Actively helping customers, employees and the global community during the coronavirus SARS-CoV-2 outbreak.  Learn more >>

Quantitative Analysis of Thiols and Maleimides

Quantitative analysis is an important tool in analytical chemistry that is used to determine the concentration of one or several particular substances present in a sample. In proteomic studies, quantitative analysis can be used to detect the amount of thiol functional groups and maleimide-reactive sites in a sample protein. This analysis can be used to characterize a protein over the course of an experiment and in bioconjugation it can help determine which crosslinking techniques are most desirable. Quantitation of thiol groups also provides analysis and insight into thiol-disulfide interconversions and the relationship it has on the oxidative-stress of a protein. Before discussing the specifics of thiol and maleimide quantification, however, it is perhaps worth discussing spectroscopy as it broadly applies to quantitative analysis.

 

Spectroscopy


One common approach for quantitating thiols and maleimides is through spectroscopic analysis. Spectroscopic analysis, or spectroscopy, is a powerful quantitative tool in analytical chemistry. It can be used to determine the presence of a particular substance in a sample by measuring the absorption of radiation, as a function of wavelength. This absorption spectrum in combination with Beer-Lambert's Law reveals a directly proportional relationship between absorbance and the concentration of the attenuating moieties, or functional groups, in the material sample. As the max absorbance of a particular substance increases so does the concentration of that same substance.

The combination of spectroscopic analysis with colored reagents has led to the development of a quantitative technique known as colorimetry. In colorimetric assays, a colored reagent causes a solution to undergo a measurable color change while in the presence of its target analyte. The intensity of this color change is directly proportional to the concentration of the substance; the more intense the color, the higher the concentration of the analyte. With the help of a colorimeter, such as a colorimetric microplate reader, the concentration of the particular substance in the solution can be determined by measuring the absorbance of that solution at a specific wavelength of light known as lambda max (λmax).

Colorimetry is a commonly accepted quantitative approach due to its simplistic protocol and the ability to easily detect a color change with the naked eye, allowing for quick results. The equipment necessary to perform a colorimetric assay is relatively cheap, and there is extensive documentation and validation of colored reagents in the scientific literature. Despite the long-standing use of colorimetric assays, however, they are susceptible to several key problems. For example, background interference by protein absorbance at the same wavelength (namely, 280 nm) can be detrimental to signal quality. Additionally, colorimetric assays suffer from poor dynamic range, leading to poor substrate sensitivity in some cases as well as easy signal saturation. If these problems are of concern, the use of a fluorimetric quantitative approach is recommended.

Fluorimetric assays are chosen for their extraordinary sensitivity and high specificity. This sensitive technique allows for accurate detection of fluorescent material in a relatively minute sample size which is perfect when the protein of focus is relatively expensive or available in small quantities. Fluorimetric assays can detect and quantitate moieties in a similar manner as colorimetric assays, albeit with the use of a fluorescent dye rather than a colored reagent. In fluorimetric spectroscopy, a fluorimeter excites the sample of interest and measures the intensity of the emitted light, which is proportional to the concentration of the analyte.


UV absorption suffers from poor selectivity and high background interference because many biological substances have a strong UV absorption from 250 nm to 400 nm. The NADH probe absorption above experiences background interference from protein absorption such as IgG at a wavelength of 280 nm (as highlighted by the shaded region below the absorption curves).


Fluorimetry is an advantageous quantitative tool because it produces minimal background interference and a brighter signal. It has a significantly broader dynamic range, which allows for assay linearity across many orders of magnitude. It is typical for a fluorimetric assay, for example, to have a significantly lower detection limit than a colorimetric assay of the same substrate. The greater assay linearity of fluorimetric assays allows them to detect fluorescent signals of different magnitude, all within one measurement. This makes fluorimetry a highly sensitive and efficient technique for measuring an extensive number of samples at one time. Furthermore, fluorimetric assays can easily be multiplex through use of different fluorometric probes, allowing for complex studies of numerous substrates simultaneously.



