Digestion proteolytic

In contrast to the nicotinamide nucleotide dehydrogenases, the prosthetic groups FMN and FAD are firmly associated with the proteins, and the flavin groups are usually only separated from the apoen2yme (protein) by acid treatment in water. However, in several covalently bound flavoproteins, the enzyme and flavin coen2ymes are covalently affixed. In these cases, the flavin groups are isolated after the proteolytic digestion of the flavoproteins. 

The ability to identify and quantify cyanobacterial toxins in animal and human clinical material following (suspected) intoxications or illnesses associated with contact with toxic cyanobacteria is an increasing requirement. The recoveries of anatoxin-a from animal stomach material and of microcystins from sheep rumen contents are relatively straightforward. However, the recovery of microcystin from liver and tissue samples cannot be expected to be complete without the application of proteolytic digestion and extraction procedures. This is likely because microcystins bind covalently to a cysteine residue in protein phosphatase. Unless an effective procedure is applied for the extraction of covalently bound microcystins (and nodiilarins), then a negative result in analysis cannot be taken to indicate the absence of toxins in clinical specimens. Furthermore, any positive result may be an underestimate of the true amount of microcystin in the material and would only represent free toxin, not bound to the protein phosphatases. Optimized procedures for the extraction of bound microcystins and nodiilarins from organ and tissue samples are needed. 

Trypsin is a major proteolytic digestive enzyme and the identified endogenous ligand for proteinase-activated receptor 2 (PAR 2). 

The proteolytic digestion of j6-lactoglobulin was carried out with trypsin which, as indicated in Table 5.4 above, is expected to cleave the polypeptide backbone at the carboxy-terminus side of lysine (K) and arginine (R). On this basis, and from the known sequence of the protein, nineteen peptide fragments would be expected, as shown in Table 5.7. Only 13 components were detected after HPLC separation and, of these, ten were chosen for further study, as shown in Table 5.8. 

There are two main classes of proteolytic digestive enzymes (proteases), with different specificities for the amino acids forming the peptide bond to be hydrolyzed. Endopeptidases hydrolyze peptide bonds between specific amino acids throughout the molecule. They are the first enzymes to act, yielding a larger number of smaller fragments, eg, pepsin in the gastric juice and trypsin, chymotrypsin, and elastase secreted into the small intestine by the pancreas. Exopeptidases catalyze the hydrolysis of peptide bonds, one at a time, fi"om the ends of polypeptides. Carboxypeptidases, secreted in the pancreatic juice, release amino acids from rhe free carboxyl terminal, and aminopeptidases, secreted by the intestinal mucosal cells, release amino acids from the amino terminal. Dipeptides, which are not substrates for exopeptidases, are hydrolyzed in the brush border of intestinal mucosal cells by dipeptidases. 

IgG consists of four polypeptide subunits held together by disulphide bonds. Native immunoglobulins are rather resistant to proteolytic digestion but certain enzymes have been usefiil in elucidating their structure. Papain cleaves the molecule into three fragments of similar size ... 

To check if PemB is surface exposed, E. chrysanthemi cells were subjected to proteolysis. Treatment of the cell suspension with trypsin, proteinase K or chimotrypsin at a concentration of 0.1 to 1 mg/ml for 1 h did not cause PemB proteolysis or its liberation into the medium. Cell pre-treatment with EDTA-lysozyme, which renders the periplasmic proteins accessible to proteases, gave no effect. PemB was also resistant to proteolytic digestion in extract of cells disrupted by sonication or in a French press. Only addition of Triton X-100 (up to 0.1%) causing formation of the micelles with PemB lead to a quick proteolyis of this protein (data not shown). In another approach to analyse the PemB exposition, bacterial cells were labelled with sulfo-NHS-biotin. This compound is unable to cross membranes and biotinylation... 

Fig. 3. (A) Disposition of afi unit in the membrane, based on sequence information [14,15], selective proteolytic digestion of the a subunit [5,6] and hydrophobic labelling (Table 1). The model for the (S subunit is based on sequencing of surface peptides and identification of S-S bridges [64,65]. T, T2 and C3 show location of proteolytic splits. CHO are glycosylated asparagines in the P subunit. (B) Peptide fragments remaining in the membrane after extensive tryptic digestion of membrane-bound Na,K-ATPase from outer medulla of pig kidney as described by Karlish et al. [7,58].

Rohrer, J. S., Cooper, G. A., and Townsend, R. R., Identification, quantification, and characterization of glycopeptides in reversed-phase HPLC separations of glycoprotein proteolytic digests, Anal. Biodiem., 212, 7, 1993. 

Current proteomics studies rely almost exclusively on 2D gel electrophoresis to resolve proteins before MALDI-TOF or ESI-MS/MS approaches. A drawback of the 2D gel approach is that it is relatively slow and work intensive. In addition, the in-gel proteolytic digestion of spots followed by mass spectrometry is a one-at-a-time method that is not well suited for high throughput studies. Therefore, considerable effort is being directed towards alternate methods for higher throughput protein characterization. 

The second method also relies on site-specific chemical modification ofphosphoproteins (Oda et al., 2001). It involves the chemical replacement of phosphates on serine and threonine residues with a biotin affinity tag (Fig. 2.7B). The replacement reaction takes advantage of the fact that the phosphate moiety on phosphoserine and phosphothreonine undergoes -elimination under alkaline conditions to form a group that reacts with nucleophiles such as ethanedithiol. The resulting free sulfydryls can then be coupled to biotin to create the affinity tag (Oda et al., 2001). The biotin tag is used to purify the proteins subsequent to proteolytic digestion. The biotinylated peptides are isolated by an additional affinity purification step and are then analyzed by mass spectrometry (Oda et al., 2001). This method was also tested with phosphorylated (Teasein and shown to efficiently enrich phosphopeptides. In addition, the method was used on a crude protein lysate from yeast and phosphorylated ovalbumin was detected. Thus, as with the method of Zhou et al. (2001), additional fractionation steps will be required to detect low abundance phosphoproteins. 

Yao, Z. P. Afonso, C. Fenselau, C. Rapid microorganism identification with on-slide proteolytic digestion followed by matrix-assisted laser desorption/ionization tandem mass spectrometry and database searching. Rapid Comm. Mass Spectrom. 2002,16,1953-1956. 

Protein modification 36 Variable Acetylation, fatty acylation, glycosylation, methylation, phosphorylation, ADP-ribosylation, ubiquitination, proteolytic digestion... 

Proteomics ultimately hinges upon protein identification to reveal the meaning behind the masses, spots, or peaks detected by other means. Because fraction collection is a natural component of HPLC separations, intact proteins can be readily collected either for direct analysis or for proteolytic digestion and identification using peptide mass fingerprinting (PMF) in conjunction with matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). 

Pinkse, M.W., Uitto, P.M., Hilhorst, M.J., Ooms, B., Heck, A.J. (2004).. Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns. Anal. Chem. 76, 3935-3943. 

Some reports even indicate that the conjugation of proteins or peptides with carbohydrates can increase dramatically their activity compared to that of the native state (Susaki et al., 1998). Carbohydrates also can provide a protective effect on modified peptides toward proteolytic digestion (Rudd et al., 1994) or mask recognition of a peptide by the immune system (Harding et al., 1993). The creation of neoglycoproteins thus can affect the activity of peptides and proteins, which are not normally glycosylated in vivo. 

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