Capillary electrophoresis and capillary electrokinetic chromatography

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Capillary electrophoresis (CE) encompasses a series of a high resolution microseparation techniques carried out in fused silica capillaries having inner diameters ≤ 75 µm. The separation of peptides by free solution CE in low-pH buffer is rapid, with good selectivity, and is thus effective for peptide mapping, which we use for protein characterization and immobilized enzyme development. Selectivity can often be improved by adding a mechanism of partition to that of electromigration through the use of pseudo-stationary phases in a mode of CE called micellar electrokinetic chromatography (MEKC). We have carried out several studies of peptide separations by MEKC and by cyclodextrin EKC, probing the structure–dependent binding of peptide analogs to additives, determining association constants, and developing separation methods for a variety of applications. For example, we quantified peptide-micelle complexation and compared the results to association constants determined by absorption spectroscopy to investigate the extent of electrostatic versus hydrophobic analyte inclusion. Fundamental and applied projects involving the development of CE and EKC methods for various applications are ongoing in our research group. For example, we developed CE methods to separate oligosaccharides of chitin and chitosan (dimers to hexamers), as well as cellulose derivatives, after derivatization with the fluorophore aminopyrenetrisulfonic acid (APTS) for detection by laser induced fluorescence (LIF).

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Immobilized enzyme microreactors (IMER)

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Enzymatic digestion of proteins is an important sample preparation step in bottom-up protein sequencing. Digestion is traditionally carried out either in a homogeneous solution (liquid-phase) followed by separation and identification of peptide fragments, or in a non-homogeneous fashion “in-gel” on trapped proteins that have been separated by polyacrylamide gel electrophoresis. This is generally followed by peptide extraction and mass mapping. However, enzyme autolysis, non-reusability of enzyme and losses due to sample handling all contribute to reduced detection sensitivity in the determination of low abundance proteins. These limitations apply to protein sequencing as well, where enzymatic digestion is an integral step. By using immobilized (solid-phase) proteolytic enzymes rather than soluble formats, higher enzyme:substrate ratios can be used to accelerate digestion while avoiding unwanted autolysis products that can lead to ionization suppression in peptide mass mapping. We have been developing and characterizing enzyme microreactors (µ-IMERs) employing immobilized trypsin and chymotrypsin. Many linking chemistries and solid supports work well to immobilize enzymes. However, a simple method to achieve this in microfluidic channels is less obvious. Therefore we have been investigating methods for making glutaraldehyde-crosslinked enzyme in batch form and also by in-situ strategies to fabricate an IMER. Our long-term goal is to develop simple IMER methods for microfluidic platforms that eliminate the need to pack solid-phase particles. Peptide mapping of substrates digested by the crosslinked enzymes is achieved by CE, HPLC and MS.

The challenge of mapping very small amounts of protein is exacerbated by dilution of peptide fragments within the microreactor, leading to an overall loss in sensitivity of the mapping process. Therefore, we characterized a microscale solid-phase extraction (µSPE) device coupled to CE-UV for quantitative transfer of the tryptic fragments into the separation capillary. In another route to circumvent the UV-absorbance sensitivity limitation during the characterization of our crosslinked enzymes, we made fluorescently-labelled protein substrates so that the collected peptides could be mapped by the highly sensitive technique CE-laser induced fluorescence (LIF). Our lab is equipped with LIF detectors operating at 488 nm, 410 nm, 325 nm and 248 nm.

Microencapsulation is another means of enzyme immobilization that provides an interesting alternative to enzyme attachment on solid phases or enzyme crosslinking because the interior cavity of the capsules creates an aqueous microenvironment that protects the enzyme from the external medium to thereby maintain enzyme activity and stability. In collaboration with Prof. Dominic Rochefort’s group, we studied microencapsulated laccase, an oxidase enzyme, using o-phenylenediamine (OPD) as a model substrate. A CE-UV method was used to separate the substrate and products and to quantify the enzymatic reaction for both free and immobilized enzyme. Microcapsules were then packed into a capillary-sized microreactor and conversion of substrate to product followed by CE-UV in an off-line manner. Our goal is to develop an integrated system based on CE that can be used to evaluate the efficiency of microencapsulated enzymes in biosensor applications.

