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Benefits of High-Resolution Mass Spectrometry (HRMS)
for Peptide Quantitation

A recent study from Janssen (2019) tells us that bioanalysts are switching from triple quadrupole mass spectrometry (MS) to high-resolution mass spectrometry (HRMS). While the triple quad MS, prized for its extraordinary1 sensitivity, has long been the standard analytical instrument of the pharmaceutical industry (as noted in Vereyken, et al., 2019), modern HRMS tools are now facilitating a new segment of analysis.


Why are pharmaceutical bioanalysts increasingly choosing HRMS?


In a word: biologics.2


As experts in triple quad MS analysis, we, at Sannova, pride ourselves on producing results that lead the industry in accuracy and precision. To stay up-to-date with pharmaceutical science that is moving towards large molecules, we have carefully reviewed various high resolution mass spectrometry options and acquired the Thermo ScientificTM Orbitrap ExplorisTM 240 instrument to best meet our clients’ emerging bioanalytical needs.


For complicated targets, like peptides, HRMS is effectively more sensitive than triple quad MS.3 The difference in sensitivity arises from the fragmentation flexibility built into OrbitrapTM spectrometers (Eliuk and Makarov, 2015). For a single fragment, triple quad MS is more sensitive than HRMS. But to conduct quantitative analysis on peptides, bioanalysts typically need to work with three to six fragments. As the number of fragments increases, triple quad MS technology requires a proportional increase in the minimum sample size needed to perform quantitative analysis. HRMS, on the other hand, requires only a marginal increase in the minimum sample size (Gallien, et al., 2012).


Modern HRMS instruments have also been designed to measure intact biological molecules, such as antibodies, that triple quad instruments have a hard time detecting. Biological molecules are more challenging to analyze than smaller molecules because larger ions are less stable, which makes them harder to transmit and detect in the mass spectrometer. OrbitrapTM instruments have been designed to optimize stability of intact proteins, accelerate detection speed and improve signal-to-noise ratio (Eliuk and Makarov, 2015).


In addition to making it possible to analyze larger molecules, HRMS is desirable for its selectivity; by offering high resolution and high mass accuracy, it is easier to separate and correctly identify a wide array of molecular species in an analytical sample (Eliuk and Makarov, 2015).


HRMS at center of proteomics and pharmaceutical bioanalysis


The importance of HRMS technology for proteomics is perhaps most powerfully illustrated by the fact that the first two drafts of the human proteome, published simultaneously in the May 2014 issue of Nature, drew upon OrbitrapTM MS data (Kim, et al.; Wilhelm, et al.). While researchers continue to work on the optimizing HRMS design and analytical parameters, scientists like Ramanathan and Korfmacher (2012) have already declared that HRMS will be “the premier analytical tool in the pharmaceutical bioanalysis arena.”


Regulatory agencies, like FDA, accept HRMS


Though HRMS technology has been available since 2005, its use in pharmaceutical applications was previously limited due to lack of regulatory compatibility. However, agencies like the FDA, have published methods (1, 2) employing HRMS for the identification and quantitation of nitrosamine impurities in metformin – as part of its work to promote the control of nitrosamine impurities in human drugs (2021). The current guidance for the pharmaceutical industry on bioanalytic method validation (2018) also does not favor any specific type of mass spectrometry, but instead recommends that sponsors or applicants carefully weigh selectivity, specificity, sensitivity, accuracy, and other factors affecting quantitation. Thus, HRMS is a robust option for pharmaceutical quality control testing.


Takeaways


In conclusion, HRMS is emerging as one of bioanalysts’ top choices for peptide quantitation. For smaller molecules, the ultra-sensitive triple quad technology is still favored. But for complicated targets, like peptides and antibodies, HRMS is effectively more sensitive than triple quad MS. Additionally, HRMS can be used to quantify larger molecules than current alternatives. Furthermore, its high resolution confers greater selectivity; and the ability to more accurately capture mass leads to greater specificity and more accurate identification of analytes. As a result, scientists and regulatory agencies are increasingly referencing HRMS as an important tool for bioanalysis in the pharmaceutical industry.


1picogram/ml (pg/ml)
2Biological molecules include peptides, proteins, lipids, and nucleotides.
3Alexander Makarov, inventor of OrbitrapTM spectrometers has said (here) that with those instruments, “more proteins [are] detected from 20ng than other instruments detect from 5µg.”


