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The shielding effect of glycerol against protein ionization in electrospray mass spectrometry.
Abstract: Most commercial recombinant proteins used as molecular biology tools, as well as many academically made preparations, are generally maintained in the presence of high glycerol concentrations after purification to maintain their biological activity. The present study shows that larger proteins containing high concentrations of glycerol are not amenable to analysis using conventional electrospray ionization mass spectrometry (ESI-MS) interfaces. In this investigation the presence of 25% (v/v) glycerol suppressed the signals of Taq DNA polymerase molecules, while 1% (v/v) glycerol suppressed the signal of horse heart myoglobin. The signal suppression was probably caused by the interaction of glycerol molecules with the proteins to create a shielding effect that prevents the ionization of the basic and/or acidic groups in the amino acid side chains. To overcome this difficulty the glycerol concentration was decreased to 5% (v/v) by dialyzing the Taq polymerase solution against water, and the cone voltage in the ESI triple-quadrupole mass spectrometer was set at 80-130 V. This permitted observation of a mass spectrum that contained ions corresponding to protonation of up to 50% of the ionizable basic groups. In the absence of glycerol up to 85% of the basic groups of Taq polymerase became ionized, as observed in the mass spectrum at relatively low cone voltages. An explanation of these and other observations is proposed, based on strong interactions between the protein molecules and glycerol. For purposes of comparison similar experiments were performed on myoglobin, a small protein with 21 basic groups, whose ionization was apparently suppressed in the presence of 1% (v/v) glycerol, since no mass spectrum could be obtained even at high cone voltages.
Copyright 2003 John Wiley & Sons, Ltd.
Publication Date: 2003-03-28 PubMed ID: 12661019DOI: 10.1002/rcm.958Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
This study examines how high levels of glycerol can hinder protein analysis via electrospray ionization mass spectrometry (ESI-MS), revealing that reducing glycerol concentrations and adjusting instrument settings can improve results.
Research Background and Aim
- The study is based on the observation that large proteins often contain high concentrations of glycerol to aid in retaining their biological activity post-purification.
- The research sought to investigate how these glycerol concentrations can affect the utilization of common ESI-MS interfaces for protein analysis, and to develop strategies for mitigating such effects.
Methods and Findings
- The researchers found that glycerol’s presence in significant amounts, such as 25% of the volume for Taq DNA polymerase and 1% for horse heart myoglobin, suppressed signals in ESI-MS measurements.
- This signal suppression is linked to glycerol’s interaction with the proteins, essentially creating a shield that prevents the ionization of the acid or base groups within the proteins’ amino acid side chains.
- In order to circumvent this, the researchers dialyzed the Taq polymerase solution to decrease the glycerol concentration to 5% of the volume and adjusted the cone voltage on the ESI-MS instrument to be within 80-130 V.
- Following these adjustments, the mass spectrum showed ions corresponding to protonation of half the ionizable basic groups. In contrast, in the absence of glycerol, up to 85% of such groups in the Taq polymerase were ionized even at lower cone voltages.
Further Analysis
- The researchers also replicated their experiments on myoglobin, a smaller protein containing 21 basic groups.
- It was found that even a 1% volume of glycerol seemingly suppressed its ionization since no mass spectrum could be obtained at high cone voltages.
- This finding further reinforces the noteworthiness of the protein-glycerol interaction and its significant implications for ESI-MS protein analysis.
Conclusion
- High concentrations of glycerol in proteins can greatly hamper their ionization in ESI-MS analyses due to glycerol’s shielding effect.
- However, glycerol concentration manipulation and appropriate setting adjustments on the ESI-MS instrument can help mitigate these effects and provide accurate results, as evidenced in experiments involving Taq polymerase and myoglobin.
Cite This Article
APA
Mendes MA, Chies JM, de Oliveira Dias AC, Filho SA, Palma MS.
(2003).
The shielding effect of glycerol against protein ionization in electrospray mass spectrometry.
Rapid Commun Mass Spectrom, 17(7), 672-677.
https://doi.org/10.1002/rcm.958 Publication
Researcher Affiliations
- Laboratory of Structural Biology and Zoochemistry, CEIS/Dept Biology, Institute of Biosciences, São Paulo State University (UNESP), Rio Claro, SP-Brazil.
MeSH Terms
- Animals
- Enzyme Stability
- Glycerol / chemistry
- Horses
- Ions / chemistry
- Myoglobin / chemistry
- Recombinant Proteins / chemistry
- Spectrometry, Mass, Electrospray Ionization / methods
- Taq Polymerase / chemistry
- Thermus
Citations
This article has been cited 3 times.- Gibson BA, Blaukopf C, Lou T, Chen L, Doolittle LK, Finkelstein I, Narlikar GJ, Gerlich DW, Rosen MK. In diverse conditions, intrinsic chromatin condensates have liquid-like material properties. Proc Natl Acad Sci U S A 2023 May 2;120(18):e2218085120.
- Crack JC, Le Brun NE. Native Mass Spectrometry of Iron-Sulfur Proteins. Methods Mol Biol 2021;2353:231-258.
- Vlahakis NW, Flowers CW, Liu M, Agdanowski MP, Johnson S, Summers JA, Jacobs LMC, Keyser C, Russell P, Rose SL, Orlans J, Adhami N, Chen Y, Sawaya MR, Basu S, de Sanctis D, Chen Y, Wakatsuki S, Nelson HM, Loo JA, Tang Y, Rodriguez JA. Combining MicroED and native mass spectrometry for structural discovery of enzyme-small molecule complexes. Proc Natl Acad Sci U S A 2025 Aug 5;122(31):e2503780122.
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