Chemistry of Thiols and Maleimides


The thiolate anion is intrinsically one of the strongest nucleophiles, making thiol groups one of the most reactive functional groups found in proteins. Thiols or sulfhydryl groups (-SH) are located in the side chains of cysteine residues. They participate in thiol-disulfide exchange reactions to introduce and remove protein disulfide bonds. These thiol-disulfide interconversions contribute to protein stability and reduce oxidative-stress. Protein disulfide bonds are involved in many cellular functions from the regulation of enzyme activity and signal transduction to protein folding. They also play a critical role in maleimide-based bioconjugation systems. The importance of thiol groups and disulfides in these functions of proteins has led to the development of many methods for the quantitative determination of thiol groups and disulfide compounds.

On the topic of conjugation systems, thiol detection can serve as a useful tool to determine if maleimide crosslinkers are appropriate for protein bioconjugation. Maleimides are crosslinkers that selectively target free thiols in proteins at near neutral conditions to form irreversible, stable thioether linkages. They are extremely beneficial in developing stable protein conjugates. The distinct location of thiol groups in cysteine residues and their relatively low abundance on proteins make them advantageous for site-specific bioconjugation. This is particularly important for labeling small proteins in applications where the activity or binding affinity of the conjugate is paramount. When conjugating antibodies with other proteins such as an enzyme, it is critical to avoid conjugation at or near the epitope-binding site because of the potential risk of blocking this site and rendering the conjugate inactive. The use of maleimides for this application eliminates this risk. Reduction of disulfide bonds found in the hinge and heavy chain region of antibodies, opposite the epitope-binding sites, generate free thiol groups which maleimides can target without compromising the functionality of the newly conjugated protein. Unfortunately, a key challenge in maleimide crosslinking is the lack of rapid and sensitive assay kits available to quantify the number of maleimide groups in a biopolymer.

 

Quantitation of Thiols



The chemical structure of Ellman's reagent, a chemical used to quantify the concentration of thiol groups in a sample.
Analysis of free thiols and disulfide bonds in proteins can be quantitatively determined by colorimetric and fluorimetric assays and by a variety of reagents. Colorimetric quantitation of thiols utilizes a color reagent to determine the concentration of a particular moiety in a sample by measuring the absorption of the colored solution. This quantitative approach is great for quantifying thiols in proteins and other high molecular weight molecules. Fluorimetric quantitation of thiols is a highly sensitive approach that can detect fluorescent materials in relatively small sample sizes and small molecules. Fluorimetric assays are highly specific and less susceptible to interferences because fewer materials absorb and also emit light (ie. fluoresce). Until recently, however, not many fluorimetric assays were commercially available to quantitate thiol groups on sample proteins.
 

The chemical structure of Ellman's reagent, a chemical used to quantify the concentration of thiol groups in a sample.
Two common colorimetric approaches for thiol detection use two distinct chromogenic thiol reagents. The first approach uses the thiol reagent DTNB (5, 5'-dithiobis (2-nitrobenzoic acid)), also known as Ellman's reagent. DTNB is an aromatic disulfide that reacts with thiols by an exchange reaction to form a mixed disulfide of the protein and one molecule of TNB, which ionizes to the TNB2-dianion in water, producing a yellow color. TNB is then quantified using a spectrophotometer with a max absorption at 412 nm and an extinction coefficient of 14,100 M-1cm-1. However, the disadvantage of DTNB as some studies have shown, is that it at times underestimates the concentration of thiol groups in a protein due to incomplete reaction of TNB. A second, more promising alternative to Ellman's reagent is the use of 4, 4' dithiodipyridine (DTDP). DTDP is smaller, more hydrophobic and mostly uncharged at neutral conditions, with a max absorption at 324 nm and an extinction coefficient of 19,800 M-1cm-1. This makes it a great reagent for protein sulfhydryls in their native environment. DTDP reacts with thiols in the same way as does Ellman's reagent.

Although these reagents are useful in quantitative analysis, limitations are associated with both. DTDP and Ellman's reagent are fairly sensitive to hydrolysis at elevated temperatures and pH values greater than 7. In these conditions, decomposition of the activated disulfide of DTNB impedes quantitative analysis. While DTDP has improved upon the sensitivity of DTNB, it experiences higher background interference by protein absorbance. This is because its max absorbance is at 324 nm, which is fairly close to the 280 nm absorbance of proteins. Furthermore, colorimetric assays of this sort are in general hindered by the need for frequent calibration steps, which increase the duration of this quantitative approach as well as provide more opportunities for human error.
 

