15.2 · 15.2.2019 2 Sieving Matrices for CGE Polyacrylamide (PA ) is widely used in slab gel electrophoresis of proteins, and also frequently utilized in CGE. Initially, PA gels are

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Capillary gel electrophoresis (CGE)

-a coated capillary filled with gel (a molecular sieve) in the buffer-sample molecules move with aid of electric field through the gelnet-separation is based on sizes of compounds, and their apparentmobilities (eof is ~0)-the method is useful only for charged compounds-sample is introduced by electrokinetic injection

-chemical gels (viscose crosslinked gels)-physical gels (low viscose polymer solvents with monomers ofpolymers as the additives)-CGE is useful for peptide, protein, DNA, RNA, oligosaccharideand carbohydrate analytics (for very large molecules, molar massesmostly above 1 kDa)

CGE

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Sieving Matrices for CGE

Polyacrylamide (PA ) is widely used in slab gel electrophoresis of proteins, and also frequently utilized in CGE.Initially, PA gels are synthesized in-situ inside capillaries.Typically, the PA gel is prepared by mixing acrylamide (monomer), N,N’-methylenebis(acrylamide) (Bis, cross-linker), ammonium peroxydisulfate or ammonium persulfate (radical initiator), N,N,N’,N’-tetramethylethylenediamine (TEMED, catalyst) and other background electrolytes, introducing the mixture into thecapillary, and allowing the solution to polymerize inside the capillary.

Various polysaccharides form another important type of sieving matrices for protein separations. One advantage ofpolysaccharides is that these polymers do not absorb as much UV light as PA does.

Poly(vinyl alcohol) (PVA) gives a platform for on-line protein concentration and separation.

In poly(2-ethyl-2-oxazoline) gel the separation efficiencies are at ∼10 million plates per meter.

Polyethylene oxide (PEO) gives also high plate numbers for proteins.

CGE

The interior walls of capillaries used in CGE are often coated for two purposes:1) reducing protein-wall interactions and 2) suppressing electroosmotic flow(EOF).

Running CGE at low or zero EOF is important for achieving reproducibleresults. If an uncoated capillary is used to run CGE, the EOF will carry thesieving matrix from anode to cathode while SDS-protein complexes migrate inthe opposite direction.

Some of the proteins will never pass the detector, unless the EOF is so large thatit brings all SDS-protein complexes to the detector.

Covalent coatings are generally more stable than dynamic coatings. Thesecoatings are obtained by chemically bonding desired substances to capillarywalls.

Capillary coatings in CGE

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Nucleosides and nucleotides

Nucleotide is a nucleoside, where in each place of 5´ or 3´ the hydroxyl (-OH)group is attached to phosphoric acid.

Nucleotide RNA: the sugar is D-ribose =ribonucleotide

Nucleotide DNA: the sugar is 2´-deoxy-D-ribose = deoxyribonucleotide

Hydroxyethyl cellulose (HEC) with anaverage molecular weight of1,300,000 was selected as a sievingpolymer. The sieving polymer solutioncontained 0.5 wt % HEC solution, 0.5×TBE buffer, and 2× SYBR Green II

Tris(hydroxymethyl)aminomethane (Tris)-borate-ethylenediaminetetraacetic acid (EDTA) buffer: (TBE buffer) ---(44.5 mM Tris, 44.5 mM borate, 1.0 mM EDTA, pH 8.3)

Separation of Small DNAs by Gel Electrophoresis in a Fused Silica Capillary Coated with a Negatively Charged Copolymer

capillary coated with an acrylamide (AM)/acrylic acid (AA) copolymer(poly(AM-co-AA)) Separations 2017, 4(3), 28; doi:10.3390/separations4030028

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Electroosmosis aided gel separation with surface coatedcapillaries

Separation of Small DNAs by Gel Electrophoresis in a Fused Silica Capillary Coated with a Negatively Charged Copolymer

Separations 2017, 4(3), 28; doi:10.3390/separations4030028

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Nucleotides are biological molecules that form the building blocks ofnucleic acids (DNA and RNA) and serve to carry packets of energywithin the cell (ATP). In the form of the nucleoside triphosphates (ATP,GTP, CTP and UTP), nucleotides play central roles in metabolism.Inaddition, nucleotides participate in cell signaling (cGMP and cAMP), andare incorporated into important cofactors of enzymatic reactions (e.g.coenzyme A, FAD, FMN, NAD, and NADP+).

