Conventional isoelectric focusing with the Agilent 3100 OFFGEL Fractionator

Transcription

1 Conventional isoelectric focusing with the Agilent 3100 OFFGEL Fractionator Technical Overview Introduction This Technical Overview demonstrates the ability of the Agilent 3100 OFFGEL Fractionator to fractionate proteins or peptides based on their isoelectric points (pi) in liquid phase (OFFGEL mode). It also shows that the fractionator can be used to perform standard isoelectric focusing with immobilized ph gradient gel strips (INGEL mode.) The alternative INGEL mode provides great flexibility to further downstream sample processing, such as classical 2D gel electrophoresis and MALDI-TOF analysis. It profits from ease-of-use and reproducibility of the 3100 OFFGEL Fractionator. Overview OFFGEL fractionation is an advancement in separation technology that fractionates proteins or peptides according to their isoelectric points (pi) using immobilized ph gradient gel (IPG) strips. 1,2 The pi-based separation using the OFFGEL Fractionator takes place in a two-phase system, with an upper liquid phase divided in multiple compartments and a gel phase used as a conventional IPG strip. In contrast to traditional isoelectric focusing (IEF), the fractionated sample is delivered in liquid phase (OFFGEL mode). This is advantageous for downstream sample processes that require liquid samples, such as LC-MS. However, for other subsequent applications

2 such as classical 2D gel electrophoresis and MALDi TOF analysis it may be advantageous to process the sample in the IPG strip gel phase. In addition to the OFFGEL mode, the OFFGEL Fractionator can also be operated in the INGEL mode. Several preconfigured methods for protein and peptide fractionations are delivered with the system, including two methods for traditional IEF that are dependent upon the length of the IPG strip (Table 1). The data presented here focuses on the alternative INGEL mode of the Agilent 3100 OFFGEL Fractionator for standard IEF. Method name IPG strip length Recommended use IG_24_00 24 cm IPG-IEF in-gel default method for proteins and peptides for use with IPG-strips ph 3-10 or ph 4 7. IG_13_00 13 cm IPG-IEF in-gel default method for proteins and peptides for use with IPG-strips ph 3-10 or ph 4 7. Table 1 The Agilent 3100 OFFGEL Fractionator provides two INGEL default methods. Experimental Materials and equipment Proteins were obtained from Sigma- Aldrich Inc. (St. Louis, MO, USA). Unless otherwise noted, the following protein stocks were prepared in water at 4 mg/ml: soybean trypsin inhibitor (TI), bovine milk β-lactoglobulin A (βlg), bovine serum albumin (BSA), bovine erythrocytes carbonic anhydrase II (CAII), human erythrocytes carbonic anhydrase I (CAI), 5 mg/ml chicken egg ovalbumin (OA), chicken egg conalbumin (ConA), 0.8 mg/l rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in 7 M urea, 2 M thiourea, 1 % DTT, 5 % glycerol and bovine pancreas trypsinogen (TG). E. coli protein sample (lyophilized, 2.7 mg) was purchased from Bio-Rad Laboratories Inc. (Hercules, CA, USA). SDS-PAGE mini-gels, buffers and equipment were from Invitrogen (Carlsbad, CA, USA), and PhastGel Blue R-350 gel stain from GE Healthcare (Freiburg, Germany). The Agilent 3100 OFFGEL Fractionator, 3100 OFFGEL kits and the automated lab-on-a-chip platform were all provided by Agilent Technologies (Waldbronn, Germany). TIBCO Spotfire Decision Site software was purchased from TIBCO Software Inc. (Sommerville, MA, USA). Sample preparation Seven individual marker proteins, TI, βlg, OA, BSA, CAII, CAI, and TG (Table 2), were diluted using 20 µg protein and 250 µl protein OFFGEL buffer (7 M urea, 2 M thiourea, 1 % DTT, 5 % glycerol, 1 % IPG buffer ph 3 10). A standard protein mix containing nine different proteins listed in Table 2 was prepared by diluting 40 µg of each protein per lane in OFFGEL protein buffer resulting in a final concentration of 5 µg/ml each. The E. coli cell lysate was diluted using 0.25 mg protein in 250 µl buffer as described above but with IPG buffer ph

3 Protein Abbreviation pi MW [kda] Trypsin inhibitor TI from soybean β-lactoglobulin A βlg from bovine milk Albumin OA 4.5, from chicken egg white Albumin BSA 5.4, from bovine serum Carbonic anhydrase II CAII 5.4, from bovine erythrocytes Carbonic anhydrase I CAI from human erythrocytes Conalbumin ConA 6.0, 6.3, from chicken egg white Glyceraldehyde-3-phosphate dehydrogenase GAPDH 8.3, from rabbit muscle Trypsinogen TG from bovine pancreas Table 2 Mixture of 9 standard proteins with isoelectric point (pi) and molecular weight (MW). Isoelectric focusing in OFFGEL and INGEL mode The protein samples were focused using the Agilent 3100 OFFGEL Fractionator in the OFFGEL mode and the Agilent 3100 OFFGEL High Resolution kit as described in the user manual. 3 For standard IEF in INGEL mode, the following protocol was used: The seven individual proteins were loaded on 13 cm IPG strips ph 3 to 10 by rehydration for 1 hour at ambient temperature, using the tray of the 3100 OFFGEL Fractionator to hold the sample; the 9-protein mixture was loaded by overnight rehydration on 24-cm IPG strips, ph 3 to 10; and the E. coli sample was loaded by overnight rehydration on 13-cm IPG strips, ph 4 to 7. Subsequently, the following steps were performed, as detailed in the user man- ual. 3 After rehydration and sample loading, the IPG strip was placed in a new tray lane. Electrode pads were placed at each end of the IPG strip. Electrodes were attached to the tray, and the tray placed in the instrument as described in the Quick Start Guide 4. The tray lane was covered evenly with 1.5-mL mineral oil per IPG strip before starting the run. Focusing was performed using the IG_13_00 method for the 13 cm IPG strips and the IG_24_00 method for 24 cm IPG strips. Runs were stopped after achieving 20 kvh for the individual proteins, 64 kvh for the protein mixture, and 16 kvh for the E.coli cell lysate. IPG strips were either stained directly using Coomassie Brilliant Blue stain or transferred onto 4% to 12% Tris-Tricine gels for second dimension SDS-PAGE analysis. 3

