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TMT or iTRAQ Analysis

TMTs (tandem mass tags, 6-plex or 10-plex)Isobaric mass tags, and iTRAQ (isobaric tag for relative and absolute quantitation, 4- and 8-plex) directly compare up to ten different samples in a single tandem MS experiment. These mass tags add only one step to the protein identification procedure. After trypsin digestion, all N-termini and lysines of peptides in a sample are labeled with an isobaric tag consisting of a reporter and a balance region. Each sample is labeled with a unique tag before all samples are combined, fractionated and analyzed by tandem MS. When an isobaric tagged peptide is fragmented the reporter and balance region cleave. The balance region is neutral and not detected. The reporter region carries a charge and is detected in the low mass region (e.g. 113-121 Da for 8-plex iTRAQ). Comparing the reporter intensities provides relative quantification. Because the total mass of the each reporter plus their respective balance mass is always the same or isobaric (e.g. 305 Da for 8-plex iTRAQ), the mass added to the same peptide present in all samples is the same. There is no peak or mass separation of different isobaric tagged but identical peptides. Thus, a single fragmentation spectrum contains both the peptide sequence and reporter ion data to quantify the same peptide from different samples. Protein identification and quantification is based on as many tagged peptides from the same protein that can be detected. Coupled with enrichment stragies, post-translational modifications can also be quantified. Sensitivity is limited by sample complexity, which typically is reduced by multi-dimensional chromatography. 

SILAC

The stable isotopic labeling of amino acids in cell culture (SILAC) technique metabolically labels proteins with Lys or Arg containing 13C or 15N stable isotopes. To eliminate technical variation between samples, labeled cells are harvested and mixed with non-labeled cells before extracting proteins. Proteins are then digested and analzyed by tandem MS. Peptide masses from labeled cells are shifted by the mass of the stable isotope (typically 9 Da) from the same peptides from unlabeled cells resulting in peak pairs. Comparing the areas of each peak in a pair determines the relative change in that protein’s expression.  This technique is designed for cell culture. The Core routinely analyzes SILAC samples. However, the Core does not have cell culture equipment and will advise investigators on methods to metabolically label proteins with stable isotopes. 

Non-Labeling Quantification by MudPIT

Changes in protein expression across many samples can be determined by comparing peptide intensities and the number peptide spectra from a protein. Peptides from proteolytic digests of complex mixtures are first separated in using strong cationic exchange (SCX) chromatograph followed by C18 reverse chromatography. The Core prefers off-line linear salt gradients versus on-line step salt gradients because it does not tie up MS instrument time, provides flexibility to customize fraction pooling, and can prevent overloading the subsequent C18 column during nanoLCMS/MS analysis. This technique routinely identifies >3000 proteins from 5 to 10 ug of protein extracts from cells or tissues at a 1% false discovery rate with sub 3 ppm mass accuracy and is also used with labeling methods (e.g. iTRAQ).

 

Location

The Johns Hopkins Mass Spectrometry and Proteomic Facility

725 N. Wolfe Street
Wood Basic Science Building, Room 504
Baltimore, MD 21205

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