Microchip Electrophoresis for Glycoprotein Separation

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Analytical Strategy,

September 30th, 2014

Karina Hasler & Adrian Müller

30.09.2014 Karina Hasler & Adrian Müller 1

Microchip Electrophoresis for Glycoprotein Separation

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• Glycoproteins

• Available techniques

• Electrophoresis microchip design

• Technical issues of channels in microchip electrophoresis

• Workflow from sample to analysis

• Microchip electrophoresis and related microfluidic methods – promising solution for point-of-care diagnostics?


Content

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• Protein that contains an poly-saccharide chain which is covalently attached to a polypeptide side chain

• Glycosylation takes place in the ER and is a co- or post-translational modification

• N-linked (e.g. asparagine) or O-linked (e.g. serine, threonine)


Glycoproteins – Structure & Synthesis

http://www.foodnetworksolution.com/wiki/word/1595/glycoprotein

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• Important function as integral membrane proteins especially in the cell-cell, hormone-cell, bacterium-cell and virus-cell interactions


Glycoproteins – Functions & Examples

http://de.wikipedia.org/wiki/Zellmembran

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Glycoproteins – Functions & Examples

Function Glycoprotein

Structural Molecule Collagens

Hormone HCG (Human chorionic gonadotropin) TSH (thyroid-stimulating hormone)

Enzyme e.g. alkaline phosphatase, patatin

Receptor Various proteins involved in hormone and drug action

…

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• Definition: A biomarker is a molecule that allows the detection and isolation of a particular cell type. In this case, glycoproteins are used to detect different diseases in the blood serum.

• Diseases: Ovarian cancer, chronic liver disease, esophageal adenocarcinoma…


Glycoproteins – Biomakers

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• Structural information must be obtained from small quantities of isolated glycoproteins.

• Resolution of different glycan isomers

• They do not contain good chromophores. A chromophore is the part of a molecule which is responsible for its colour. To solve this problem, a glycan can be labelled.


Glycoproteins – Analytical challenges

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• e.g. MALDI-TOF-MS

• A powerful tool for the identification of cancer-linked markers by comparative glycomic analysis

• Capable for structural characterization of unknown glycans through the molecular mass

• High-throughput analysis of known glycan structure


Available techniques - MS

+ small sample volumes and quantities - resolution of structural isomers

+ sensitive - expensive

+ effective in glycan profiling - too sophisticated for routine clinical work

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• Group of electrokinetic separation methods which are performed in submillimeter capillaries.


Available techniques – Capillary electrophoresis (CE) with laser-induced fluorescence detection

http://en.wikipedia.org/wiki/Capillary_electrophoresis

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• The analytes migrate through the electrolyte solution under the influence of an electric field.

• The separation of the compounds is dependent on the different migrations of the analytes in the applied electrical field.

• E.g. capillary gel electrophoresis


Available techniques – Capillary electrophoresis (CE) with laser-induced fluorescence detection

+ highly efficient in isomers separation (e.g. positional and linkage)

- capability of structural identification

+ practical for clinical analysis

+ tremendous detection selectivity

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Microchip - Microfluidics

• High-throughput • Low cost • Reduced sample volumes (10-9-10-18 L) • Increased efficiency in separation methods • Improved heat transfer • Simplicity of the planar design parallelization • Separation time • Serial processing Materials:

• Mostly polymers: PDMS • Or glass, silicon etc.

Dittrich and Manz, Nature Drug Disc 5 (2006), 210-218 Whitesides, Nature 442 (2006), 368-373 http://de.wikipedia.org/wiki/Datei:PmdsStructure.png

Karina Hasler & Adrian Müller 30.09.2014 11

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Electrophoresis Microchip Designs

Serpentine channel Spiral channel

Mitra et al. Anal. Chem. 84 (2012), 3621-3627 Zhuang et. al., Anal. Chem. 79 (20107) 7170-7175


• Minimized racetrack effect • Potentials are applied simultaneously to sample buffer and waste volumes

Incorporate low dispersion turns

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• «Racetrack» effect

• Channel length

• Coating

• Sample introduction

• Detection


Technical Issues of Channels in Microchip Electro-phoresis

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• While passing the U-turn, the fluid changes its velocity distribution to non-homogeneous

• The change in fluid arrangement changes the results of the measurements


«Racetrack» Effect

http://www.google.com/patents/US6186660

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Channel Length & Electric Field Strength

