The World of Acronyms - Mass Spectrometry (MS): Post-Translational Modifications (PTMs)

Mass spectrometry is a commonly used technique in analytical chemistry. But, what can mass spectrometry tell us about protein post-translational modifications? How could you distinguish between two proteins of identical mass using this technique?

Note: A brief introduction to mass spectrometry, and how it can be used in biochemical contexts as a start to a series discovering more of the chemistry behind biochemistry and biological systems in general.

Mass spectrometry (MS) is the study of matter through the formation of gas-phase ions. The procedure measures the mass-to-charge ratio (m/z) through the detection, and consequently characterisation of these ions. Post-translational modifications (PTMs) are those which occur after a protein has been synthesised - there are hundreds of different types of PTMs, such as acetylation, glycosylation and phosphorylation. Can you see where the world of acronyms is coming from already?

PTMs almost always change the m/z, and as a result, MS can be used to detect and identify particular PTMs within a protein. PTMs can all be monitored through the use of MS - notably, this can be done simultaneously, and hence there is no need (at least in a theoretical sense) to target and identify each modification individually. In proteomic studies (looking at the whole proteome (complete set of proteins) within an organism), PTMs are present in stoichiometric amounts in the peptide stage. However, it would be found that there are more peptides to detect because the same peptides will be present in the samples both with and without PTMs. As a result of this, before the process of liquid chromatography - mass spectrometry/mass spectrometry (LC-MS/MS), an additional step is required - namely another PTM enrichment step, related to the type of modification which you are trying to match - one example would be a glycopeptide enrichment. Approximately 50% of human proteins are glycosylated, however, glycans are highly hydrophilic which means that their ionisation efficiency is greatly reduced. The enrichment stage is thus deemed essential in order to ensure that they appear on the spectra, and the false impression that they are not abundant is not encountered.

In the process of data analysis, matches get determined as a result of carefully considering the raw data present against the database it is compared to since, otherwise, possibilities including a phosphorylated protein may be missed in the overall output reading from the spectra.

To further identify and analyse which amino acid residue has been post-translationally modified, mass spectrometry/mass spectrometry (MS/MS) is used. In this process, through the splitting of the overall peptide into different fragments, it is then possible to identify which has the modification as a result of comparing results between the fragmentation patterns produced to find a modification which would be deemed consistent with the data presented.

MS/MS has been found to be particularly useful in the identification of specific amino acids within key proteins known to regulate mutate in cancer. One such example is p53 (tumour protein 53 / TP53) which is found to be a common mutant, with a mutated form of the protein being present in approximately 50% of cancer cases throughout all types of cancer. MS/MS can then be used to develop targeted drug therapeutics aimed at the amino acid residues, which could then in turn improve the treatment quality for patients through the reduction of side effects including fever.

When reading mass spectrometry outputs, it is worth noting that components will separate different in a native MS spectrum. A small molecule (such as ATP) is only likely to result in one peak, whereas a small peptide (circa. 1kDa) is likely to have a few charges present and hence some of these charges may give rise to different peaks - so you could have three peaks for example. A large protein complex (such as green fluorescent protein (GFP)) will have numerous charges induced through the electrospray ionisation in the machine, and hence will separate differently leading to the reader seeing a distribution of charges all corresponding to the same protein, on a greater scale than that experienced with the peptide, and hence a larger number of peaks.

As mentioned, two proteins of identical mass can be distinguished through the use of tandem mass spectrometry (MS/MS). If there are two possible sequences given for a peptide, both of which would have the same molecular weight, MS/MS can be used in order to determine the order and/or positioning of the amino acid residues if they are unknown. Upon fragmentation, and then further MS (second MS after determining the molecular weight of the original peptide), it is possible to determine the order of the amino acid sequence of the peptide, as outline as an example in Figure 1.

Figure 1: schematic representing the process of identifying which amino acid sequence order of a peptide is correct from two possibilities, given that the molecular weight is the same. The peptide will have been fragmented using Collision Induced Dissociation (CID) to give numerous fragments of different masses. Fragment masses can be calculated from their respective m/z and intensities. Hence, the order of the peptide can be determined.

The m/z values for each peak can be used to calculate the fragment mass, and hence be able to identify which amino acid has been added on each time when aligned with the MS spectrum through the known theoretical molecular weights for each amino acid, and comparison.

