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Terminal amine isotopic labeling of substrates (TAILS) is a method in quantitative proteomics that identifies the protein content of samples based on N-terminal fragments of each protein (N-terminal peptides) and detects differences in protein abundance among samples. Like other methods based on N-terminal peptides, this assay uses trypsin to break proteins into fragments and separates the N-terminal peptides (the fragments containing the N-termini of the original proteins) from the other fragments (internal tryptic peptides). TAILS isolates the N-terminal peptides by identifying and removing the internal tryptic peptides. This negative selection allows the TAILS method to detect all N-termini in the given samples. Alternative methods that rely on the free amino group of the N-terminus to identify the N-terminal peptides cannot detect some N-termini because they are "naturally blocked" (i.e. the natural protein does not have a free amino group). The TAILS method has a number of applications including the identification of new substrates and proteases (including those that have an unknown and broad specificity) and as a way to define the termini of proteins that enables protein annotation. TAILS can also be used to link proteases with a variety of defined biological pathways in diseases such as cancer, in order to gain a clearer understanding of the substrates and proteases involved in the disease state. ==Method== TAILS is a 2D or 3D proteomics based assay for the labeling and isolation of N-terminal peptides, developed by a group at the University of British Columbia.〔 The TAILS method is designed for comparison of multiple protease treated cells and control proteome cells.〔 Samples can be derived from a variety of sources including tissue, fibroblasts, cancer cells and from fluid effusions. This assay isolates the N-terminal peptides by removing the internal tryptic peptides via ultrafiltration leaving the labeled mature N-terminal and neo-N-Terminal peptides to be analyzed by tandem mass spectrometry (MS/MS). This negative selection allows the TAILS method to detect all N-termini in the given samples. Alternative methods that rely on the free amino group of the N-terminus to isolate the N-terminal peptides cannot detect naturally blocked N-termini because they do not have a free amino group. TAILS requires only small sample of peptide for experimentation (100-300 ug), can be used with proteases which have unknown or broad specificity and supports a variety of methods for sample labeling. However, it identifies ~ 50% of proteins by two or more different and unique peptides (one of the original mature N-termius and/or one or more neo-N-terminal peptide via cleavage site) that do not represent independent biological events thus cannot be averaged for quantification. It also has difficulty validating results for single peptide-base N-terminome analysis.〔 The following steps are for the dimethylation-TAILS assay, comparing a control sample (exhibiting normal proteolytic activity) and a treated sample (which in this example exhibits an additional proteolytic activity). # Proteome-wide proteolysis occurring in both the treated and control samples with additional proteolytic activity in the treated sample. # Inactivation of the proteases and protein denaturation and reduction. # Labelling with stable isotopes. This allows peptides that originated in the control sample to be distinguished from those that originated in the treated sample so their relative abundance can be compared. In this example, the labelling is applied by reductive dimethylation of the primary amines using either heavy (d(2)C13)-formaldehyde for the treated samples or light (d(O)C12)-formaldehyde for the controls. This reaction is catalyzed by sodium cynoborohydride and attaches the labeled methyl groups to lysine-amines and the free (∝)- amino groups at the N-termini of the proteins and protease cleavage products. # Blocking of reactive amino groups. This allows the internal tryptic peptides to be identified later in the process because they will be the only peptides with reactive amino groups. In this example the labelling reaction (reductive dimethylation) also blocks the reactive amino groups. # Pooling. The two labeled proteomes are now mixed. This ensures that the samples are treated identically at all subsequent steps allowing the relative quantities of the proteins in the two samples to be more accurately measured. # Trypsinization. This breaks each protein into fragments. The labeled N-termini of the original proteins remain blocked, while the new internal tryptic peptides have a free N-terminus. # Negative selection. A hyperbranched polyglycerol and aldehyde (HPG) polymer specific for tryptic peptide binding is added to the sample and reacts with the newly generated tryptic peptides through their free N-termini. As in step 3 above, this reaction is catalyzed by sodium cynoborohydride. The dimethylated lysine's acetylated and isotopically labeled protein peptides and neo(new)-N-terminal peptides are unreactive and remain unbound and can be separated from the poly-internal tryptic peptide complexes using ultrafiltration. # The eluted unbound proteins are highly concentrated with the N-terminal peptides and neo-N-terminal peptides. # This eluted sample is then quantified and analysis completed by MS/MS. # The final step in TAILS involves bioinformatics. Using a hierarchical substrate winnowing process that discriminates from background proteolysis products and non-cleaved proteins by a peptide isotope quantification and certain bionformatic search criteria.〔〔 抄文引用元・出典: フリー百科事典『 ウィキペディア（Wikipedia）』 ■ウィキペディアで「Terminal amine isotopic labeling of substrates」の詳細全文を読む スポンサード リンク
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