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Analysis of Proteins with Capillary Gel Electrophoresis Fabulous Precision

Cianciulli, C.; Wätzig, H.

Innsbruck, Tuesday, September 20th

20th September 2011 | C. Cianciulli | Analysis of Proteins with CGE | Page 2

Quantitative Protein Analysis

Protein Quantitation

Gel Electrophoresis

Mass Spectrometry

High Performance Liquid

Chromatography

Capillary Electrophoresis

20th September 2011 | C. Cianciulli | Analysis of Proteins with CGE | Page 3

Quantitative Protein Analysis

Capillary Electrophoresis

cIEF

CZE CGE

20th September 2011 | C. Cianciulli | Analysis of Proteins with CGE | Page 4

CE system Agilent HP3D CE

Capillary Fused-silica, 50 µm ID, 375 µm OD; 33 cm Leff, 24.5 cm Ltot

Sample buffer 100 mM Tris-HCl, 1% SDS, pH 8.0

Separation buffer SDS Gel Buffer (Beckman Coulter)

Injection -5 kV for 20 sec

Temperature 25°C

Detection UV, 220 nm

Rinsing 0.1M NaOH, 0.1M HCl, H2O, SDS Gel Buffer 4 bar external pressure

Voltage / current -16.5 kV / -30 µA

Separation time 30-60 min

Sample Myoglobin Carbonic anhydrase Ovalbumin BSA

1 mg/ml 0.5 mg/ml 1 mg/ml 1 mg/ml

Application note

CE system Agilent HP3D CE

Capillary Fused-silica, 50 µm ID, 375 µm OD; 33 cm Leff, 24.5 cm Ltot

Sample buffer 100 mM Tris-HCl, 1% SDS, pH 8.0

Separation buffer SDS Gel Buffer (Beckman Coulter)

Injection -5 kV for 20 sec

Temperature 25°C

Detection UV, 220 nm

Rinsing 0.1M NaOH, 0.1M HCl, H2O, SDS Gel Buffer 4 bar external pressure

Voltage / current -16.5 kV / -30 µA

Separation time 30-60 min

Sample Myoglobin Carbonic anhydrase Ovalbumin BSA

1 mg/ml 0.5 mg/ml 1 mg/ml 1 mg/ml

CE system Agilent HP3D CE

Capillary Fused-silica, 50 µm ID, 375 µm OD; 33 cm Leff, 24.5 cm Ltot

Sample buffer 100 mM Tris-HCl, 1% SDS, pH 8.0

Separation buffer SDS Gel Buffer (Beckman Coulter)

Injection -5 kV for 20 sec

Temperature 25°C

Detection UV, 220 nm

Rinsing 0.1M NaOH, 0.1M HCl, H2O, SDS Gel Buffer 4 bar external pressure

Voltage / current -16.5 kV / -30 µA

Separation time 30-60 min

Sample Myoglobin Carbonic anhydrase Ovalbumin BSA

1 mg/ml 0.5 mg/ml 1 mg/ml 1 mg/ml

CE system Agilent HP3D CE

Capillary Fused-silica, 50 µm ID, 375 µm OD; 33 cm Leff, 24.5 cm Ltot

Sample buffer 100 mM Tris-HCl, 1% SDS, pH 8.0

Separation buffer SDS Gel Buffer (Beckman Coulter)

Injection -5 kV for 20 sec

Temperature 25°C

Detection UV, 220 nm

Rinsing 0.1M NaOH, 0.1M HCl, H2O, SDS Gel Buffer 4 bar external pressure

Voltage / current -16.5 kV / -30 µA

Separation time 30-60 min

Sample Myoglobin Carbonic anhydrase Ovalbumin BSA

1 mg/ml 0.5 mg/ml 1 mg/ml 1 mg/ml

CE system Agilent HP3D CE

Capillary Fused-silica, 50 µm ID, 375 µm OD; 33 cm Leff, 24.5 cm Ltot

Sample buffer 100 mM Tris-HCl, 1% SDS, pH 8.0

Separation buffer SDS Gel Buffer (Beckman Coulter)