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RNADNA

Nucleotides

Adenosine 5´monophosphate

A ribonucleotide

Paternity Test from DNA with capillary gel electrophoresis

CGE

PCR (polymerase chain reaction) technique is used for manifolding individual genes or anyDNA fragments exponentially. The growth specific and it is done by denaturation (94–96 °C),annaling (45–65 °C), and elongation (72 °C).

DNA fingerprinting involves electrophoretic analysis of DNA fragment sizes.

Restriction enzymes are endonucleases which catalyze the cleavage of the phosphate bondswithin both strands of DNA. They require Mg2+ for activity aand generate a 5 prime (5´) anda 3 prime (3´) hydroxyl group at point of cleavage.

The distinquishing feature of restriction enzymes is that they only cut at very specificsequences of bases called recognition sites. Restriction enzymes are produced by manydifferent species of bacteria (incl. Blue-green algae).

Over 3000 restriction enzymes have been discovered.

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Applied Biosystems 3730xl DNA Analyzer 96-Well Capillary SequencerCE

DNA

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A simple example to evaluate the data

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Amino acids:

20 pieces

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CAPILLARY ISOELECTRIC FOCUSING (CIEF)

In CIEF, substances are separated on the basis of their isoelectric points (pI) by pHvalues.The most common type of compound that utilizes this analytical method is protein.The protein samples and a solution that forms a pH gradient are placed inside acapillary.The anodic end of the column is placed into an acidic solution, and the cathodicend in a basic solution.Under the influence of an applied electric field, charged proteins migrate throughthe medium until they reside in a region of the pH, where they become electricallyneutral and therefore stop migrating.Consequently, zones are focused until a steady state condition is reached.

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After focusing, the zones are migrated (mobilized) from thecapillary by a pressurized flow.

Alternatively, after focusing, salt (e.g. NaCl) can be added to theanolyte (acid reservoir) or catholyte.

By the principle of electroneutrality, Na+ is exchanged for H+ in thetube, generating a pH imbalance gradient which causes themigration of the components.

Sharp peaks are obtained with good resolution, and a large peakcapacity is observed mainly because the whole tube issimultaneously used for focusing.

CIEF

CAPILLARY ISOELECTRONIC FOCUSSING

• Used for zwitterionic and amphoteric compounds such as proteins and peptides• Separation is based on the isoelectric points (pI values) which indicate the pH at

which the compounds are externally electrically neutral and no longer migrate inan electric field

• pH gradients are generated by using in a capillary an electrolyte solution ofamphoteric substances. The pI values of the substances cover the pH rangeneeded.

• Aliphatic aminocarboxylic acids with different ratios of amino to carboxylic acidgroups are used as the ampholytes.

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CIEF•Separation of proteins and peptides•Separation is on based on pI (buffer contains pH-gradient)•Resolution of DpI < 0.005

• Ampholytes Ax (proteins)• Focusing ~ 6 kV - ~ 5 min - sharp boundaries• Mobilization ~ 7 kV ~ 5 - 15 min.

t = 0(capillary filled with sample

and ampholyte solution)

t > 0(pH gradient)

A1

A1

A1

A2

A2A2

A3

A3 A3

A4

A4

A4

S1S1

S1

S2 S2S2

S3

S3S3

A5

A5

A5

migration time (s)

resp

onse

S3S2S1

Mobilization

+ -

Detection

S1S1S1

S2S2S2

S3S3

S3 A1A1A1

A2A2A2

A3A3A3

A4A4A4

A5A5A5

Low pH High pH

CIEF

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-+

-+ Low pH buffer High pH buffer

CIEF

CIEF

pH = 3 pH = 9

pI 3.90 5.01 6.05 7.81

Separation is based on differences in isoelectric points (pI)

