Learn More About Isobaric Labeling

Mass spectrometry is the most powerful tool for proteomics to assess the relative abundance of proteins among biological samples. Numerous methodologies now support relative quantification measurements, providing a routine means to analyze protein expression patterns and post-translational modification states as a function of biological perturbation. Label-free quantification, SILAC labeling based quantification, and TMT isobaric multiplex (up to 18 plex) labeling quantification is available.

The most cost-efficient methods for relative quantification through mass spectrometry are isobaric mass tags. In isobaric labeling-based quantification, each sample is derivatized with a different isotopic variant of an isobaric mass tag from a set, and then the samples are pooled and analyzed simultaneously in MS. Since the tags are isobaric, peptides labeled with isotopic variants of the tag appear as a single composite peak at the same m/z value in an MS1 scan with identical liquid chromatography (LC) retention time. The fragmentation of the modified precursor ion during MS/MS (MS2) event generates two types of product ions: (a) reporter ion peaks and (b) peptide fragment ion peaks. The quantification is accomplished by directly correlating the relative intensity of reporter ions to that of the peptide selected for MS/MS fragmentation. The fragment ion peaks observed at higher m/z are specific for peptide amino acid sequence and are used for peptide identifications, which are eventually assigned to the proteins that they represent. Since every tryptic peptide can be labeled in an isobaric labeling method, more than one peptide representing the same protein may be identified, thereby increasing the confidence in both the identification and quantification of the protein. This technology has proved to be successful in numerous experimental contexts for comparative analysis upon perturbation. The Thermo Scientific™ TMT™ Mass Tag Labeling Kits and Reagents enable multiplex relative quantitation up to eighteen different peptide samples prepared from cells or tissues.

Learn More About Pathway Analysis

Pathway analysis (PA) is widely used to interpret large “omics” data and is based on existing biological knowledge from databases with statistical testing, mathematical analyses and computational algorithms. In principle, biological information flows from the DNA/genome to protein/proteome automatically, regulating live cell status in response to internal or external signals. The genome encoding the proteome is relatively static, while the proteome is much more dynamic allowing proteins to orchestrate all biological processes precisely, ranging from central metabolism to cell structure, maintenance, and replication. It is speculated that all biological processes (on/off) are regulated and operated by the dynamics of proteins such as modifications and changes in abundance. The regulation of different cellular functions has been categorized into a number of pathways, such as the Wnt signaling pathway and the TGF signaling pathway. In each pathway, the components are generally named according to their function, including ligands, receptors, activating regulators, inhibitory regulators, and effectors. To measure the activation strength of a pathway, the protein molecules that belong to ligands, receptors, activating regulators, or inhibitory regulators are grouped as the pathway protein ontology chain (POC), and their relative abundances (ppm) are summed. Based on the summed abundance of each POC, the activation strength or activation status of a pathway is compared between two proteome profiles. Based on these scientific assumptions Poochon developed a novel pathway tool called PROMICPATH.