Data analytics software (such as Proteome Discover) can be used in order to accelerate this process since the software can provide a list of peptides that match that it has found within data. Typically, if 2 or more unique peptides are identified, the presence of a protein within a sample can be confirmed.

This can be further used to compare a normal MS with one where a drug has been applied to trigger the increased production of a certain peptide. This would lead to an increased intensity for the signal of that peptide, and hence an increase in the amount of protein identified, which can be particularly useful when analysing the differences in cell responses between cancerous and non-cancerous proteins, as well as their effects on the proteome, amongst numerous other things.

References

Aebersold, R. and M. Mann. 2003. Mass spectrometry-based proteomics. Nature 422:198–207.

Cantin, G.T. and J.R. Yates 3rd. 2004. Strategies for shotgun identification of post-translational modifications by mass spectrometry. J. Chromatogr. A. 1053:7–14.

Fenn, J.B., M. Mann, C.K. Meng, S.F. Wong, and C.M. Whitehouse. 1989. Electrospray ionization for mass spectrometry of large biomolecules. Science 246:64–71.

Hoffman, M.D. and J. Kast. 2006. Mass spectrometric characterization of lipid-modi-fied peptides for the analysis of acylated proteins. J. Mass Spectrom. 41:229–241

Huddleston, M.J., M.F. Bean, and S.A. Carr. 1993. Collisional fragmentation of gly-copeptides by electrospray ionization LC/MS and LC/MS/MS: methods for selective detection of glycopeptides in protein digests. Anal. Chem. 65:877–884

Jensen, O.N.2004. Modification-specific proteomics: characterization of post-translational modifications by mass spectrometry. Curr. Opin. Chem. Biol. 8:33–41

Kim, J.Y., K.W. Kim, H.J. Kwon, D.W. Lee, and J.S. Yoo. 2002. Probing lysine acetyla-tion with a modification-specific marker ion using high-performance liquid chromatog-raphy/electrospray-mass spectrometry with collision-induced dissociation. Anal. Chem. 74:5443–5449

Larsen, M., Trelle, M., Thingholm, T. and Jensen, O., 2006. Analysis of posttranslational modifications of proteins by tandem mass spectrometry. BioTechniques, 40(6), pp.790-798.

Mann, M. and O.N. Jensen. 2003. Proteomic analysis of post-translational modifications. Nat. Biotechnol. 21:255–261.

Molloy, M.P. and P.C. Andrews. 2001. Phosphopeptide derivatization signatures to identify serine and threonine phosphorylated peptides by mass spectrometry. Anal. Chem. 73:5387–5394.

Riboni, N., Quaranta, A., Motwani, H.V. et al. Solvent-Assisted Paper Spray Ionization Mass Spectrometry (SAPSI-MS) for the Analysis of Biomolecules and Biofluids. Sci Rep9, 10296 (2019). https://doi.org/10.1038/s41598-019-45358-x

Ryan CM, Souda P, Bassilian S, et al. Post-translational modifications of integral membrane proteins resolved by top-down Fourier transform mass spectrometry with collisionally activated dissociation. Mol Cell Proteomics. 2010;9(5):791-803. doi:10.1074/mcp.M900516-MCP200

Sadygov, R.G., D. Cociorva, and J.R. Yates 3rd. 2004. Large-scale database searching using tandem mass spectra: looking up the answer in the back of the book. Nat. Methods 1:195–202

Tao, W.A., B. Wollscheid, R. O'Brien, J.K. Eng, X.J. Li, B. Bodenmiller, J.D. Watts, L. Hood, and R. Aebersold. 2005. Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry. Nat. Methods 2:591–598

Urban PL. Quantitative mass spectrometry: an overview. Philos Trans A Math Phys Eng Sci. 2016;374(2079):20150382. doi:10.1098/rsta.2015.0382

Zhang, H., E.C. Yi, X.J. Li, P. Mallick, K.S. Kelly-Spratt, C.D. Masselon, D.G. Camp 2nd, R.D. Smith, et al.. 2005. High throughput quantitative analysis of serum proteins using glycopeptide capture and liquid chro-matography mass spectrometry. Mol. Cell. Proteomics 4:144–155

Zubarev, R.A.2004. Electron-capture dissociation tandem mass spectrometry. Curr. Opin. Biotechnol. 15:12–16