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CIEF - Principle

-ampholytes + sample+

anode cathodeH3PO4 NaOH

H+ -OHA+- + H+ A+ A- -OH + A+-

A+ A+- A-

pK1 pK2

-ampholytes + sample+

anode cathode

ampholytes + sample+

ampholytes + sample+

anode cathodeH3PO4 NaOH

H+ -OHA+- + H+ A+ A- -OH + A+-

A+ A+- A-

pK1 pK2

H3PO4 NaOHH+ -OH

A+- + H+ A+ A- -OH + A+-

A+ A+- A-

pK1 pK2

-focused molecules+

anode cathodeH3PO4 H3PO4

H+ 2H+ + 2e H2

or NaOH NaOH4-OH 2H2O + O2 + 4e -OH

or pressure + E detector

-focused molecules+

anode cathode

focused molecules+

focused molecules+

anode cathodeH3PO4 H3PO4

H+ 2H+ + 2e H2

or NaOH NaOH4-OH 2H2O + O2 + 4e -OH

or pressure + E detector

H3PO4 H3PO4H+ 2H+ + 2e H2

or NaOH NaOH4-OH 2H2O + O2 + 4e -OH

or pressure + E detector

Focusing Mobilization

A

For detection the focused analytes(proteins) have to be delivered to thedetection window (this stage is calledmobilization). Mobilization reduces theresolution.

CIEF

In this case, the electroosmotic force is weaker than elctrophoretic force.

OHNH3

O

+

O

NH3

O

+

pH pH

iso-electric

zwitterionic

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Creation of pH gradient by anolyte and catholyte underapplied electric field (migration of OH- and H30+ ions)

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Routine• Proteins in serum, urine, CSF (=Cerebrospinal fluid; in the brain and spinal cord)• Hemoglobins• Isoenzyme patterns• Carbohydr. Def. Transferrin (CDT)àmonitoring of alcohol abuse and cancer• Lipoproteins

Special• Metabolic disorders• Drugs (therapeutic, illicit) Forensics• Complex carbohydrates• DNA-Fingerprinting Forensics• Tissue Biopsies

Clinical applications

0 time (min) 12

1

2

3 4

56

7 8

•pH 3 - 10 Ampholytes•Compounds 1 - 8: pI 8.6 - 5.1

CIEF of protein standards

1. Lentil lectins (8.6, 8.4, 8.2)2. Human hemoglobin C (7.5)3. Human hemoglobin A (7.1)4. Equine myoglobin (7.0)5. Equine myoglobin, minor band (6.8)6. Human carbonic anhydrase (6.5)7. Bovine carbonic anhydrase (6.0)8. b-lactoglobulin B (5.1)

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CIEF – Human MonoclonalImmunoglobulin G (IgG2)

Minutes0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0

AU

-0.05

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

AU

-0.05

0.00

0.05

0.10

0.15

0.20

Blank

Calibrator blank

Calibrator blank + IgG2

IgG2 lot 1

7.3 5.9

Þ Importance of calibration to get accurate pI determination

IgG2 Lot 1 in Biolyte 3-10, Injection: 10kv/99s; 30kV/0.5 psi

CIEF –IgG2 Lot 1 vs Lot 2

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28

0.000

0.025

0.050

0.075

0.100

0.125

0.150

0.175

0.200

0.225 7.3

5.9

IgG2 lot 2

7.3 5.9

IgG2 lot 1

6.88

6.79 6.68

6.55

6.94

6.83

6.60

6.38

The resolving power of CIEF clearlyidentifies differences in the 2 lots

IgG immunoglobulin

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An example of measuring migration of amino acids in CE

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Amino acids:

20 pieces

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