Enzyme digestion amino acid sequencing

If a protein is digested into peptide fragments with an enzyme, the amino acid sequence of the individual fragments can be determined by MS-MS. The MS-MS analysis step can be done directly from the mixture, or this sample can be separated by capillary liquid chromatography. In the MS-MS technique, two mass analysis steps are performed (see triple quadrupole section). First, an ion is selected or isolated for MS-MS analysis. In the ESI process at 0.1 M acid, the most abundant peptide molecular ion for a molecular weight between 700 to 2400 amu is usually the [M + 2H]+ or the [M + H]+ ion, so one of these two ions is typically chosen. Because the [M + H]+ ion is the base peak in MALDI-MS, it... 

Confirmation of Amino Acid Sequence Using the Analysis of LC-MS Data from an Enzyme Digest of a Protein 152... 

Amino Acid Sequencing of Polypeptides Generated by Enzyme Digestion Using MS-MS 166... 

The presence of three polypeptides in Table 5.8 tliat were not predicted from the relationship between the amino acid sequence and the enzyme used for digestion is worthy of note when interpretation of data of this sort is undertaken. The MALDI data showed six further unexpected polypeptides, none of which were detected in the LC-MS data ... 

Peptide mapping The process of considering the amino acid sequence information from peptides obtained by enzyme digestion in an attempt to derive the (amino acid) sequence of the parent protein. 

Retention of viscous purulent secretions, which contain high concentrations of extracellular DNA—released by degenerating leukocytes that accumulate in response to infection [24]—in the airways contributes both to reduced pulmonary function and to exacerbations of infection [24,25], Digestion of DNA polymers in purulent secretion with DNAse (dornase-a or Pulmozyme) has been shown to reduce sputum viscosity in cystic fibrosis patients. The availability of recombinant DNAse has allowed its use in an aerosol formulation to deliver the enzyme into the deep lung alveoli of CF patients. The purihed glycoprotein contains 260 amino acids with an approximate molecular weight of 37,000 daltons [26], The primary amino-acid sequence is identical to that of the... 

Among proteases, the digestive enzyme trypsin catalyzes the hydrolysis of only those peptide bonds in which the carbonyl group is contributed by either a Lys or an Arg residue, regardless of the length or amino acid sequence of the chain. The number of smaller peptides produced by trypsin cleavage can thus be predicted... 

The considerable detail to which we now can understand enzyme catalysis is well illustrated by what is known about the action of carboxypeptidase A. This enzyme (Section 25-7B and Table 25-3) is one of the digestive enzymes of the pancreas that specifically hydrolyze peptide bonds at the C-terminal end. Both the amino-acid sequence and the three-dimensional structure of carboxypeptidase A are known. The enzyme is a single chain of 307 amino-acid residues. The chain has regions where it is associated as an a helix and others where it is associated as a /3-pIeated sheet. The prosthetic group is a zinc ion bound to three specific amino acids and one water molecule near the surface of the molecule. The amino acids bound to zinc are His 69, His 196, and Glu 72 the numbering refers to the position of the amino acid along the chain, with the amino acid at the /V-terminus being number l. The zinc ion is essential for the activity of the enzyme and is implicated, therefore, as part of the active site. 

Schematic diagrams of the amino acid sequences of chymotrypsin, trypsin, and elastase. Each circle represents one amino acid. Amino acid residues that are identical in all three proteins are in solid color. The three proteins are of different lengths but have been aligned to maximize the correspondence of the amino acid sequences. All of the sequences are numbered according to the sequence in chymotrypsin. Long connections between nonadjacent residues represent disulfide bonds. Locations of the catalytically important histidine, aspartate, and serine residues are marked. The links that are cleaved to transform the inactive zymogens to the active enzymes are indicated by parenthesis marks. After chymotrypsinogen is cut between residues 15 and 16 by trypsin and is thus transformed into an active protease, it proceeds to digest itself at the additional sites that are indicated these secondary cuts have only minor effects on the enzymes s catalytic activity. (Illustration copyright by Irving Geis. Reprinted by permission.)...

Proteolytic enzymes cleave specific amino acid sequences within the peptide chain of the digested protein since proteases differ in their cleavage specificities, they can have markedly different effects upon tissues depending upon the type of fixative used for processing the tissue, the antigenic target, and the epitope recognized by the antibody. 

Because of the importance of ADPGlc synthetase for plant starch synthesis, efforts have been made to determine the structure of the enzyme and to relate catalytic and allosteric function to structure. The spinach leaf enzyme is composed of two subunits of 51 and 54 kd mass (5,6). The molecular mass of the native enzyme is 206,000 and presumably is a tetramer composed of two of each subunit. The subunits are antigenically dissimilar, exhibit different peptide patterns on HPLC after trypsin digestion and their N-terminal amino acid sequences are different (7). Thus it is likely that the peptide subunits are products of different genes. 

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