Spectrometry In Biotechnology
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Mass Spectrophotometer applications are discussed.
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Application of Mass Spectrometry In Biotechnology
- 1. Applications of Mass Spectrometry Bhanu Krishan
- 2. Contents: Introduction to MS General Scheme Applications References
- 3. Introduction A powerful analytical technique used to quantify materials or to identify unknown compounds within a sample, elucidate the structure and different chemical properties. Involves generation of multiple ions from the sample under investigation Separation of ions according to their specific mass to charge ratio (m/z) Mass spectrometry plays a potential role in fields of food safety, proteomics, glycomics and metabolics (Aprea et.al., 2020).
- 4. Figure: GC- MS Instrumentation Figure: Mass Spectrometry Instrumentation at Mellon College of Science , C. Mellon University, Pittsburg
- 5. General scheme of Mass Spectrometer
- 6. Applications Hyphenated Technologies In Mass Spectrometry GC-MS Combined feature of Gas Chromatography and Mass spectrometer, simplest and has highest separation power other than LC- MS. GC-MS is considered a gold standard for substance identification and the method of choice, for analysis of pharmaceuticals/drugs of abuse, steroids and hormones, contaminants, and other analytes. GC is the classical method for the determination of fatty acid composition, but only the coupling with MS, allowing identification of the number of double bonds, can provide peak assignment and compound identification. For the detection and quantification of target compounds in complex matrices at trace levels, GC coupled to triple quadrupole-MS is a very powerful instrument providing greater selectivity and sensitivity than single quad MS (Aprea et.al., 2020).
- 7. Comprehensive two-dimensional GC (GC GC) coupled to fast acquisition MS (mostly TOF-MS) facilitates the analysis of very complex matrix, that is components of essential oils, residual pesticides, or contaminants in food products. GC GC provides much higher peak capacity, better separation power, and enhanced sensitivity. TOF-MS provides high data acquisition speed suitable to GC GC. Recent applications in food-related topics include the investigation of the impact of malolactic fermentation on the volatile composition of Pinotage wines (Vestner et al., 2011) and the analysis of volatile compounds in cacao beans and its quality control (Humston et al., 2009).
- 8. Figure 1: A Mass spectrum of Xenobiotics, which are generally an Environment pollutant. Source: https://doi.org/10.1007/978-3-319-39312-4_159
- 9. LC MS Involves the combination of Liquid chromatography technique with Mass Spectrometry. Used for the Analysis of contaminants (veterinary drugs, growth promoters, mycotoxins, organic contaminants, pesticides) and nutrients (vitamins, flavonoids, amino acids, terpenoids, saponins) in food. LC MS involves is the need for an interface between the two systems such as the ionspray interface particularly useful for protein and peptide samples.
- 10. Figure 2: CE-MS electrospray interface Source: D. Sheehan, Physical Biochemistry
- 11. Identification of proteins The majority of proteomics applications involve peptide sequencing by LC-MS/MS. Proteins are enzymatically digested to their peptide components, and analyzed by LC-MS/MS. The resulting sequence data is used to determine the original protein components of the sample. Information on posttranslational modifications (PTMs) and stoichiometry can also be obtained with this approach. For single proteins, the sequence can confirm identity, characterize PTMs. For protein complexes, this approach determines the identity of the individual proteins that make up the complex, stoichiometry of the subunits and PTMs.
- 12. Edman Degradation Phenyl isothiocyanate is reacted with an uncharged N-terminal amino group, under mildly alkaline conditions, to form a cyclical phenylthiocarbamoyl derivative. Then, under acidic conditions, this derivative of the terminal amino acid is cleaved as a thiazolinone derivative. The thiazolinone amino acid is then selectively extracted into an organic solvent and treated with acid to form the more stable phenylthiohydantoin (PTH)- amino acid derivative that can be identified by using chromatography or electrophoresis. This procedure can then be repeated again to identify the next amino acid.
- 13. Figure 3: Protein ladder sequencing by partial Edman degradation.
- 14. Analysis of Peptide synthesis Solid-phase peptide synthesis is used for the generation of model peptides which can be used in a wide variety of studies (e.g. synthesis of epitopes for antibody preparation, design of novel antibiotics and identification of novel protein ligands in drug screening programs). This technique involves the use of amino acids which are initially chemically protected NH2 and other functional groups (e.g. 竜-NH2, SH) while chemically activated at their COOH group. Amino acids readily reacts with the exposed NH2 group of an amino acid immobilized on a solid phase, thus forming a peptide bond. De-blocking of the blocked NH2 group allows a second cycle of reaction leading to formation of a second peptide bond and, in this way, a polypeptide chain of 20 30 residues may be synthesized from the C-terminus. When synthesis is complete, the peptide is cleaved from the resin (exposing a C- terminal COOH) and functional groups are deblocked. This procedure is readily automated and peptides may be routinely synthesized by adding commercially-available activated/ blocked amino acid derivatives in the desired sequence.
