Mass Spectrometry (MS) in Protein biomarker discovery

 

-By Dr. Akash G Prabhune

Proteomic is the branch which works on the study of proteins and their interactions in human physiology(Carol E. Parker et al., 2010).The field is relatively new field (around 25 years old) but it has established itself as the backbone of modern day clinical chemistry. Proteomics deals with characterizing the proteins in human body, these characteristics help clinical chemists the understand the interplay of different protein structure in our body(Wasinger et al., 1995). Mass Spectrometry is the tool which helps in characterizing the proteins, it measures the mass of protein molecules through three main steps using different components, as shown in the Image 1. Below:

Mass_Spectrometry

Image 1: Steps in Mass Spectroscopy

The principle of mass spectrometry is based on these three components, where the source produces ions that are analysed according to their mass: charge ratio by the mass analyzers. This analysis creates signals, which are then amplified by the detector to be measured easily. The mass gives information on the protein identity, its chemical modifications, and its structure. The table below lists the different techniques that have evolved over time in these three basic components of MS.

Table 1. Techniques in MS (Carol E. Parker et al., 2010; William Reusch, 2013)
Source techniques Analyzer techniques Detector techniques
Electrospray ionization (ESI): For studying particles of molecular masses 1000 Da or below Time-of-Flight Analysers (TOF): Has ability to analyse proteins of mass up to 20kDa
Matrix-assisted laser desorption/ionization (MALDI): Particles with molecular mass of 38kDa can be analysed. Fourier transform (FT):

The most powerful with high sensitivity of upto 0.5 ppm.

Multichannel plate (MCP) detectors:

These are latest technological advancement where in the device is capable of ion detection.

 

Around 2002, the biomarkers research was in boom and researchers needed a well-established technique which would rapidly screen large number of proteins for selected target for oncology research. Anderson used MS to screen protein molecules for prostate cancer, he collected samples from 30 cases of lump in the breast cases and subjected them to a pool of proteins using mass spectrometry. A protein “Pro2PSA “would express itself differently in cancer cases than in non-cancer cases. He further went on the research on the molecule and finally FDA validated “Pro2PSA” as biomarker to differentiate between benign lumps in breast and breast cancer(Anderson and Anderson, 2002). For biomarker research two approaches of MS are applied

  • non-targeted approach (shotgun proteomics)
  • Targeted approach

Non-Targeted approach is one where large number of proteins are subjected to spectrometry on small number of samples of some certain diseases, this process helps to select initial candidates which show different expression in disease positive and disease negative samples. These initial candidate proteins are then subjected to samples of disease positive patients which filters out 2-3 proteins with higher affinity towards disease targets. The filtered out proteins are then used in clinical studies and the randomized samples are subjected to qualification wherein 1-2 proteins can be designated as biomarkers for a specific disease(Paulovich et al., 2008). Table 2. Lists the FDA approved protein biomarkers discovered using the non-targeted approach.

Table 2. List of FDA-approved protein tumor markers currently used in clinical practice (Füzéry et al., 2013)
Biomarker Cancer type Clinical use Specimen
Pro2PSA Prostate Discriminating cancer from benign disease Serum
p63 protein Prostate Aid in differential diagnosis FFPE tissue
c-Kit Gastrointestinal stromal tumors Detection of tumors, aid in selection of patients FFPE tissue
CA19-9 Pancreatic Monitoring disease status Serum, plasma
Estrogen receptor (ER) Breast Prognosis, response to therapy FFPE tissue
Nuclear Mitotic Apparatus protein (NuMA, NMP22) Bladder Diagnosis and monitoring of disease (professional and home use) Urine

Targeted mass spectrometry is a different approach designed to overcome the sampling limitation of non-targeted mass spectrometry. In targetedapproach, instead of blind search of molecules form the protein pool a protein of specific class is screened for specific class of receptors. As compared to conventional method, targeted MS enables to scan to multiple permutations and combinations of protein assay against selected disease targets (Gillette and Carr, 2013a). Enzyme linked immunosorbent assays (ELISAs) are best examples of targeted MS, where only a few biomarkers need to be verified or validated on a large number of disease positve samples. ELISA assays also have limited multiplexing capabilities and can exhibit cross-reactivity (Hoofnagle and Wener, 2009a). While ELISAs are useful for the final clinical validation assays, i.e., assays where there are fewer protein targets, ELISA technology is not well-suited for quantitating a large number of candidate biomarker proteins (Gillette and Carr, 2013b)

To summarize, MS has led to large number of possibilities in biomarkers research and has helped in validation of large number of biomarkers which have played role in different aspects of oncology research. Targeted MS is the newer approach which aims at specific biomarker discovery for specific disease protein(Gillette and Carr, 2013b; Hoofnagle and Wener, 2009b). MS and targeted MS has played key role in developing proteomics to a field which extensively works in biomarkers discovery and also helps in achieving the long-fetched dream of personalized medicine.

References

  • Anderson, N.L., Anderson, N.G., 2002. The human plasma proteome: history, character, and diagnostic prospects. Mol. Cell. Proteomics MCP 1, 845–867.
  • Carol E. Parker, Maria R. Warren, Viorel Mocanu, 2010. Neuroproteomics, Chapter 5 Mass Spectrometry for Proteomics. Taylor and Francis Group, LLC.
  • Füzéry, A.K., Levin, J., Chan, M.M., Chan, D.W., 2013. Translation of proteomic biomarkers into FDA approved cancer diagnostics: issues and challenges. Clin. Proteomics 10, 13. doi:10.1186/1559-0275-10-13
  • Gillette, M.A., Carr, S.A., 2013a. Quantitative analysis of peptides and proteins in biomedicine by targeted mass spectrometry. Nat. Methods 10, 28–34. doi:10.1038/nmeth.2309
  • Gillette, M.A., Carr, S.A., 2013b. Quantitative analysis of peptides and proteins in biomedicine by targeted mass spectrometry. Nat. Methods 10, 28–34. doi:10.1038/nmeth.2309
  • Hoofnagle, A.N., Wener, M.H., 2009a. The fundamental flaws of immunoassays and potential solutions using tandem mass spectrometry. J. Immunol. Methods 347, 3–11. doi:10.1016/j.jim.2009.06.003
  • Hoofnagle, A.N., Wener, M.H., 2009b. The fundamental flaws of immunoassays and potential solutions using tandem mass spectrometry. J. Immunol. Methods 347, 3–11. doi:10.1016/j.jim.2009.06.003
  • Paulovich, A.G., Whiteaker, J.R., Hoofnagle, A.N., Wang, P., 2008. The interface between biomarker discovery and clinical validation: The tar pit of the protein biomarker pipeline. Proteomics Clin. Appl. 2, 1386–1402. doi:10.1002/prca.200780174
  • Wasinger, V.C., Cordwell, S.J., Cerpa-Poljak, A., Yan, J.X., Gooley, A.A., Wilkins, M.R., Duncan, M.W., Harris, R., Williams, K.L., Humphery-Smith, I., 1995. Progress with gene-product mapping of the Mollicutes: Mycoplasma genitalium. Electrophoresis 16, 1090–1094.
  • William Reusch, 2013. Mass Spectrometry [WWW Document]. URL https://www2.chemistry.msu.edu/faculty/reusch/virttxtjml/spectrpy/massspec/masspec1.htm (accessed 9.16.17).

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