2D-DIGE
Label Free Protein Expression
SILAC
Proteolytic 18O Labeling
Two dimensional difference gel electrophoresis (2D-DIGE) is an established
analytical technique for differential proteome profiling. Like a
traditional 2D-PAGE, the technique involves separation of the proteins
according to their charge in the first dimension by isoelectric focusing
(IEF) and size in the second dimension by SDS-PAGE. 2D-DIGE differs from
2D-PAGE in that each sample is pre-labeled with a fluorescent dye prior to
isoelectric focusing (called Cy2, Cy3 and Cy 5). The three dyes are mass
and charge matched N-hydroxy succinimidyl ester derivative of fluorescent
dyes but with different spectral characteristics (distinct excitation and
emission spectra) and therefore up to 3 samples can be run together on the
same gel at the same time. The ability to run and analyze up to three
samples on the same gel reduces spot pattern variation and also reduces the
number of gels that are required in an experiment to measure statistical
significant changes. The most common stains used to visualize proteins on
the gel, with traditional 2D-PAGE method, are coomassie or silver. These
dyes either have low detection limits or poor linear responses. Gel to gel
variations could be introduced either due to the fact that the staining
technique does not have a real end point (in the case of silver) or have
high background (in the case of coomassie) which makes the traditional
2D-PAGE less suitable for quantitative analysis. However, cyanine dyes
have very good sensitivity like silver and wider dynamic range (4-5 order).
Basically, the dyes spectral properties and very good sensitivity as well
as very good dynamic range of detection make 2D-DIGE much more powerful
platform than traditional 2D-PAGE for quantitative proteomics study.
Currently, no other technique can match 2D-DIGE in terms of resolution, and
sensitivity, in the analyses of large mixtures of proteins.
Most importantly 2D-DIGE technique is enabled to incorporate the internal standard (Alban et al, Proteomics 2003, 3, 36-44). The internal standard is a pool mix of equal aliquots of protein sample from each biological replicates used in the experiment in this way, every protein from all sample will be represented in the internal standard. Running the internal standard on every single gel along with each individual samples links every sample in-gel to a common internal standard and has the following advantages: 1) each sample within a gel can be normalized, 2) the abundance of each protein spot in a biological sample can be measured as ratio to its corresponding spot present in the internal standard, 3) experimental variation can be separated from the biological variation and therefore it enable accurate quantitation between gels.
Contact Person:
Elizabeth Yohannes, Ph.D. (elizabeth.yohannes@case.edu)
Case Center for Proteomics and Bioinformatics
Case Western Reserve University
10900 Euclid Avenue BRB 935
Cleveland, Ohio 44106
(phone) 216-368-0655
(fax) 216-368-6846
Label free protein expression utilizes a bottom-up proteomic strategy. Bottom-up proteomics differs from top down approaches (i.e. 2D-DIGE) as they analyze a proteome at the peptide level by cleaving proteins into peptide fragments via enzymatic digestion. One advantage of bottom up proteomics is increased overall proteome coverage. Moreover, most bottom up methods simultaneously provide both qualitative and quantitative data in a single run which improves throughput, and quantifying at the peptide level leads directly into a bottom up confirmation/validation analysis thereby avoiding the peptide selection step in this procedure. The label free approach capitalizes on the highly reproducible chromatography and accurate mass accuracy available in the Center.s current LC/MS systems. This method observes all detectable peptides and, if interrogated by MS/MS, their corresponding fragment ions. This approach quantifies a peptide by its intensity and groups each peptide across individual samples based on its accurate mass and retention time. Once compiled, these intensities associated with accurate mass and retention time are organized into a peptide array which can be subsequently analyzed by statistical techniques that accommodate high dimensional data. As with other bottom up approaches, this method is suited to pre-fractionation strategies but it is not limited in the comparisons it can make as isotope labels are not used. Therefore, this is a very attractive alternative to bottom up label and top down proteomic platforms when analyzing samples from complex experimental designs such as those derived from clinical studies.
Contact Person:
Danie Schlatzer (daniela.schlatzer@case.edu)
Senior Research Associate
Case Center for Proteomics and Bioinformatics
Case Western Reserve University
10900 Euclid Avenue, BRB 947
Cleveland, Ohio 44106
(phone) 216-368-4014
(fax) 216-368-6846
Stable isotope labeling with amino acids in cell culture (SILAC) is a simple and straightforward approach for in vivo incorporation of a mass tag into proteins for mass spectrometry (MS)-based quantitative proteomics. SILAC relies on metabolic incorporation of a given 'light' or 'heavy' form of the amino acid into the proteins. The method relies on the incorporation of amino acids with substituted stable isotopic (e.g. deuterium, 13C, 15N). Thus in an experiment, two cell populations are grown in culture media that are identical except that one of them contains a 'light' and the other a 'heavy' form of a particular amino acid (e.g. 12C and 13C labeled L-lysine, respectively). When the labeled analog of an amino acid is supplied to cells in culture instead of the natural amino acid, it is incorporated into all newly synthesized proteins. After a number of cell divisions, each instance of this particular amino acid will be replaced by its isotope labeled analog. Since there is hardly any chemical difference between the labeled amino acid and the natural amino acid isotopes, the cells behave exactly like the control cell population grown in the presence of normal amino acid. It is efficient and reproducible as the incorporation of the isotope label is 100%. The benefit of labeling proteins metabolically is that .light.- and .heavy.-labeled cells can be mixed together for subsequent proteomic analysis, therefore, variations in protein extraction, fractionation, and digestion can be minimized, and the obtained quantification will be more accurate and reliable.
This technique utilizes a protease and H218O to produce labeled peptides, with subsequent chromatographic and mass spectrometric analysis to identify and quantify (relative) the proteins from which the peptides originated. The technique determines the ratio of individual protein.s expression level between two samples relative to each other, and can be used to quantitatively examine protein expression (comparative proteomics) and post-translational modifications, and to study protein-protein interactions.
The proteolytic 18O labeling method achieves isotope labeling concurrent with the proteolytic digestion of proteins in both sample groups; one sample incorporates 16O by digestion in H216O solvent, and the other sample incorporates 18O by digestion in H218O. The same peptides generated from the same protein from two different sample groups differ only in their molecular weights: one has .16O. in the C-terminal carboxyl groups of the peptides, and the other incorporates .18O.. Because the peptide labeled with 16O and the same peptide labeled with 18O co-elute from the chromatography step in the LC/MS/MS analysis, a constant difference in molecular weight (2 Da in the case of a single 18O atom incorporation, and 4 Da with two 18O atom incorporation) exists in the 16O- and 18O-labeled peptides; that difference can be readily distinguished in the mass spectrum. The relative abundance of the two peptides can be determined by comparing the peak intensities of the 16O- and 18O-labeled peptides in the mass spectrum, the relative abundance also equals the relative abundance of the protein from which the peptide was generated in the original samples. The identity of the peptide, which in most cases identifies the protein, can be determined by subjecting one of the peptide ions (16O- or 18O-labeled peptide) to tandem mass spectrometry to obtain the amino acid sequence from a MS/MS spectrum.
Contact Person:
Masaru Miyagi, Ph.D. (masaru.miyagi@case.edu)
Case Western Reserve University
Center for Proteomics and Bioinformatics
10900 Euclid Ave., BRB 928
Cleveland, OH 44106-4988
Phone: (216) 368-5917
Fax: (216) 368-6846