Injection -5 kV for 20 sec

Temperature 25°C

Detection UV, 220 nm

Rinsing 0.1M NaOH, 0.1M HCl, H2O, SDS Gel Buffer 4 bar external pressure

Voltage / current -16.5 kV / -30 µA

Separation time 30-60 min

Sample Myoglobin Carbonic anhydrase Ovalbumin BSA

1 mg/ml 0.5 mg/ml 1 mg/ml 1 mg/ml

Peak area: RSD≈4% (n=6)

20th September 2011 | C. Cianciulli | Analysis of Proteins with CGE | Page 5

Optimisation

Reasons for the poor RSD% Signal-to-noise ratio <100 Increasing the injection volume Increasing the protein concentration Injection error Hydrodynamic injection Integration with CISS1 Evaluation of the results with the 100% method

1 Schirm, B., Wätzig, H., Chromatographia 1998, 48, 331-346

20th September 2011 | C. Cianciulli | Analysis of Proteins with CGE | Page 6

Optimisation – long series

Migration time

Mean [min] RSD% Myoglobin 14.33 0.76 Carbonic anhydrase 17.03 0.77 Ovalbumin 18.56 0.84 BSA 20.61 0.84

Relative migration time

Mean RSD% 0.841 0.11 Internal standard 1.09 0.13 1.21 0.13

20th September 2011 | C. Cianciulli | Analysis of Proteins with CGE | Page 7

Comparision of the injection modes

Hydrodynamic injection: 50 mbar 240 sec (n=48) Electrokinetic injection: -10 kV 30 sec (n=48)

Evaluated over the total area

Hydrodynamic injection

Mean [%] RSD% Myoglobin 28.0 2.24 Carbonic anhydrase 15.6 1.15 Ovalbumin 20.9 2.20 BSA 35.5 1.57

Electokinetic injection

Mean [%] RSD% 30.5 3.74 14.7 1.65 21.1 2.66 33.7 3.29

20th September 2011 | C. Cianciulli | Analysis of Proteins with CGE | Page 8

Application

20th September 2011 | C. Cianciulli | Analysis of Proteins with CGE | Page 9

165 kDa

173 kDa

LC: 25 kDa

HC: 54-62 kDa 110-156 kDa

Application

20th September 2011 | C. Cianciulli | Analysis of Proteins with CGE | Page 10

Summary

Fabulous precision

if the signal-to-noise ratio is greater than 100 if the sample is injected

hydrodynamically if CISS is used as integration software if the results are evaluated with the

100% method High selectivity

Precise tool for quality control of proteins

and antibodies instead of Gel Electrophoresis and Chromatographique Techniques

Hydrodynamic injection

Mean [%] RSD% Myoglobin 28.0 2.24 Carbonic anhydrase 15.6 1.15 Ovalbumin 20.9 2.20 BSA 35.5 1.57

20th September 2011 | C. Cianciulli | Analysis of Proteins with CGE | Page 11

Acknowledgment

Donia Elfahem Jorrit Maul Franziska Steinicke Postersession am Donnerstag: PO-135 PO-136 PO-141 PO-142 PO-178

20th September 2011 | C. Cianciulli | Analysis of Proteins with CGE | Page 12

Summary

Fabulous precision if the signal-to-noise ratio is greater

than 100 if the sample is injected

hydrodynamically if CISS is used as integration

software if the results are evaluated with the

100% method High selectivity

Precise tool for quality control of

proteins and antibodys instead of Gel Electrophoresis and Chromatographique Techniques

Hydrodynamic injection

Mean [%] RSD% Myoglobin 28.0 2.24 Carbonic anhydrase 15.6 1.15 Ovalbumin 20.9 2.20 BSA 35.5 1.57