Protein into Peptides

Proteins can be cleaved at specific sites by enzymes or by chemical reagents. The enzyme trypsin cleaves peptide bonds preferentially at amino acids that have positively charged R groups, such as lysine and arginine. The cleavage takes 

FIGURE 5.15 HPLC chromatogram of amino acid separation. 

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T cells all have the ahility to internalize and degrade proteins into peptide fragments and ean all therefore aet as APCs. 

The data obtained in MS after the proteolytic digestion of gel-separated proteins into peptides and mass analysis of the peptides will enable the researcher to search protein and nucleotide sequences, which can then be checked against their theoretical fingerprints in databases. 

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... 

Under the proper stimulus renin is released into the circulating blood where it can be identified, especially in that from the renal vein. It acts rapidly upon its specific substrate, splitting the protein into peptides, one or more of which have been called hypertensin or angiotonin. Hypertensin has been concentrated but not obtained in pure form. It is the effector substance of renin, constricting arterioles and raising blood pressure. The action of hypertensin is abolished by hypertensinase, an enzyme found in blood and renal extracts. Fortunately, the latter is destroyed by heat and alkalinity. 

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 major application of liquid chromatography (LC) thus far is as nano-LC after digestion of proteins into peptides. 

Fig. 8.9 Schematic of the processing of a protein into peptide fragments for presentation by MHC. (a) The synthesized protein ( ) is present in the cytoplasm of the cell and is degraded into peptide by the proteasome. The derived peptide fragments are presented by MHC I to cytotoxic CD8+ T lymphocytes, (b) Protein ( ) is endocytosed by an APC (antigen-presenting cell) and processed into peptide within lysosomes. The derived peptide fragments are presented by MHC II to helper CD4+ T lymphocytes.

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. 

Hydrolysis of a protein into peptides can be accomplished by group-specific chemical and enzymatic reagents (Table 3-2). N-Bromosuccinimide and cyanogen bromide hydrolyze proteins at tryptophan and methionine (Figure 3-12) residues, respectively. Trypsin hydrolyzes... 

The membrane reactor concept was demonstrated in laboratory scale a decade ago by Butterworth et al. (15) and by Chose and Kostick (16) in studies on the hydrolysis of starch and cellulose, respectively. Later on several publications have appeared describing the analogous, continuous conversion of various proteins into peptides intended for human nutrition (17-22). Among these works only that of laccobucci et al. (18) presents a quantitative model of the membrane reactor in continuous protein hydrolysis, and it is also the only demonstration of the practical feasibility of the concept in pilot plant scale. 

If your protein sample has an acetylated N-terminus, it will not yield any information from Edman degradation of the intact protein. Enzymes have been described that remove N-acetyl groups from peptide chains, but, in general, these have been found to function well only on peptides and not on intact proteins. Procedures have been described for cleavage of blocked proteins into peptides followed by modihcation of all newly revealed peptide N-termini (e.g., by succiny-lation) and then incubation of the mixture with a deacetylase. The entire mixture can then be subjected to Edman degradation. Only the deacetylated peptide representing the original N-terminus will provide sequence. 

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. 

Do you know the amino acid sequence (e.g., from cDNA cloning) and do you have purified protein Then you can determine the MW of the protein via MALDI-TOF and compare to the MW calculated from the cDNA sequence. If the MW are identical, the primary structure of your protein is correct. Deviations are evidence of modifications (e.g., phosphorylations, glycosylations, point mutations). With smaller proteins, the acetylation of the N-terminal amino acid becomes apparent in the MALDI-TOF. You determine the position of a modification by cutting the protein into peptides, measuring the MW of the individual peptides, and comparing to the MW predicted from the sequence. 

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). 

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. 

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. 

Pepsin (EC 3.4.23.1) has heen the protease of choice since the hegimiing of HX-MS. It digests food proteins into peptides in the stomach. Extracted from the porcine gastric mucosa, it is commercially avaUahle and cheap. The activity of this protease reaches a maximum between pH 2 and 4, and the optimum tenperature is around 37°C. However, its activity around pH 2.5 and at 0°C is still high... 

The analysis of proteins by MALDI-MS peptide mass mapping involves the proteolytic degradation of proteins into peptides. Prior to such proteolytic diges-... 

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