- 16. Figure 4: Synthesis of Octa-arginine using double coupling. Source: https://selekt.biotage.com
- 17. Determination of Disulphide Bonds The structural integrity of many proteins is achieved and stabilized by the formation of intra- and interchain disulfide bonds (Weinfurtner, 2018). The sulphydryl groups of cysteine are among the most chemically-reactive side-chains found in proteins which is used for conversion of disulphides to sulphydryl. Disulfides can be identified in proteins and allocated to regions of primary structure based on m/z values. Different agents such as Idoacetate, Performic acid and 硫- Mercaptoethanol generates protein into 2 forms Reduced and Non reduced forms.
- 18. Figure 5: Determination of Disulphide bonds by Cysteine leading to formation of two forms of peptide forms i.e. Reduced and Non reduced forms
- 20. Determination of Post Translation Modification and Microheterogeneity in Proteins Sources of possible heterogeneity within polypeptide chains include glycosylation,phosphorylation, acylation, disulfide bond formation, charge isomerization and endoprotease activity. Covalent modifications result in mass alterations which is detected by MS on comparing them by m/z values of proteins and thus the proteins can be detected based upon the PTMs. This determination is independent of the chemical basis of heterogeneity and this is a major reason for the versatility of MS.
- 21. Figure 6: Post Translation Modifications of Proteins
- 22. Figure 7: Proteome-wide profiling and mapping of post translational modifications in human hearts (Bagwan et.al., 2021). Source: https://doi.org/10.1038/s41598-021-81986-y
- 23. Analysis of mutagenic component in DNA Mutagens cause a cytotoxic effects to the DNA by forming covalent adducts with DNA bases such as Guanine Ethylene Oxide. ESI/MS techniques have been extensively applied to the characterization of such DNA chemical adducts. Other non covalent adducts and duplex formation in Oligonucleotides can be studies via GC/MS. Apart from Molecular sciences Mass spectrometry has been helpful in various fields such as Parasitological and microbiological studies.
- 24. Figure 8: Mass Spectrometry Based Ultrasensitive DNA Methylation Profiling Using Target Fragmentation Assay Source: doi: 10.1021/acs.analchem.5b04247
- 25. References Bagwan, N., El Ali, H.H. & Lundby, A. (2021). Proteome-wide profiling and mapping of post translational modifications in human hearts. Sci Rep 11, 2184 https://doi.org/10.1038/s41598-021-81986-y Freitas, M. A., Sklenar, A. R., & Parthun, M. R. (2004). Application of mass spectrometry to the identification and quantification of histone post-translational modifications. Journal of Cellular Biochemistry, 92(4), 691 700. doi:10.1002/jcb.20106 Weinfurtner D, CHAPTER 1.4:Analysis of Disulfide Bond Formation in Therapeutic Proteins , in Oxidative Folding of Proteins: Basic Principles, Cellular Regulation and Engineering, 2018, pp. 81-98 DOI: 10.1039/9781788013253-00081 eISBN: 978-1-78801-325-3 Medeiros P.M. (2018) Gas ChromatographyMass Spectrometry (GCMS). In: White W.M. (eds) Encyclopedia of Geochemistry. Encyclopedia of Earth Sciences Series. Springer, Cham. https://doi.org/10.1007/978-3-319-39312- 4_159 Vestner, J., Malherbe, S., Du Toit, M., Nieuwoudt, H. H., Mostafa, A., G坦recki, T., et al. (2011). Investigation of the volatile composition of pinotage wines fermented with different malolactic starter cultures using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GCGC-TOF- MS). Journal of Agricultural and Food Chemistry, 59, 1273212744. Humston, E. M., Zhang, Y., Brabeck, G. F., McShea, A., & Synovec, R. E. (2009). Development of a GCGC TOFMS method using SPME to determine volatile compounds in cacao beans. Journal of Separation Science, 32, 22892295. Lin XC, Zhang T, Liu L, Tang H, Yu RQ, Jiang JH. Mass Spectrometry Based Ultrasensitive DNA Methylation Profiling Using Target Fragmentation Assay. Anal Chem. 2016 Jan 19;88(2):1083-7. doi: 10.1021/acs.analchem.5b04247. Epub 2016 Jan 4. PMID: 26710177. Sheehan, D. Phyical Biochemistry Principles and Applications. John Wiley & Sons Ltd, 2009.