Quantitation of Thiols with Amplite® Technology



Cysteine or GSH dose response
A key goal in colorimetric thiol quantitation has been the development of a rapid and accurate method to quantify the amount of free thiols in a specific protein. AAT Bioquest's Amplite® Rapid Colorimetric Protein Thiol Quantitation Kit (Cat# 5529) addresses this challenge with the uses of its proprietary thiol sensor, Thiol Blue™. Thiol Blue™ reacts with a protein sample that contains free thiol groups. Upon completion of the reaction, the resulting thiol adduct runs through a single spin column to remove the excess Thiol Blue™. The absorption spectrum of the purified product can then be measured at a max absorbance of ~680 nm with use of a colorimeter. The thiol to protein ratio is calculated from the ratio of absorbances at 680 nm (sensor max) and 280 nm (protein max).

For small molecule thiol quantification, such as cysteine and glutathione, AAT Bioquest offers a fluorimetric quantitative analysis kit called Amplite® Fluorimetric Total Thiol Quantitation Assay Kit (Cat# 5524). This kit utilizes a proprietary dye which, while initially non-fluorescent, emits a strong fluorescence upon reacting with thiols. It can detect as little as 1 picomole of cysteine. The absorption and emission spectra of the thiol adduct are pH-independent, making this assay kit highly robust.

 

Applications for Thiol Quantitation


One of the key applications for quantification of thiols is in relation to reactive oxygen species. Reactive oxygen species (ROS) are highly reactive and naturally occurring intracellular byproducts of cellular metabolism. Increases in the production of ROS in cells are related to the overconsumption of oxygen. When the production of ROS in cells far exceeds its neutralization by antioxidants, the cell experiences oxidative stress. Prolonged or excessive oxidative stress is detrimental, causing significant damage to DNA or RNA, deactivation of enzymes, and damage to cell structures which can lead to apoptosis. The natural production of an antioxidant, glutathione (GSH), in cells acts as a defense mechanism to counteract the adverse effects of elevated cellular ROS. Amplite® Thiol Quantitation Kits can be used to detect thiol groups in glutathione and monitor its rate of production in cells as a response to elevated levels of oxidative stress from cellular activities. GSH also plays an important role in the detoxification of a variety of compounds and regulation of protein and gene expression via thiol-disulfide exchange reactions. AAT Bioquest's Amplite® Thiol Quantitation Kits provide a rapid and sensitive approach for the detection and analysis of thiols resulting from thiol-disulfide exchange reactions. This can be a powerful tool in studying the effects of thiol oxidative-stress in areas such as immunochemistry.

In immunochemistry, phagocytic cells have been shown to regulate redox by producing antioxidants. For example, murine macrophages and dendritic cells secrete cysteine, which are taken up by T cells and converted to intracellular GSH. Furthermore, these phagocytic cells utilize GSH precursors, such as N-acetyl-L-Cysteine (NAC), to increase surface protein thiol expression. Through colorimetric and fluorimetric quantitative analysis, these increases in intracellular and surface-expressed thiols can be detected in mononuclear cells. This analysis allows for the study of thiol concentration and its effects on T cell reactivity and proliferation. AAT Bioquest's Amplite® Rapid Colorimetric Total Protein Thiol Quantitation Kit (Cat# 5529) can be used to investigate the role of protein surface thiol groups during redox regulations. To investigate the role of intracellular, small molecule thiols during redox regulations, AAT Bioquest offers a convenient and ultrasensitive kit- Amplite® Fluorimetric Total Thiol Quantitation Kit (Cat# 5524).

