Cleaving the Protein into Peptides

My recommendation protect the N-terminal ends during purification. But what if the N-terminus is already blocked You can try sequencing C-terminally (see Section 7.6.5) or with the mass spectrometer (see Section 7.6.6). If you cannot do this or do not want to, you have no choice but to give up and cleave the protein into peptides, and separate and sequence them. Which of the three methods is to be preferred depends on the local climate. If you can find someone in the institute who knows a lot about sequencing by mass spectrometer, enlist the services of this person. For one or a few sequences it is not worth studying the peptide ladder technique for yourself (see Section 7.6.6), especially if you have to purchase a mass spectrometer first. 

Cleaving peptides, on the other hand, does not require big equipment purchases (every lab has an HPLC nowadays), and it works almost always. However, the cleaving and separating is laborious. As a beginner, you should plan on three months. 

Doolittle, R., and Armentrout, R. (1968). Pyrrolidonyl Peptidase An Enzyme for Selective Removal of Pyrrol-idone Carboxylic Acid Residues from Polypeptides, Biochemistry 1-. 516-521. 

Georghe, M., et al. (1997). Optimized Alcoholic Deacetylation of N-acetyl-blocked Polypeptides for Subsequent Edman Degradation, Anal. Biochem. 254 119-125. 

Wellner, D., et al. (1990). Sequencing of Peptides and Proteins with Blocked N-terminal Amino Acids N-acetylserine or N-acetylthreonine, Proc. Natl. Acad. Sci. USA 87 1947-1949. 

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For microsequencing of a protein, you must perform several preparation steps. The protein has to be purified and prepared, and for the most important methods the protein must have a free N-terminus (or you have to cleave the protein into peptides). 

You have been unlucky and your N-terminus is blocked You need the sequence of a protein and are not able to clone or are not allowed to Then you have no choice but to cleave the protein into peptides. I know, this causes an inner struggle. There you have paid attention to the proteases, added inhibitors, shivered in the cold room, and worked through the night until you had rings under your eyes—only to get it done faster you are supposed to add proteases and intentionally destroy the precious product But there s nothing you can do if you want to get a sequence, you have to cleave, cleave, cleave. 

Cleavage of disulfide bonds occurs before hydrolysis of the protein into peptides. Disulfide bonds may be cleaved oxidatively, or they may be reduced and alkylated. Treatment of the native protein with performic acid, a powerful oxidizing agent, breaks disulfide bonds and converts cystine residues to cysteic acid (Figure 3-11). Reduction of the disulfide linkage by thiols, such as d-mercaptoethanol, yields reactive sulfhydryl groups. These groups may be stabilized by alkylation with iodoacetate or ethyleneimine to yield the carboxymethyl or aminoethyl derivative, respectively. 

Having cleaved a protein into peptides of manageable length for Edman analysis, and determined the seqnence of each peptide, next we need to order the peptides correctly. For this, at least two sets of peptide seqnences, from different selective cleavage reactions, are reqnired. 

Peptide mapping is a powerful tool for the analysis of the primary structure of a protein. This method typically takes advantage of one or more specific proteases that cleave the protein into smaller peptides, which are then separated and analyzed by reversed-phase liquid chromatography (RP-HPLC), sometimes in conjunction with mass spectrometry (MS). 

In the life cycle of HIV, its RNA is translated into a polypeptide chain that is composed of several individual proteins including protease, integrase and reverse transcriptase, but in this form these enzymes are not functional. They must be cleaved by viral proteases from the assembled sequence in order for them to become functional. These posttranslational modifications allow the enzymes to facilitate the production of new viruses. The protease itself is made up of two 99-amino-acid monomers, and an aspartic acid residue in the monomer is required for the cleavage. The protease inhibitors inhibit the enzyme protease and consequently interfere with viral replication and maturation by preventing proteases from cleaving proteins into peptides. In humans, these drugs inhibit cleavage of HIV gag and pol polyproteins, which are part of the essential viral structural components, P7, P9, P17 and P24, and... 

