The glycans present in a glycoprotein are complex structures and their characterization typically involves a multistep process: glycoprotein affinity purification, sample concentration, protein denaturation, glycan release, glycan purification, and analysis by MS/LC-MS. This process results in long lead times for analysis, as well as significant costs for skilled personnel and sophisticated instrumentation. A simple and reliable laboratory kit to give researchers a snapshot of the terminal glycosylation patterns would be a complementary and extremely useful research tool for studying natural and engineered glycoproteins. Lectenz® Bio is developing a quick, robust and easy to use method, called GlycoSenseTM, to determine relative amounts of key glycosylation features. The GlycoSenseTM method does not supplant a complete analysis of glycan structures but instead provides an overview of the glycosylation state at a fraction of the cost and time of current approaches. This approach can be thought of as a soluble analog of a 2D lectin microarray, offering unique and important advantages over either 2D microarrays or lectin blotting.
By combining the established capabilities and benefits of flow cytometry, with the use of bead-based carbohydrate-specific reagents (lectins, antibodies, and Lectenz®), the company is developing the GlycoSenseTM technology that is complementary to current MS- or LC-based glycoprofiling. While MS-based methods are highly sensitive, they require expensive instrumentation, extensive technical training, and sample purification, generally including glycan release. In contrast, flow cytometry is high-throughput, relatively low cost in terms of instrumentation and personnel, generates statistically robust data, and, in the GlycoSenseTM approach, sample purification is not required. The GlycoSenseTM approach requires no additional processing of the glycoprotein and is not limited to a single detection method (e.g.: streptavidin-PE) or instrument.
The GlycoSenseTM method extends 2D lectin microarray technologies into the domain of suspension arrays that can be analyzed with a flow cytometer, resulting in several advantages (see table). In contrast to 2D microarrays, suspension array approaches do not require lengthy biotin/fluorophore/nanomaterial labeling of the glycoprotein sample, repeated washing and drying steps, or specialized scanning equipment, and they have advantages in terms of the solution chemistry employed to immobilize the detection reagent uniformly on the bead surface, and in terms of the very large numbers of replicate measurements achievable. The cost and complexity associated with printing, storing and reading the flat arrays has inhibited the adoption of lectin screening as a technique for characterizing glycosylation.
|Microsphere Array (GlycoSenseTM)||Microarray||Microplate Array|
|Array element distinction||Microsphere (bead) fluorescence||Position||Well|
|Array preparation||Lectins conjugated to beads||Lectins spotted on the array||Lectins coating wells|
|Instrumentation||Flow cytometer||Microarray scanner||Plate reader|
|Multiplexing level||Low 100s (practically, 10)||1000s||1 per well|
|Replicates||Each bead||Multiple spots||Multiple wells|
|Sample volume||50-100µL||50-100 µL||20-250 µL|
|Washes||Not required||Sometimes required||Required|
|Time||1-2 hours||3-6 hours||4-5 hours|
|Throughput||High (with a microplate loader)||Low||Low|
The presentation of the glycoprint data is similar to that obtained from 2D arrays of lectins, however, using flow cytometry provides the crucial advantages of statistical reliability, low standard deviations, the ability to mix and match reagents on-the-fly to suit particular applications, and to easily detect and replace degraded reagents. These features make the GlycoSenseTM technology well-suited to use in independent research laboratories, particularly in applications where a detailed glycosylation analysis is neither necessary nor practical.
Performing a typical GlycoSenseTM analysis of a glycoprotein is outlined schematically in Figure 1. In a typical assay, a glycoprotein is combined with a multiplexed assortment of GlycoSenseTM beads. The glycoprotein is either intrinsically fluorescent, as in a fluorophore labelled glycoprotein, or more commonly will be labeled with a fluorescent secondary reagent that binds the glycoprotein. Such reagents are routinely employed for detection in Western blots, etc. The multiplexed mixture is prepared by combining an approximately equal number of spectrally-unique beads, conjugated to different carbohydrate-binding reagents, as desired. The fluorescence intensity, corresponding to glycoprotein bound to each bead, is measured for each multiplex bead by flow cytometry. Signals are corrected for any non-specific binding by subtracting signals from the glycoprotein in the presence of a set of BSA-conjugated control beads.
Figure 1. Schematic of representation of the multiplex interactions between multiple GlycoSenseTM beads and a glycoprotein analyte. Glycan-specific reagents are conjugated to microspheres (beads) with discrete levels of red fluorescence intensity. The beads can be mixed immediately prior to use to create a custom multiplex array specific to the target analyte, and then incubated with a green fluorescently labeled glycoprotein (either by direct labeling or through a labeled secondary antibody directed against the glycoprotein). The amount of glycoprotein recognized and bound by each bead is measured using flow cytometry and converted to a “glycoprint”, which can be monitored over time.
To ensure that the GlycoSenseTM reagents are capable of detecting and discriminating between the relevant glycan structures they are selected on the basis of their reported binding profiles as determined by screening against the large glycan microarrays available through the Consortium for Functional Glycomics. Furthermore, the company’s proprietary Lectenz® reagents are also used to detect specific glycan biomarkers of biological relevance for which existing lectins or antibodies are ineffective.