4 On-chip electrophoresis of OFFGEL fractions As an alternative to the traditional electrophoresis in the second dimension, microfluidic on-chip electrophoresis was performed to separate the liquid OFFGEL proteins fractions according to their molecular weight. After focusing, 4 µl of each OFFGEL protein fraction were analyzed with an automated labon-a-chip platform. Data analysis was done using corresponding software in conjunction with the TIBCO Spotfire Decision Site. TI βlg OA BSA CAII CAI TG ph ph Figure 1 Coomassie stained IPG strips demonstrating the IEF of seven individual proteins (for protein abbreviations and pi see Table 2). 4

5 Results and discussion The IEF-based protein separation in the INGEL mode is a very reproducible method that returns sharply focused protein bands (Figure 1). Seven individual IEF marker proteins (Table 2) were analyzed separately on IPG strips in traditional INGEL mode, and Coomassie stained. Some proteins such β-lactoglobulin or carbonic anhydrase II focus in a single sharp band. Other proteins with multiple isoforms or multimers, such as ovalbumin or bovine serum albumin, focus in several very defined bands. In a separate experiment, a mixture of nine IEF standard proteins differing by molecular weight and pi (Table 2) were separated in the first dimension according to their pi with the 3100 OFFGEL Fractionator in INGEL mode as shown in Figure 1. The separation in the first dimension was followed by a molecular weight based separation using traditional SDS-PAGE. The obtained 2D gel was stained with Coomassie (Figure 2A). For comparison, the same protein mixture was fractionated using the OFFGEL mode followed by an on-chip protein electrophoresis with an automated labon-a-chip platform for the second dimension (Figure 2B). Proteins that focus in a single spot are found in one or two fractions only (for example, β- lactoglobulin), whereas those displaying multiple isoforms are detected in several fractions (like conalbumin). The data demonstrates an excellent correlation between the INGEL and OFFGEL modes using the Agilent 3100 OFFGEL Fractionator. For example, trypsin inhibitor is predominantly detected in fraction 4 and carbonic anhydrase II in fraction 14 using both approaches. Finally, the combination of protein focusing in INGEL-mode followed by conventional SDS-PAGE as second dimension was applied to a complex sample such as an E. coli cell lysate. Figure 3 demonstrates that this separation approach provides the resolution required by proteomics applications and is comparable to IEF with standard equipment. 5

6 A ph % of input MW (kda) B TI βlg OA TI βlg OA BSA BSA ConA CAII CAI CAI CAII ConA GAPDH Fraction TG TG GAPDH Figure 2 A mixture of nine standard proteins (Table 2) was separated using two different approaches: (A) IEF in the INGEL mode followed by SDS-PAGE and Coomassie staining. The red grid indicates the approximate locations of the 24 OFFGEL fractions. (B) OFFGEL fractionation followed by on-chip electrophoresis using an automated lab-on-a-chip platform providing details on protein content of a fraction. ph (A) OFFGEL Fractionator (B) Standard IEF Equipment Figure 3 E. coli lysate was separated in the first dimension with the Agilent 3100 OFFGEL Fractionator in the INGEL mode and for comparison with standard IEF equipment, followed by a SDS-PAGE for the separation in the second dimension. The resulting 2D-gels were stained with Coomassie. 6

7 Conclusions The Agilent 3100 OFFGEL Fractionator is a versatile instrument for isoelectric focusing of proteins and peptides, permitting both INGEL mode and the OFFGEL mode. The OFFGEL protein fractionation correlates well with the results obtained using the INGEL isoelectric focusing. Proteins or peptides are reproducibly recovered in gel or liquid phase, providing great flexibility to further downstream sample processing. References 1. P. E. Michel, F. Reymond, I. L. Arnaud, J. Josserand, H. H. Girault, and J. S. Rossier, Protein fractionation in a multicompartment device using Off-Gel isoelectric focusing, Electrophoresis, 24, 3 11, P. Hörth, C. A. Miller, T. Preckel, and C. Wenz C, Efficient fractionation and improved protein identification by peptide OFFGEL electrophoresis. Mol. Cell. Proteomics 5, , Agilent 3100 OFFGEL Fractionator, Agilent Technologies Manual, Agilent part number G , Agilent 3100 OFFGEL Fractionator Kit Quick Start Guide, Agilent Technologies Manual Agilent publication number ,

8 Agilent Technologies, Inc., 2010 July 1, 2010 Publication Number EN