Peptide mapping studies, generated by the cleavage of a protein into peptide fragments, must be highly reproducible and quantitative. Several electropherograms of protein digests have been obtained when chicken ovalbumin was cleaved by trypsin... 

The product of gene 8 is the major capsid protein of the filamentous phage. The protein is synthesized as a precursor with an N-terminal extension of 23 amino acids, which is necessary for the insertion of the protein into the bacterial cytoplasmic membrane where the leader peptide is cleaved. During the process of phage assembly, about 2700 copies aggregate around the virus DNA to form a helical array with the amino terminus exposed to the... 

Although any of several combinations of proteases can be used, ideally, one or more non-specific endopeptidases should be used first to convert the protein into many small peptides. These small peptides can then be degraded to amino acids by aminopeptidases and prolidase (hydrolyzes X-Pro bonds). Sometimes, carboxypeptidases are also used. Although leucine aminopeptidase has been used as the amino-peptidase (see Hill and Schmidt 1962), it may be preferable to use aminopeptidase M (Rohm and Haas, supplied by Henley and Co. of N.Y.), since this enzyme removes most residues at acceptable rates. Leucine aminopeptidase removes hydrophobic residues most rapidly, whereas some other residues are removed very slowly. Most procedures should probably include the use of prolidase (Miles) since many aminopeptidases do not cleave X-Pro bonds at appreciable rates. If it is found that proline is not released quantitatively by these procedures, the use of citrus leaf carboxypeptidase C (Rohm and Haas) can be tried after the initial endopeptidase hydrolysis and before the addition of aminopeptidase M and prolidase. Carboxypeptidase C (also yeast carboxypeptidase Y - see Hayashi et al. 1973) hydrolyzes proline bonds (as well as all others), but if proline is at or adjacent to the NH2 terminus of a peptide, it would probably not be released. In all procedures a control consisting of the enzymes only should be run in parallel with the hydrolyzed sample, and corrections should be made for any amino acids found by analysis of the control. suhic / /< > , mi... 

With respect to the use of ESI-MS, this approach was pioneered by the group of Henion [57-58]. The intact protein is digested using trypsin into smaller peptides under neutral pH conditions at 37°C. Trypsin selectively cleaves the protein at the C-terminal side of Lys or Arg (Ch. 17.4.4). The tryptic digest is separated by RPLC-MS. ESI-MS generates abundant double-charge ions for the tryptic peptides. 

In principle, it should be possible to sequence an entire protein by using the Edman method. In practice, the peptides cannot be much longer than about 50 residues, because not all peptides in the reaction mixture release the amino acid derivative at each step. For instance, if the efficiency of release for each round were 98%, the proportion of "correct amino acid released after 60 rounds would be (0.98 ), or 0.3—a hopelessly impure mix. This obstacle can be circumvented by cleaving a protein into smaller peptides that can be sequenced. In essence, the strategy is to divide and conquer. 

The space between the inner and outer mitochondrial membrane can be reached by proteins that have two signal peptides. The first inserts the protein into the membrane and is cleaved in the matrix the second remains and directs the protein to the intermembrane space. 

Trypsin, chymotrypsin, and thrombin are all proteases. Trypsin cleaves peptide bonds where there are amino acids with positively charged side chains (Lys and Arg). Chymotrypsin cleaves peptides at amino acids with aromatic side chains. Thrombin cleaves the protein fibrinogen into fibrin. 

Protein sequences can be determined directly or from the DNA that encodes them. The sequence of amino acids in a protein determines its biological function. Direct determination of the amino acid sequence of an unknown protein is accomplished first by cutting the protein into smaller peptides at specific residues. For example, cyanogen bromide cleaves proteins after methionine residues, and the... 

That amyloid fibrils can be formed from Vl peptides was demonstrated by Glenner et al. (127) and by Linke et al. (128). Glenner et al. treated Bence Jones proteins of types k and X with pepsin in such a manner as to cleave the chains into V and C segments (129). During the incubation, precipitates formed in many of the solutions. Under the microscope some of these precipitates (nearly all from X chains) were seen to consist of fibrils resembling those of amyloid deposits. Furthermore, the X-ray diffraction pattern had a gross appearance characteristic... 

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