Posted by & filed under Enzymology.

Beta Glucuronidase (β-D-Glucuronidase) is an enzyme regularly used for in vitro drug metabolism studies, as well as in routine drug testing applications. β-Glucuronidase is an enzyme classified as a hydrolase, as it is actively involved in the hydrolytic cleavage of drug-glucuronide conjugates, also known as cleaving glucuronide tails. Glucuronide conjugate formation is a common metabolic process utilized by numerous organisms, and is called glucuronidation.

Glucuronidation plays many important roles in the human body, including often being a critical component of drug metabolism. Glucuronidation is an enzymatic process that takes place primarily in the liver, and is involved in the removal of toxins, drugs or other xenobiotics from the body. Glucuronidation is the enzyme facilitated process of transferring a glucuronic acid from UDP-glucuronic acid to the xenobiotic substance (Figure 1). Often this results in increased water solubility of the drug or other foreign molecule, allowing for the more efficient excretion from the organism.

Glucuronidation Reaction Mechanism

Figure 1. General mechanism for glucuronidation of morphine from UDP-Glucuronic acid.

For this reason, when a patient ingests a controlled substance, either legitimately or illegally, glucuronide conjugates, such as Morphine-6-glucuronide will be present in the subject’s blood as well as urine. This fact makes it possible for drug testing laboratories to perform routine screening of urine or blood for the presence of these controlled substances. The glucuronide tag complicates the analysis, and makes direct analysis and quantification difficult. Therefore, it is usually preferable to cleave the conjugate, resulting in the release of the drug compound or metabolite. There are multiple ways in which these conjugates can be cleaved, primarily consisting of chemical or enzymatic processes. The enzymatic approach is typically preferred for complex biological samples or in specific cases where the chemical approach is known to cause the formation of multiple by-products for a given conjugate.

Cleavage of the glucuronide-drug conjugate with the β–Glucuronidase enzyme renders the intact de-glucuronated organic compound that has better solubility properties and can more effectively be extracted from the sample. This glucuronide removed compounds can be readily detected by definitive analytical methods, such as liquid chromatography with a mass spectrometer detector (LCMS). Figure 2 illustrates the cleavage of the 6-Morphine-6-glucuronide (M6G) conjugate. After de-conjugation, and prior to analysis, it is often routine for the sample to be further processed through ion exchange chromatography or extraction before the final LCMS analysis is performed.

Beta-Glucuronidase Hydrolysis Reaction Mechanism

Figure 2. Beta Glucuronidase mediated hydrolysis of Morphine-6-glucuronide

It is important to note, that not all glucuronide-drug conjugates cleave at exactly the same rate, and some conjugates can hydrolyze at rates considerably different than other seemingly similar compounds. For example, it is known that Morphine forms multiple metabolic glucuronides (M3G and M6G). The M6G conjugate has an enzymatic hydrolysis rate similar to that of codeine, and roughly 1/4th that of the M3G glucuronide conjugate.1

Therefore, when developing a method for the cleavage of a wide range of substrates, it is worthwhile to explore conditions that will adequately account for these variations in conjugate hydrolysis rates. For this reason, protocols are often slightly overdeveloped, insuring that a single set of reaction conditions will fully cleave a wide range glucuronide-drug conjugates. Furthermore, depending on the application, it may also be beneficial to experiment with slight changes in temperature (up to about 65 ⁰C) when exploring optimal conditions.

β-Glucuronidase is obtained through its isolation from its natural biological source. There are many organisms that are rich in Beta-Glucuronidase, and several of these can be used to extract this enzyme. Some examples include: Bovine Liver, E. coli, Helix pomatia, limpets, Escherichia, and Abalone. While there have been a handful of studies comparing β–Glucuronidase sources, it is clear that Abalone tends to give some of the most favorable conversion results, and the least amount of chromatographic interference.2 Abalone as a β–Glucuronidase source is also one of the most economical options, which can be of particular concern in high throughput applications, such as routine drug screening.3

CovaChem produces some of the industries most active Beta-Glucuronidase. This product is available as both a 100,000 U/mL liquid (CovaChem ) and as a lyophilized powder. For product information, see the links below:

Beta-Glucuronidase Liquid (CovaChem 20101)
Beta-Glucuronidase Powder (CovaChem 20102)

For more general information regarding this enzyme, see our Beta-Glucuronidase page.

References

1. Comparison of the hydrolysis rates of morphine-3-glucuronide and morphine-6-glucuronide with acid and beta-glucuronidase. Romberg RW, Lee, L. J. Anal Toxicol. (1995) 19(3):157-162.
2. Enzymatic hydrolysis of conjugated steroid metabolites: search for optimum conditions using response surface methodology. Ferchaud V, Courcoux P, Le Bizec B, Monteau F, André F. Analyst. (2000) 125 (12): 2255-2259.
3. Evaluation of abalone β–Glucuronidase substitution in current urine hydrolysis procedures. Malik-Wolf B, Vorce, S, Holler, J, Bosy, T. J Anal Toxicol. (2014) 38 (3); 171-176.

