Mass Spectrometry: Advancing Protein Biomarker Assays

Detection of protein biomarkers is a primary goal in proteomics research. Their quantification can play a key role in the measurement of both the presence and progress of a wide variety of diseases. Thus, many researchers are turning to mass spectrometry, a solution that is much more sensitive than previous biomarker assays and has very high quantitative capability. The mass spectrometric immunoassay (MSIA) technology of Thermo Scientific has helped to increase protein biomarker assay accuracy with a fully integrated, high-throughput workflow for research laboratories.

A fundamental challenge in proteomics research is gaining the capability to differentiate between — and accurately quantify — numbers of intact proteins and their variants. This is essential for a more complete view of the proteome and in the search for protein biomarkers — characteristic proteins or segments that can be used to measure the presence or progress of a disease.¹ Conventional protein biomarker assays, such as enzyme-linked immunosorbent assays (ELISA), used antibodies that bind to the protein of interest.² However, this technique was limited by the inability to produce antibodies specific enough to recognize all variants of a protein and so to quantify their abundance.²

A solution to this problem was to use mass spectrometry, as this technology has a much higher specificity than any previous assay techniques. Mass spectrometry measures the mass-to-charge ratio of molecules or molecule fragments in a sample, therefore allowing the protein composition of the sample to be much more accurately characterized.³ However, target proteins are often low abundance in a sample, and increasing the concentration of these proteins to get more accurate measurements has been a significant challenge. Traditional approaches involved the depletion of other interfering proteins that are in higher abundance. However, this was often time consuming and could introduce significant analytical variability.

Thermo Scientific and Intrinsic Bioprobes (IBI) formed an alliance to try and increase the accuracy of protein biomarker assays using mass spectrometry and to give more complete, higher resolution views of the proteome.⁴ Their solution involved combining the high specificity immunoenrichment technology of IBI with the high sensitivity and quantitative capability of the mass spectrometry technology of Thermo Fisher. Immunoenrichment technology is based on the use of pipette tips, which allow the enrichment and concentration of target proteins. The pipette tips integrate a high-binding capacity microcolumn activated with antibodies, which allow these target proteins to be isolated and analyzed. Moreover, this technology works with very low abundance proteins in complex biological matrices. Thus, the combination of this technology with the highly sensitive protein analysis that mass spectrometry provides offers much more effective detection and analysis of low abundance proteins than had ever been available before.

In the past, there was always a distinct lack of a robust mass-spectrometry workflow that was reproducible from laboratory to laboratory. The alliance between Thermo Scientific and IBI has helped to tackle this considerable problem in developing their MSIA workflow for research laboratories. Moreover, the technologies discussed above have been combined with Thermo Scientific’s automated sample-handling technology, which has significantly increased sample throughput and productivity.

The detection and quantification of human parathyroid hormone (PTH) and its variants has been the initial target of this MSIA solution. PTH is associated with a variety of bone- and kidney-related diseases, and thus, it has the potential to serve as a biomarker for such diseases.⁵ Thermo Scientific’s MSIA workflow is being used in the quantification of very low abundance PTH. However, vitally, the workflow is also being used to help identify novel PTH variants that could not have otherwise been detected using conventional assays. Identifying new PTH variants means PTH screening tests can be made more reliable, with reliable screening key in monitoring PTH-associated diseases in patients.⁶

References

1. Rifai, N., Gillette, M.A., and Carr, S.A. (2006) ‘Protein biomarker discovery and validation: The long and uncertain path to clinical utility‘, Nature Biotechnology, 24 (8), (pp. 971-983)

2. Vieira, J.G.H. (2012) ‘PTH assays: Understanding what we have and forecasting what we will have‘, Journal of Osteoporosis, 2012, (p. 523246)

3. Yates, J.R. (2000) ‘Mass spectrometry: From genomics to proteomics‘, Trends in Genetics, 16 (1), (pp. 5-8)

4. Thermo Fisher Scientific. http://www.thermoscientific.com/ecomm/servlet/newsdetail?contentId=51476&&storeId=11152

5. Aloia, J. F., Feuerman, M., and Yeh, J.K. (2006) ‘Reference range for serum parathyroid hormone‘, Endocrine Practice, 12 (2), (pp. 137-144)

6. Lopez, M.F., et al. (2010) ‘Selected reaction monitoring – mass spectrometric immunoassay responsive to parathyroid hormone related variants‘ Clinical Chemistry, 56 (2), (pp. 281-290)

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