We offer label-based quantitative proteomics using DIGE (2D-gel based), 4-plex/7-plex-iTRAQ and 6-plex TMT (LC based), as well as label free (LC-based) quantitative proteomics for smaller size projects.
- Difference Gel Electrophoresis (DIGE) (GE-Healthcare) is a modification of 2-D PAGE. We use CyDyeTM DIGE Fluor dyes (Cy2, Cy3, and Cy5). Two separate protein samples are labeled with different fluorescent dyes Cy3 and Cy5 prior to separation. Cy2 is used to label the internal standard which contains half of the proteins from each sample. The three samples are then combined and run on a single gel, enabling accurate analysis of differences in protein abundance between samples. Associated technologies available in the facility include: Cy-dye labeling, isoelectric focusing using Immobilized pH gradient strips, 2nd dimension SDS gel electrophoresis, fluorescence scanning, image analysis with SameSpots, and protein identification of desired spots by MS/MS.
- iTRAQ (AB Sciex) is a non-gel based technique used to identify and quantify proteins from different sources in one single experiment. It uses isotope coded covalent tags. The method is based on the covalent labeling of the N-terminus and side chain amines of peptides from protein digests with isotopically labeled tags. The facility is set up to run the 4-plex (114, 155, 116 and 117 labels) or 7-plex (113, 114, 115, 166, 117, 118 and 119 labels) iTRAQ which can be used to label all peptides from up to 4 or 7 different samples/treatments. After labeling, the samples are pooled and fractionated by OFFGEL isoelectric focusing (Agilent). Each fraction is then analyzed by LC-MS/MS. A database search is then performed using Mascot Server v2.4 (Matrix Science). The relative abundances of the proteins in a sample are calculated using ProteoIQ (PREMIER Biosoft). The fragmentation of the attached tag generates a low mass reporter ion that can be used to relative quantitation of the peptides and the proteins from which they originated.
- TMT (Thermo-Fisher Scientific) enables labeling of proteins extracted from cells and tissues for relative quantitation and uses LC-based separation followed by tandem mass spectrometry. The labeling chemistry of the 6-plex tags (126, 127, 128, 129, 130 and 131) is the same as for iTRAQ labeling. The reagents label peptides prepared from up to 6 samples. The following steps to the identification and quantification of the proteins present in the samples are the same as previously described for iTRAQ.
- Label free quantitation is LC-based.We offer quantitation based on precursor ion intensity for relative quantitation of proteins between samples. Because it does not use a stable isotope tag, the comparison between samples is limited to small experiments of 2 samples including a minimum of 3 biological/technical replicates to reduce the experimental variation. Pre-fractionation using OFFGEL (Agilent) is also recommended to increase proteome coverage. The data acquired is analyzed using the Progenesis LC-MS (NonLinear Dynamics) for chromatogram alignment and protein quantitation. Protein identification is done using Mascot Server v2.4 (Matrix Science).