 

Quantitation of Maleimides


For a long time, the challenge of quantifying maleimide groups was the lack of commercially available colorimetric and fluorimetric quantification tools. A rudimentary approach for the quantitation of maleimides can be performed by spectrophotometrically assaying maleimides at a wavelength of 302 nm with an extinction coefficient of 620 M-1cm-1. Unfortunately, this approach is a very insensitive assay technique complicated by the interference of protein absorbance at a nearby wavelength (ie. 280 nm). To eliminate this complication, AAT Bioquest invested in developing the most comprehensive set of solutions for maleimide quantification. Utilizing proprietary technology, AAT Bioquest provides two highly sensitive colorimetric maleimide quantitation kits that provide a rapid and robust colorimetric method for quantifying maleimides as well as an ultrasensitive fluorimetric maleimide quantitation kit.

 

Quantitation of Maleimides with Amplite® Technology



4,4'-DTDP assay principle for quantifying maleimides.
AAT Bioquest's Amplite® colorimetric and fluorimetric quantitation kits are designed using proprietary technology to ensure rapid, robust and efficient quantitative analysis of maleimide groups. The quantitation of maleimides is an excellent intermediate step to verify the successful initial linkage of maleimides to the sample protein prior to bioconjugation with its target.

Colorimetric maleimide quantitation can be used as an intermediate step in bioconjugation involving the use of heterobifunctional crosslinker. Heterobifunctional crosslinkers are designed to have two different reactive-moieties at respective ends of the crosslinker. The bioconjugation of an antibody with an enzyme can be facilitated by heterobifunctional crosslinkers containing a NHS-ester and a maleimide moiety. Following the heterobifunctional-antibody reaction, the rate of successful linkage can be estimated by quantitative analysis of excess maleimides. AAT Bioquest offers an Amplite® Colorimetric Maleimide Quantitation Kit (Cat# 5525) that may be used for such purposes.



Amplite® Colorimetric Maleimide Quantitation Kit is a robust spectrophotometric assay that quantifies maleimide-reactive groups by reverse glutathione (GSH) assay with use of DTDP. This kit takes advantage of the high reactivity of GSH thiols with the maleimide moiety. GSH with a known amount of excess thiol is reacted with the sample to form stable thiosuccinimidyl linkages with GSH. The excess unreactive thiols of GSH are reacted with a DTDP and then assayed with a max absorption at 324 nm and a molar extinction coefficient of 19,800 M-1cm-1.

For a faster colorimetric maleimide assay, AAT Bioquest offers an Amplite® Rapid Colorimetric Maleimide Quantitation Kit (Cat# 5526). This kit utilizes our proprietary Maleimide Blue™ probe, which significantly simplifies the maleimide quantification process. The target sample simply needs to be incubated with the Maleimide Blue™ sensor and passed through a spin column. The elutant can then be directly read in a colorimeter at a max absorbance of about 780 nm. The large separation between sensor max and protein max ensures minimal spectral interference and clean assay results, and the simple protocol allows for quick, convenient maleimide quantification.

Both Amplite® colorimetric assays can be used to draw observations or make changes that will improve the success of bioconjugation. For example, a low max absorbance indicates a low concentration of maleimide moieties. This can be visualized with little to no color change in the assay solution. Since antibodies contain amine and thiol groups, a little to no color change can be indicative of self-polymerization, protein precipitation or antibody aggregation. This would indicate that changes should then be made to the bioconjugation protocol to in order to improve conjugation yield. An intense color change indicates a high concentration of maleimide-reactive sites after the initial crosslinking, and the antibody is ready for further bioconjugation with an enzyme or other protein.


N-ethylmaleimide dose response was measured in a 96-well black plate with Amplite® Fluorimetric Maleimide Quantitation Assay Kit (Cat# 5523) using a NOVOstar microplate reader (BMG Labtech). As little as 0.1 nM (10 picomol/well) maleimide can be detected with 10 minutes incubation time (n=3).
In sensitive and selective situations where the protein of interest is expensive or only available in relatively small amounts and successful bioconjugation is paramount, use of fluorimetric quantitative analysis is recommended. AAT Bioquest's Amplite® Fluorimetric Maleimide Quantitation Kit (Cat# 5523) is a convenient and ultrasensitive approach that can detect as little as 10 picomoles of maleimides. It can be used in a broad array of applications where the quantification of maleimide groups is essential. This fluorimetric assay has a wide dynamic range allowing for the detection of very weak and very strong signals in a large sample at one time. These benefits make fluorimetric analysis a sensitive and efficient approach to quantify maleimides.
 

