FaMD-HPLC

Fluorescence-assistant

Multi-dimensional HPLC

 

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Through the use of fluorescent dyes to label protein samples prior to 2-D PAGE, the DIGE technique allows multiple samples to be co-separated and visualized on one 2-D gel. Up to three protein extracts, for example one control and two treated, are labeled with different fluorescent dyes (Cy2, Cy3 and/or Cy5), then combined and separated by 2-D PAGE (see Figure). Up to three images of the gel are captured – using the Cy2, Cy3 and/or Cy5 excitation wavelengths. The images are then merged, and differences between them can be determined using image analysis software. The dyes are purported to have a linear response to variation in protein concentration over five orders of magnitude, offer sub-nanogram sensitivity, and are compatible with MS analysis. A main disadvantage to this technology is the high cost involved in acquiring equipment as well as expendable supplies, such as the fluorescent dyes.

Selected References:
Unlu, M, Morgan, ME, and Minden, JS. 1997. Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis, 18:2071-77.

Tonge, R, Shaw, J, Middleton, B, Rowlinson, R, Rayner, S, Young, J, Pognan, F, Hawkins, E, Currie, I, and Davison, M. 2001. Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology. Proteomics, 1:377-396

FaMD HPLC Protocol:

1.      Please see the Ettan DIGE User Manual for all buffer recipes and for more detailed directions - the following provides only an overview.

2.      Resuspend cell pellets in 1 ml Standard Cell Wash Buffer in a 1.5 ml microcentrifuge tube. Note: 1 x 106 tissue culture cells or 3 x 108 bacterial cells contain approximately 50 protein.

3.      Pellet the cells (12,000 g for 4 min in a refrigerated bench top microfuge) and repeat steps 2 and 3 at least once more to remove all media which in the case of cell culture media may contain large amounts of albumin.

4.      Lyse the cells with a volume of Lysis Buffer (7M urea, 2M thiourea, 4% CHAPS, in 25mM tris, pH 8.6, @ 40C) such that the final protein concentration should be from 5-10 mg/ml

5.      Centrifuge the cell lysates and confirm with pH paper that the pH is still at 8.6.

6.      Store cell lysates in aliquots at -70 degrees C while protein assays are carried out on aliquots of all lysates.

7.      Use hydrolysis/amino acid analysis (available through the Keck Laboratory) or the PlusOne 2-D Quant Kit (GE Healthcare), which is compatible with detergents, to determine the protein concentrations in all samples.

8.      In vitro label 50ug of the control protein extract and 50ug of the experimental protein extract with GE Healthcare Cy-3 and Cy-5 N-hydroxysuccinimidyl ester dyes. These dyes have been matched with respect to charge and mass - with the single positive charge of the dye replacing the charge lost by the modified lysine or N-terminus of the protein. Cy-3 and Cy-5 labeled proteins co-migrate - with the dye label adding approximately 450 Da to the proteins in each sample.

9.      Optional (highly recommended) use of a third dye (Cy-2) as an internal (pooled 25ug control + 25ug experimental) standard to permit normalization of multiple gels and for internal normalization.

10.  Mix control, experimental, and internal standard samples together (i.e., 150ug total protein) and then add an equal volume of 2X Sample Buffer.

11.  Bring volume to 450 ul with Rehydration Buffer, rehydrate 24 cm Immobiline (IPG) Drystrips (GE Healthcare) for 10-24 hrs, and carry out isoelectric focussing. Currently, the Keck Laboratory is using pH 3-10 IPG strips unless requested otherwise. Note that other pH ranges that are available include: 3-7, 4-7, 3.5-4.5, 4.0-5.0, 4.5-5.5, 5.0-6.0, 5.5-6.7, and 6-9.

12.  If necessary, after isoelectric focusing the IPG strip may be stored in an equilibration tube (GE Healthcare) at -70 degrees C for at least several months.

13.  Carry out the SDS polyacrylamide gel electrophoresis (second) dimension on a 10 inch wide by 7.5 inch tall by 1.0mm thick gel with one side coated with Gelbond. Currently, the Keck Laboratory routinely uses a 12.5% polyacrylamide gel which will optimally separate 12-100 kD proteins.

14.  Immediately after SDS PAGE, the gel (which is still held between two glass plates) is scanned at all 3 wavelengths simultaneously on a GE Healthcare Typhoon 9410 Imager. After scanning, 16 bit tiff files of each color channel are exported for image analysis using the differential in-gel analysis module of the GE Healthcare DeCyder software package. After spot detection (which includes automatic background correction, spot volume normalization and volume ratio calculation), a user defined "dust filter" may be applied to each gel. This has the effect of automatically removing non-protein spot features from the gel and is followed by recalculation of experimental parameters.

15.  The front glass plate is removed and the gel is then fixed and stained with Sypro Ruby, which is the fluorescent stain that will be used as a guide to excise spots of interest from the gel. The reason for using Spyro Ruby, which stains all protein in the gel, is that the Cy-dye labeling is carried out such that the extent of incorporation will be <5% in terms of mole Cy-dye/mole protein. Since the Cy-dye has a MW of about 580 Da, low MW proteins (e.g., 10 Kd) labeled with Cy-dyes will not exactly co-migrate in the SDS PAGE dimension with their non-labeled counterparts.

16.  GE Healthcare DeCyder software is used to quantify the gel image and to identify a "pick list" of differentially expressed protein spots to be excised and subjected to MS-based protein identification. The DeCyder software can analyze any two Cy-dyed gel images, either on the same gel or on different gels, match the spots between the two images, and then identify differentially expressed protein spots. The DeCyder software automatically outputs a listing of statistically significant differences in protein expression including t-test values, using the Cy-2 internal standard. Please note, however, that replicate samples are required for statistical analysis. Differentially expressed spots may be identified using a number of criteria including area, volume, 3D peak slope, 3D peak height, and/or statistical variation. Protein spots that show different degrees of intensity between the two samples will be highlighted by the software so they can be manually confirmed. The DeCyder software can also analyze Sypro Ruby images, match the spots found with Sypro staining to those identified with the Cy-dye stains, and then choose a ick list?from the Sypro stained gel image. DeCyder data can be read by labs without the DeCyder software using an HTML format.

17.  The protein spot pick list is transferred to the Ettan Spot Picker instrument (GE Healthcare) which automatically excises the selected protein spots from the gel and transfers them into a 96-well microtiter plate.

18.  The excised protein spots are then subjected to automated in-gel tryptic digestion on the Ettan TA Digester.

19.  An aliquot of each digest is spotted (along with matrix) onto a MALDI-MS target.

20.  High mass accuracy, automated MALDI-MS/MS spectra are acquired on each target (using the Keck Laboratory Applied Biosystems 4800 Tof/Tof instrument) and the resulting peptide masses are subjected to database searching using Mascot algorithms.

21.  The remaining aliquots of digests of protein spots that are not identified by this approach may be subjected to nanospray or LC/MS/MS analysis (Micromass Q-Tof) with the resulting MS/MS spectra then being subjected to Sequest database searches to identify proteins present in the sample.

22.  Amersham Biosciences DIGE Instruction Manual