Trypsin, peptide cleavage with

Triterpenoid, 1071 tRNA, see Transfer RNA Trypsin, peptide cleavage with, 1033 Tryptophan, pKa of, 52... 

From a rare fungus you have isolated an octapeptide that prevents baldness, and you wish to determine the peptide sequence. The amino acid composition is Lys(2), Asp, Tyr, Phe, Gly, Ser, Ala. Reaction of the intact peptide with FDNB yields DNP-alanine plus 2 moles of e-DNP-lysine on acid hydrolysis. Cleavage with trypsin yields peptides the compositions of which are (Lys, Ala, Ser) and (Gly, Phe, Lys), plus a dipeptide. Reaction with chymotrypsin releases free aspartic acid, a tetrapeptide with the composition (Lys, Ser, Phe, Ala), and a tripeptide the composition of which, following acid hydrolysis, is (Gly, Lys, Tyr). What is the sequence ... 

TEV) protease cleavage site is inserted between the binding/reactive group and the analytical handle. Labeling of proteomes with these probes followed by (strept) avidin resin incubation yields an enriched proteome. Trypsin digest followed by filtration yields a supernatant that can be analyzed by multidimensional LC-MS/MS analysis as in the MudPIT-ABPP experiment above. In the last step, the probe-labeled active site peptides are released from the resin by cleavage with TEV protease and subjected to MudPIT analysis (Lig. 5). 

Various trypsin-like serine proteases such as kallikreins [37] and plasmin or plasminogen activator [38] have been suggested to be involved in the cleavage of hormone precursors. However, it is not clearly known whether any of these proteases can correctly process proinsulin to generate both native insulin and C-peptide. Cleavage of proinsulin by plasmin is shown to result in equal amounts of insulin and a form of insulin with a residual Arg residue before the Gly at the N-terminus of the A-chain [39]. There may possibly be a novel amino peptidase for removing this... 

Note Some of this information is redundant peptides Tryp 1 and CNBr 1 would be sufficient for unambiguous sequence determination. In addition, in such a simple case, with only two peptides from each cleavage reaction, we could determine the sequence from either one of these cleavages alone using the fact that the carboxyl sides of Met and Lys are the sites of cleavage with CNBr and trypsin, respectively. 

FIGURE 2-9. This peptide profile represents 16 hours of tryptic digestion. Note that peak 6 has decreased in height while 2 and 4 have increased. The peptide corresponding to peak 6 contains an internal lysine (K) which is slowly cleaved by trypsin. Peptide 8 also contains an internal lysine (K) which is well-protected from cleavage. As a result peaks X and Y (cleavage products of peptide 8) are only produced after long exposure to trypsin. (Reprinted from reference 6 with permission.)... 

Dynorphin (D)is a neuropeptide. Indicate the -terminal end and the C-terminal end. Show the products of cleavage with (a) trypsin (b) chymotrypsin. Show the A-phenylthiohydantoin that results from treatment of D with phenyl isothiocyanate. Do you expect D to be an acidic, a neutral or a basic peptide ... 

The fragmentation of a larger peptide is acheived by chemical cleavage with cyanogen bromide or enzymatically by endopeptidase, such as trypsin or chymotrypsin. 

A protein that has 10 methionine residues will usually yield 11 peptides on cleavage with CNBr. Highly specific cleavage is also obtained with trypsin, a proteolytic enzyme from pancreatic juice. Trypsin cleaves polypeptide chains on the carboxyl side of arginine and lysine residues (Figure 4.24 and Section 9.1.4). A protein that contains 9 lysine and 7 arginine residues will usually yield 17 peptides on digestion with trypsin. Each of these tryptic... 

Most polypeptides in nature are longer than 20 to 30 amino acids, the practical limit on the number of amino acids that can be sequenced by repetitive Edman degradation. The special value of cleavage with cyanogen bromide, trypsin, and chymotrypsin is that, at specific peptide bonds, a long polypeptide chain can be cleaved into smaller polypeptide fragments, and each fragment can then be sequenced separately. 

Yet the more critical question is how can a state be ascertained by a physical measurement of a property with attached units and, in turn, f ix) and F Cx) The exercise appeals to the chemical action of trypsin. This is a much-leveraged enzyme that catalyzes peptide cleavage at the carboxyl side of R and K sites [11]. The exercise is one that randomly selects a peptide from a large population followed by trypsin application and isolation of the R- and V-containing product. For extra simplicity, the hypothetical cell never allows K (lysine) as a polypeptide component. 

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