Additional Information


 

Table 1. Thiols and Maleimides Quantitation Reagents and Assay Kits

Cat No.
Product Name
Ex (nm)
Em (nm)
Unit Size
17590Amplite® Colorimetric Maleimide Quantitation Kit324 100 Tests
5523Amplite® Rapid Colorimetric Total Protein Thiol Quantitation Assay Kit680 2 Tests
5523Amplite® Rapid Fluorimetric Total Thiol Quantitation Assay Kit *Green Fluorescence*510524200 Tests
21507Thiolite™ Blue3354605 mg
21506Thiolite™ Blue, AM3354601 mg


Further Reading

  1. Chen, Xinfeng, Mengjia Song, Bin Zhang, and Yi Zhang. "Reactive Oxygen Species Regulate T Cell Immune Response in the Tumor Microenvironment." Oxidative Medicine and Cellular Longevity 2016 (2016): 1-10.
  2. Cummins, Timothy D., Ashlee N. Higdon, Philip A. Kramer, Balu K. Chacko, Daniel W. Riggs, Joshua K. Salabei, Louis J. Dell'italia, Jianhua Zhang, Victor M. Darley-Usmar, and Bradford G. Hill. "Utilization of fluorescent probes for the quantification and identification of subcellular proteomes and biological processes regulated by lipid peroxidation products." Free Radical Biology and Medicine 59 (2013): 56-68.
  3. Habeeb, A.f.s.a. "[37] Reaction of protein sulfhydryl groups with Ellman's reagent." Methods in Enzymology Enzyme Structure, Part B (1972): 457-64.
  4. Hansen, Rosa E., and Jakob R. Winther. "An introduction to methods for analyzing thiols and disulfides: Reactions, reagents, and practical considerations." Analytical Biochemistry 394.2 (2009): 147-58.
  5. Henneman, Dorothy H., Mark D. Altschule, and Rose Marie Goncz. "Glutathione in Human Disease." Glutathione (1954): 299-310.
  6. Kim, Younggyu, Sam O. Ho, Natalie R. Gassman, You Korlann, Elizabeth V. Landorf, Frank R. Collart, and Shimon Weiss. "Efficient Site-Specific Labeling of Proteins via Cysteines." Bioconjugate Chemistry 19.3 (2008): 786-91.
  7. Laragione, T., V. Bonetto, F. Casoni, T. Massignan, G. Bianchi, E. Gianazza, and P. Ghezzi. "Redox regulation of surface protein thiols: Identification of integrin -4 as a molecular target by using redox proteomics." Proceedings of the National Academy of Sciences 100.25 (2003): 14737-4741.
  8. Maeda, Kenji, Christine Finnie, and Birte Svensson. "Cy5 maleimide labelling for sensitive detection of free thiols in native protein extracts: identification of seed proteins targeted by barley thioredoxin h isoforms." Biochemical Journal 378.2 (2004): 497-507.
  9. Martner, A., J. Aurelius, A. Rydstrom, K. Hellstrand, and F. B. Thoren. "Redox Remodeling by Dendritic Cells Protects Antigen-Specific T Cells against Oxidative Stress." The Journal of Immunology 187.12 (2011): 6243-248.
  10. Pires, Marcos M., and Jean Chmielewski. "Fluorescence Imaging of Cellular Glutathione Using a Latent Rhodamine." Organic Letters 10.5 (2008): 837-40.
  11. Singh, Rajeeva. "A Sensitive Assay for Maleimide Groups." Bioconjugate Chemistry 5.4 (1994): 348-51.
  12. Trivedi, Maulik, Jennifer Laurence, and Teruna Siahaan. "The Role of Thiols and Disulfides on Protein Stability." Current Protein & Peptide Science 10.6 (2009): 614-25.
  13. Winther, Jakob R., and Colin Thorpe. "Quantification of thiols and disulfides." Biochimica et Biophysica Acta (BBA) - General Subjects 1840.2 (2014): 838-46. Web.