Mass spectrometry (MS) in protein biomarker discovery
Mass spectrometry is a tool that helps in characterizing the proteins. It measures the mass of protein molecules through different steps and using different components and show a positive response to a protein biomarker research. Proteomics is a branch that works on the study of proteins and their interactions in human physiology (Parker et al., 2010). However, the field has established itself as the backbone of modern day clinical chemistry. Proteomics deals with characterizing the proteins in the human body. Furthermore, these characteristics help clinical chemists the understand the interplay of different protein structure in our body. The mass of protein molecules undergoes three main steps of identification and measurement.
Principle of mass spectrometry
The principle of mass spectrometry is based on three components, where the source produces ions that get analysed according to their mass: charge ratio by the mass analyzers and creates signals. The signals get amplified by the detector and measured easily. The mass gives information on the protein identity, its chemical modifications, and its structure.
|Techniques in MS (Carol E. Parker et al., 2010; William Reusch, 2013)|
|Electrospray ionization (ESI): For studying particles of molecular masses 1000 Da or below.||Time-of-Flight Analysers (TOF): Has the 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 a high sensitivity of up to 0.5 ppm.
|Multichannel plate (MCP) detectors:
These are the latest technological advancement where the device is capable of ion detection.
Around the year 2002, the biomarkers research was in boom and researchers needed a well-established technique. This technique would rapidly screen large number of proteins for a selected target for oncology research. In one research by Anderson, in the late millennium year used mass spectrometry to screen protein molecules for prostate cancer. The researcher 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. The researcher 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).
Most applied approaches of mass spectrometry
For biomarker research two approaches of mass spectrometry most applied;
- Non-targeted approach (shotgun proteomics)
- Targeted approach
The non-targeted approach comprises of large number of proteins subjected to spectrometry on a small number of samples of some certain diseases. Furthermore, this process helps to select initial candidates which show different expression in disease positive and disease negative samples. These initial candidate proteins subjected to samples of disease positive patients and filters out 2-3 proteins with higher affinity towards disease targets. The filtered out proteins used in clinical studies and the randomized samples subjected to qualification. Moreover, from the samples wherein 1-2 proteins are designated as biomarkers for a specific disease (Paulovich et al., 2008).
Targeted mass spectrometry uses different approaches designed to overcome the sampling limitation of non-targeted mass spectrometry. In targeted approach, instead of blind search of molecules from the protein pool, a protein of specific class screened for a 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, 2013). Enzyme-linked immunosorbent assays (ELISAs) use targeted MS. A few biomarkers assure verification or validation on a large number of disease positive samples. ELISA assays also have limited multiplexing capabilities and can exhibit cross-reactivity (Hoofnagle and Wener, 2009). ELISA is also applicable in final clinical validation assays, i.e., assays where there are fewer protein targets. However, ELISA technology is not well-suited for quantifying a large number of candidate biomarker proteins.
|List of FDA-approved protein tumour markers currently used in clinical practice (Füzéry et al., 2013)|
|Pro2PSA||Prostate||Discriminating cancer from benign disease||Serum|
|p63 protein||Prostate||Aid in differential diagnosis||FFPE tissue|
|c-Kit||Gastrointestinal stromal tumours||Detection of tumours, aid in the 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|
Furthermore, targeted mass spectrometry is the newer approach which aims at specific biomarker discovery for specific disease protein (Gillette and Carr, 2013; Hoofnagle and Wener, 2009). Lastly, mass spectrometry and targeted mass spectrometry has played a 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.
- 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
- 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
- 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
- William Reusch, 2013. Mass Spectrometry [WWW Document]. URL https://www2.chemistry.msu.edu/faculty/reusch/virttxtjml/spectrpy/massspec/masspec1.htm (accessed 9.16.17).