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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