Posted by & filed under Protein Crosslinking.

There are numerous biochemical reagents that utilize the NHS ester functional group. This efficient N-Hydroxysuccinimide reactive group was first introduced in 1975 by Bragg and Hou1. NHS esters, with a few exceptions, tend to be insoluble in aqueous buffers, which generally require them to first be dissolved in an anhydrous organic solvent, such as DMSO or DMF, prior to being added to the buffered aqueous solution containing the protein molecule of interest.
NHS / Sulfo-NHS Ester Reaction With Amines
Another variety of these reagents, the N-Hydroxysulfosuccinimide group (Sulfo-NHS), does possess a water soluble property, allowing these reagents to be dissolved directly in aqueous buffer. However, many users still dissolve Sulfo-NHS containing crosslinkers and protein modifiers in organic solvents prior to use. The reason for this is twofold. First, when dissolved in organic solvent, the propensity for the reagent in the stock solution to hydrolyze is much lower than when dissolved in water. Secondly, the solubility of Sulfo-NHS containing reagents is considerably higher in DMSO or DMF than in aqueous buffers. For example, in DMSO, many reagents with the Sulfo-NHS ester functional group can be dissolved at concentrations as high as 50 mg/mL, whereas concentrations of 5 to 10 mg/mL are more typical for Sulfo-NHS esters in water. It is important to note that in order to maintain NHS and Sulfo-NHS activities in organic solvent, the solvents should have very low water content, such as CovaChem’s Molecular Biology Grade DMSO (CovaChem 18252) or DMF (CovaChem 18251).
Many of these amine containing sample reactions with Sulfo-NHS and NHS esters are performed directly in an organic solvent, which often simplifies the procedure and gives the scientists more product isolation options. When carried out in dry solvent, these reactions tend to eliminate the hydrolysis by-products, and often result in the higher modification efficiencies, and require less NHS or Sulfo-NHS ester reagent. It is commonplace for the scientist to utilize a Lewis base, such as triethylamine to catalyze this reaction in organic solvent.
One of the primary benefits of NHS esters and Sulfo-NHS esters is that they tend to yield reaction products that are specific for the reaction with primary amine residues, such as those found on the N-terminus and on lysine residues in proteins. While other reactions, such as those with hydroxyl groups (-OH) and sulfhydryl groups (-SH) can occur, they tend to occur at a significantly slower rate, and yield reaction products that are easily hydrolyzed or otherwise exchanged for the more stable amide linkage. The reaction selectivity of NHS and Sulfo-NHS esters tends to be for primary amines (-NH2), and this is maximized under pH 7-9 aqueous reaction conditions.
It is important to note that any buffers used in these reactions should not contain an interfering nucleophile, such as a Tris or Tris-HCl buffering system. In fact, the addition of Tris-HCl (pH 7.5) or Glycine is an excellent way to quench the reaction, after the reaction has run its course, deactivating the remaining active ester. A common buffering system for these reactions is 100 mM Sodium phosphate with 150 mM Sodium Chloride pH 7.2, which comes pre-formulated and aliquoted in PBS DryBlend pouches (CovaChem 19213).
As shown by Sélo et al, the NHS and Sulfo-NHS ester is also capable of being used to more specifically target the N-terminus, rather than Lysine (K) amino groups. This is accomplished by taking advantage of the pKa differences of the two amino (-NH2) groups. The pKa of the ε–amino group of Lysine (K) is about 10.5, while that of the N-terminus is roughly 8.9. By lowering the buffer pH to 6.5, one can take advantage of this difference in protonation state. At pH 6.5, the Lysine ε–amino group would be more fully protonated (-NH3+) and non-nucleophilic, while the protein or peptides N-terminus would still be sufficiently available in the nucleophilic, non-protonated (-NH2) form2.
CovaChem manufactures a wide range of protein crosslinkers and protein modification reagents that contain this useful NHS or Sulfo-NHS ester reactive group. Many of our crosslinkers have an additional reactive group, making these molecules heterobifunctional, allowing for the targeting of an additional protein functional group. This gives the user even more control over their protein investigational studies, or bioconjugate formation.  Our technical representatives are readily available to assist in finding the right crosslinker or modifier and to aide in developing application specific procedures.

References
1. Bragg, P.D, Hou, C. European Journal of Biochemistry. 106, 495-503 (1975).
2. Sélo, I. Negroni, L, Creminon, C. Grassi, J., Wal, J.M. J Immunol. Methods. 199, 127-138 (1996).