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Development of UV laser-based detection methods for CE

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The short optical pathlengths (≤ 50 µm) encountered in CE for on-column UV absorbance result in poor concentration detection limits (ca. 10 µM), an inconvenience that has plagued CE since its commercialization in the early 1990s. On the other hand, superb detection sensitivity for CE can be achieved using laser induced fluorescence (LIF); however, this method is limited by the minimum analyte concentration (10-100 nM) that can be effectively conjugated to an appropriate fluorophore unless a pre-concentration step is first incorporated. Our group is interested in laser-based methods that eliminate the need for fluorescent labelling. This has been carried out using an inexpensive KrF excimer laser (λ=248 nm) for a) thermo-optical absorbance (TOA) detection of organic acids and native or derivatized peptides, and b) laser-induced native fluorescence (LINF) detection of proteins and smaller organic acids in biofluids, pharmaceuticals and tryptophan-containing peptides. The TOA detector, which is a pump-probe beam refractive index technique that is essentially pathlength independent, has demonstrated detection limits 10-100 times lower than by traditional absorbance with CE, particularly for weakly absorbing species. The KrF excimer laser provides the latitude to study non-fluorescing species that have even marginal absorption in the range 220 to 280 nm by TOA, or species that naturally fluoresce when excited at 248 nm by LIF using this single-wavelength source.

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Development of chromatographic methods

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Our group has been developing LC and GC methods for a wide variety of applications in collaboration with the Regional Centre for Mass Spectrometry at UdeM. For example, we developed and validated two rapid LC-MS/MS methods for screening consumer products containing illegal and/or counterfeit drugs. More specifically, 82 erectile dysfunction-related species in one method and 24 cannabinoid species in the other could be simultaneously and accurately determined in less than 10 min. The proliferation of counterfeit and illegal drugs combined with their ease of procurement on the internet is an ongoing problem for food and drug security in all countries. Accurate and rapid determination of the drug components in products ranging from pills to smoking mixtures can be a challenge; standard GC-MS and HPLC-MS methods used by food and drug agencies can be slow and/or require knowledge about the analytes, i.e., the spectra must be in searchable databases or targeted methods like MRM must be used. Our rapid LC-MS/MS methods were based on solid-core particle chromatography coupled with a high resolution orbital ion trap mass spectrometry, which permitted full scan acquisition in an untargeted approach.

We developed a hydrophilic chromatography (HILIC)-mass spectrometry method to screen and sequence chitosan oligosaccharides produced by deacetylase enzymes as part of a collaborative project with Prof. Joelle Pelletier’s group on directed evolution of enzymes for green-chemistry. Chito-oligosaccharides (COS), which are the fully or partially deacetylated form of chitin (polyß-(1→4)-N-acetyl-D-glucosamine) found in crustacean shells and thus a waste fisheries product, are biocompatible materials used in the pharmaceutical and cosmetic industries. Using HILIC-MS with a polar cyano column, we separated up to 9 different forms of COS arising from varying degrees of enzymatic deacetylation of unlabeled chitin hexa-saccharide. By tandem MS we identified over 20 different deacetylation products resulting from positional isomers for a given degree of deacetylation.

HILIC-based methods are also used in a project aimed at separating the constituents in aqueous solvents used for carbon capture. These solvents are typically amine-based so classical reversed phase chromatography methods are not suitable to efficiently separate the highly polar species.

We are developing microscale LC methods to characterize new stationary phase materials based on cholic acid dimers. These materials, which demonstrate amphiphilic properties and potential as mixed-mode phases, are synthesized in small batches. Therefore, we pack 0.250 mm ID capillaries with a few mg of material to make micro-columns 10-15 cm in length. These are tested on a capillary-LC system equipped with a 500 nL UV flow-cell.    

We developed preparative scale LC and analytical LC-MS methods to fractionate and determine the chemical composition of the most biotoxic extracts associated with the combustion products of chlorogenic acid and of whole tobacco smoke. Chlorogenic acid is the most abundant naturally occurring polyphenol found in leaf tobacco. We identified by LC-MS over forty compounds in the dimethyl sulfoxide (DMSO) extract of the combustion products. The DMSO extract was then fractionated and sub-fractionated, with toxicity studies carried out on all fractions. The most toxic response was determined to contain catechol. A GC-MS method coupled to stir-bar sorptive extraction was applied to identify the most hydrophobic gas-phase components of whole tobacco smoke.

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