References:


Eliuk, S. and Makarov, A. Evolution of Orbitrap Mass Spectrometry Instrumentation. Ann. Rev. Anal. Chem. [Online] 2015, 8, 61-80. https://www.annualreviews.org/doi/10.1146/annurev-anchem-071114-040325 (accessed June 25, 2021).


Gallien, S.; Duriez, E.; Crone, C.; Kellmann, M.; Moehring, T.; Domon, B. Targeted Proteomic Quantification on Quadrupole-Orbitrap Mass Spectrometer. MCP [Online] 2012, 11, 1709-1723. https://doi.org/10.1074/mcp.O112.019802 (accessed June 21, 2021).


Kim, M-S.; Pinto, S. M.; Getnet, D.; Nirujogi, R. S.; Manda, S. S.; Chaerkady, R.; Madugundu, A. K.; Kelkar, D. S.; Isserlin, R.; Jain, S.; Thomas, J. K.; Muthusamy, B.; Leal-Rojas, P.; Kumar, P.; Sahasrabuddhe, N. A.; Balakrishnan, L.; Advani, J.; George, B.; Renuse, S.; Delvan, L. D. N.; Patil, A. H.; Nanjappa, V.; Radhakrishnan, A.; Prasad, S.; Subbannayya, T.; Raju, R.; Kumar, M.; Sreenivasamurthy, S. K.; Marimuthu, A.; Sathe, G. J.; Chavan, S.; Datta, K. K.; Subbannayya, Y.; Sahu, A.; Yelamanchi, S. D.; Jayaram, S.; Rajagopalan, P.; Sharma, J.; Murthy, K. R.; Syed, N.; Goel, R.; Khan, A. A.; Ahmad, S.; Dey, G.; Mudgal, K.; Chatterjee, A.; Huang, T-C.; Zhong, J.; Wu, X.; Shaw, P. G.; Freed, D.; Zahari, M. S.; Mukherjee, K. K.; Shankar, S.; Mahadevan, A.; Lam, H.; Mitchell, C. J.; Shankar, S. K.; Satishchandra, P.; Schroeder, J. T.; Sirdeshmukh, R.; Maitra, A.; Leach, S. D.; Drake, C. G.; Halushka, M. K.; Prasad, T. S. K.; Hruban, R. H.; Kerr, C. L.; Bader, G. D.; Iacobuzio-Donahue, C. A.; Gowda, H.; Pandey, A . A draft map of the human proteome. Nature [Online] 2014, 509, 575–581. https://doi.org/10.1038/nature13302 (accessed June 25, 2021).


Ramanathan, R. and Korfmacher, W. The emergence of high-resolution MS as the premier analytical tool in the pharmaceutical bioanalysis arena. Bioanalysis [Online] 2012, 4, 467–469. https://www.future-science.com/doi/pdfplus/10.4155/bio.12.16 (accessed June 14, 2021).


Vereyken, L.; Dillen, L.; Vreeken, R. J.; Cuyckens, F. High-Resolution Mass Spectrometry Quantification: Impact of Differences in Data Processing of Centroid and Continuum Data. J. Am. Soc. Mass. Spectrom. [Online] 2019, 30, 203-212. https://link.springer.com/article/10.1007/s13361-018-2101-0 (accessed June 14, 2021).


Wilhelm, M.; Schlegl, J.; Hahne, H.; Gholami, A. M.; Lieberenz, M.; Savitski, M. M.; Ziegler, E.; Butzmann, L.; Gessulat, S.; Marx, H.; Mathieson, T.; Lemeer, S.; Schnatbaum, K.; Reimer, U.; Wenschuh, H.; Mollenhauer, M.; Slotta-Huspenina, J.; Boese, J-H.; Bantscheff, M.; Gerstmair, A.; Faerber, F.; Kuster, B. Mass-spectrometry-based draft of the human proteome. Nature [Online] 2014, 509, 582–587. https://doi.org/10.1038/nature13319 (accessed June 25, 2021).


Other Resources:


Makarov, A. Orbitrap Mass Spectrometry: Past, Present, and Future. In 9th International Symposium on Recent Advances in POPs Analysis. April 28-29, 2014. Bremen, Germany. youtube.com/watch?v=pvgnOvhZU2A (accessed June 21, 2021).


Makarov, A. Orbitrap Instrumentation: the First Decade and Beyond. In Orbitrap and Science: 10 years together. 2015. youtube.com/watch?v=jijxTW1NbTg (accessed June 21, 2021).