Long channels >20 cm

Higher electric fields >1000 V/cm

Rapidly and efficiently separate N-glycans

Analysis time < 100s

Short channels <10 cm Modest electric field strengths <500 V/cm


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Coating with Linear Polyacrylamide (PAM)

Minimize electroosmotic flow

Prevent analyte adsorption

http://upload.wikimedia.org/wikipedia/commons/6/6b/Polyacrylamide.png 30.09.2014 Karina Hasler & Adrian Müller 16

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Sample Introduction

Standard or modified pinched injection

Zhuang et al., Anal. Chem. 79 (20107) 7170-7175 30.09.2014 Karina Hasler & Adrian Müller 17

a) Transmitted light image of the cross intersection b) Sample loading c) Injection d) Analysis

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Labelling & Detection

• Label with APTS fluorophor to permit fluorescence detection

• Most popular approach is fluorescence analysis for detection

• Fluorescence detection with the use of an inverted optical microscope is usual

Vanderschaeghe et. al., Anal. Chem. 82 (2010), 7408-7415 Dittrich and Manz, Nature Drug Disc 5 (2006), 210-218 30.09.2014 Karina Hasler & Adrian Müller 18

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Workflow from Sample to Analysis

Dittrich and Manz, Nature Drug Disc 5 (2006), 210-218 Vanderschaeghe et. al., Anal. Chem. 82 (2010), 7408-7415 30.09.2014 Karina Hasler & Adrian Müller 19

• µTAS • Sampling, sample pre-treatment & transport, chemical reactions,

analyte separation, product isolation and detection

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Patients serum

Vanderschaeghe et. al., Anal. Chem. 82 (2010), 7408-7415

• Collection of blood samples

• Take 3µL of blood serum


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Protein Denaturation

• Dilution of samples

• Denaturation of proteins

Vanderschaeghe et. al., Anal. Chem. 82 (2010), 7408-7415 30.09.2014 Karina Hasler & Adrian Müller 21

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N-Glycan Release

• PNGase F (reagent B) specifically removes the N-glycans from the denatured proteins to which they are attached.


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Desialylation

• Remove sialyl group for simplifying the capillary electrophoresis output.


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Evaporate to Dryness

• The samples were then evaporated to dryness in a thermo-cycler at 80°C.


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APTS Labelling

Labelling of the N-glycans with APTS:

• Labelling by reductive amination • Increase of sensitivity • Provides the analytes with a charge and fluorescence

Vanderschaeghe et. al., Anal. Chem. 82 (2010), 7408-7415

8-aminopyrene-1,3,6-trisulfonic acid


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Electrophoretic Separation

Nomenclature and structures of the most abundant N-glycans in the glycome profile:

Mmitra et. al. J. Proteome Res. 12 (2013), 4490-4496

Electrophoresis profile:


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• Potential to be used as a simple and robust method for routine analysis of clinical samples

• Low costs of mass producing microchips (1 CHF)

• Short times for analysis

• Simplicity of the set-up & easy to bring to field

• Ability to use very small quantities of samples and reagents

• Carry out separations and detections with high resolution and sensitivity

• Physiologically relevant environments can be achieved

• Parallel reactions on one chip


Microchip electrophoresis & related microfluidics methods – positive features

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• Ease of handling and robustness of systems have to be refined

• Process complex biological samples without the sophisticated sample preconditioning capabilities available in centralized labs

• All steps of their use should be simple and as culturally in-dependent as possible

• The reaction are influenced by temperature, pH, ionic concentration etc. and should be able to be used in the whole world, so it has to be stable


Microchip electrophoresis & related microfluidics methods – current challenges

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• Definition: Point-of-care testing or short POCT refers to the medical diagnostic tests that are performed directly in the hospital, in practice of a medical practitioner or in a pharmacy and not in a laboratory.

• Examples: Electrochemical sensors (e.g. blood chemistry, urin- alysis and small molecules analytes) and lateral- flow assays (LFAs) (e.g. pregnancy test)


Microchip electrophoresis & related microfluidics methods – Point-of-care diagnostics?

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Electrochemical sensor

(e.g. diabetes test)

Lateral-flow assays (LFAs)

(e.g. pregnancy test)


Microchip electrophoresis & related microfluidics methods – Point-of-care diagnostics?

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Thank you for your attention!

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Microchip Electrophoresis for Glycoprotein Separation