Posted by & filed under Liquid Chromatrography.

The selection of the proper mobile phase is a critical component of successfully analyzing a sample by HPLC and LCMS. Good liquid chromatography begins with high quality mobile phase additives from CovaChem.

A typical requirement for LCMS buffers is that all additives added to the mobile phase should be volatile. In addition to having all of the important properties associated with HPLC buffers and solvents that lead to good separation, the need for mobile phase volatility is equally as important. This is one of the reasons why inorganic buffers, such as phosphate buffers, do not find much utility in LCMS applications. Although phosphate buffers and other inorganic buffers are quite useful in providing good analyte separations in HPLC, and are often an excellent choice for improving peak shape and reducing tailing effects of highly polar compounds, they are typically not used in LCMS due to their lack of volatility.

Trifluoroacetic acid(CovaChem 11204), provides a good alternative when analyte resolution is difficult or polar functional groups cause poor peak shape. TFA, when added to reversed phase mobile phases (such as Water and Acetonitrile), provides an acidic pH controlled environment, where protonation states of the analyte remain consistent. Furthermore, TFA has an ion pairing effect, which is sometimes of critical importance when trying to separate polar compounds with subtle structural variations, such as peptides or amino acids.

TFA Buffers are volatile, and can be used in LCMS applications, but certain things should be considered when doing so. The ion pairing effect of TFA, which can be of great benefit to chromatographic separations, can cause decreased ionization efficiencies in the mass spectrometer, due to partial charge masking of the analyte. One very useful technique for reversing this ion pairing effect for optimal ionization is the employment of a “TFA fix.”

There are a couple of approaches one can take when trying to maximize ionization efficiency when using TFA in their HPLC-MS (LCMS) mobile phase. The first approach involves reducing the concentration of TFA in the mobile phase and increasing the concentration of another volatile organic acid, such as Formic acid, which has a weaker ion pairing effect than TFA. This is the basis behind CovaChem’s LCMS Grade Formic Acid plus TFA, 1.1 mL ampules (CovaChem 11207-10×1) and 25 mL premade formulation. This product when added to the mobile phase tends to give all the benefits of TFA in the separation, but less peak suppression in the mass spectrometer, due to the lower abundance of the trifluoroacetate anion at the ionization source.

The second approach involves the post column addition of a volatile organic acid, typically Propionic acid (CovaChem 11206), Acetic acid (CovaChem 11201) or Formic acid (CovaChem 11202), with the former being the most common. This is accomplished by using a mechanical syringe pump and a “T” fitting. While using a minimum amount of tubing, the post column eluent is directed toward the “T” fitting, which has a syringe pump containing either concentrated or dilute organic acid. The organic acid in the syringe is slowly and continuously pumped into the “T” fitting, and the post column eluent and TFA fix acid are mixed in the fitting and directed towards the electrospray ionization source for detection by mass spectrometry.

Both approaches are very effective ways of preventing peak masking and ion suppression in the ESI chamber, however because every chromatographic application is unique, requiring its own set of chromatography conditions, it is impossible to say which approach will give the best results without a direct comparison. Regardless, when TFA is deemed necessary for your LCMS separation, it is nice to know you have one more tool in the toolbox to help get the job done.

Posted by & filed under MALDI Mass Spectrometry.

MALDI Matrix: Some Important Features and the Fundamentals of Ionization

MALDI (Matrix Assisted Laser Desorption Ionization) was first introduced in 1985 and since then, MALDI-MS has been a common means of analysis for large biomolecules (proteins, peptides, sugars), as well as organic molecules such as polymers. There is also increasing interest in lipid analysis1. There is no denying that MALDI methods are revolutionary, for they allow soft ionization which permits intact analysis of compounds that would easily fragment through other analysis methods. Several matrices have been developed, to respond to various demands in the field. A couple of the more versatile matrices for MALDI-MS are 2,5-dihydroxybenzoic acid (DHB) and α-Cyano-4-hydroxycinnamic acid (CHCA). However, before you decide which matrix best suits the experiments you want to perform, there are a few things you you might want to consider. While this article is far from all inclusive, its intention is solely to help keep a few things in mind when getting started.

First, the matrix you’re using should have a strong absorbance at the laser wavelength you will use to irradiate. A strong absorbance of the wavelength used is critical to ensure optimal ionization of the compound to be analyzed. DHB and CHCA are convenient for many wavelengths, ranging from 300 to 360 nm, with λ maximums of both of these matrices at 337 nm and 355 nm. Not surprisingly, one of the most common lasers used in UV MALDI matrix is centered at 337 nm (N2 laser).

One way to aide in determining if your analyte / matrix ratio is optimal is to compare the UV-VIS spectra of the matrix to that of its analyte / matrix mixture. Ideally, the λmax should not shift significantly, or this would indicate that the ionization conditions will likely be less than optimal, as this will likely decrease energy transfer from the 337 nm N2 laser or other laser employed. Another lement to consider is the solubility of both your matrix and your analyte compound. Ideally, both should be highly soluble in one single, volatile solvent or cosolvent. While there are methods to overcome solubility problems (such as the sandwich technique2 or the overlayer method3), these are usually less than optimal and increases sample preparation time, especially when analyzing large numbers of samples. DHB is particularly useful in these circumstances, as it is quite soluble in a wide range of solvents, including water and aqueous cosolvents. Also, once the droplet is drying on the plate, the matrix should effectively co-crystallize with the analyte, which is important for spectral reproducibility1. DHB is also useful in this capacity, as it is miscible with a wide range of molecules and is highly crystalline.

The matrix should be appropriately acidic or basic, depending on whether you’re analyzing for positive or negative ions, and the pKa of the sample1. For typical protein and peptides MALDI-MS, 2,5-dihydroxybenzoic acid (DHB) and CHCA are commonly used, with pKa values of 2.95 and 1.17 respectively. Not surprisingly, other isomers of dihydroxybenzoic acid also lend themselves to other applications in MALDI-MS, where analyte ionization is more optimal with a matrix with a differing pKa. This effect has been thoroughly evaluated on a various PEG polymers, and revealed that the best ionization in fact came from 2,6- Dihydroxybenzoic acid (CovaChem 15107)4, which has a lower pKa than the more commonly used 2,5-Dihydroxybenzoic acid counterpart (CovaChem 15102).

The final characteristic which is critical to ionization is having an adequately clean sample to analyze, and a sufficiently pure MALDI matrix that is free from metals and other ionic compounds that have proven to be detrimental to ionization and M+1 ion formation. This characteristic of the sample / matrix combination is particularly important, when attempting to decipher low level analytes from the baseline. Furthermore, the presence of metal ions and other impurities can also lend themselves to false identification of ions.

While this article is very general, it is hoped that this may aide in considering the key concepts associated with MALDI ionization when preparing samples and choosing the appropriate matrix. To conclude, CHCA and DHB are very useful matrices that serve as a good starting point for your MALDI-MS analyses of peptide fragments and proteins below 5,000 Da. DHB is also a good starting point MALDI matrix for carbohydrates, lipids, and some synthetic polymers. Please stay tuned for CovaChem’s MALDI Matrix selection guide, which will provide even more of a guide towards selecting the best MALDI matrix for you samples analysis, which will be published soon.

References:

1. Schiller, J. et al. Eur. Biophys. J. 36 (2007), 517-527.

2. Liang Li, Rafael E. Golding and Randy M. Whittal. J. Am. Chem. Soc., 118 (1996) 11662-11663.

3. Yuqin Dai, Randy Whittal and Liang Li. Anal. Chem. 71 (1999) 1087-1091.

4. Aera Lee, Hyo-Jik Yang, Yangsun Kim, Jeongkwon Kim. Bull Korean Chem Soc. 30 (2009) 1127-1130.

dtssp crosslinking agent structure

Posted by & filed under Protein Crosslinking.

Crosslinking with DTSSP, separation with SDS-PAGE, digestion and subsequent MS analysis. DTSSP makes mass spectrometry analysis less complicated.

Chemically crosslinking proteins, of a cell lysate, on the surface or inside of cells, with a crosslinker, such as BS3, are common ways to perform protein crosslinking reactions. After completion of the crosslinking reaction (and cell lysis if necessary), often the crosslinked protein complexes are identified through 2D-SDS-PAGE, in gel trypsin digestion, followed by mass spectrometry, such as MALDI-MS.

This approach, can give direct evidence for specific protein-protein interactions, as it can be safely inferred that the two proteins would have to be interacting with one another in order to be crosslinked with a small molecule protein crosslinker. This approach can at times be cumbersome, and the mass spectrometry data generated can be overwhelming.

One way to simplify the data analysis would be to use a cleavable crosslinker like CovaChem’s DTSSP. After the proteins have been crosslinked with DTSSP, the lysed mixture could be immunoprecipited for the protein of interest, the precipitate washed, then reduced with 10-50 mM DTT (CovaChem 11302-10×5), separated with SDS-PAGE, digested in-gel with trypsin, followed by MALDI-MS analysis.

Using this approach with DTSSP, and cleaving the crosslink between the two proteins allows for easier identification of interacting proteins, as the proteins can be isolated from one another prior to digestion, and subsequent mass spectrometry. Sometimes a little simplification of the data can make all the difference. Best of luck in your research.