Peptides, detection disulfide bridges

Solouki, T. Emmet, M.R. Guan, S. Marshall, A.G. Detection, Number, and Sequence Location of Sulfur-Containing Amino Acids and Disulfide Bridges in Peptides by Ultrahigh-Resolution MALDI-FTICR Mass Spectrometry. Anal. Chem. 1997,69, 1163-1168. 

Marshall, A.G. Detection, Number, and Sequence Location of Sulfur-Containing Amino Acids and Disulfide Bridges in Peptides by Ultrahigh-Resolution... 

Sequence analysis is one of the most useful tools for determining disulfide connectivities by detecting the phenylthiohydantoins (PTH) of either cystine or cysteine derivatives. Sequencing can be carried out on the intact peptide, although most frequently disulfide-bridged fragments are subjected to this type of analysis. 

Solouki, T., Emmett, M. R., Guan, S., and Marshall, A. G. (1997). Detection, number, and sequence location of sulfur-containing amino acids and disulfide bridges in peptides by ultrahigh-resolution MALDI FTICR mass spectrometry. Anal. Chem. 69,1163-1168. 

Proteins and peptides are linear polymers made up of combinations of the 20 most common amino acids linked with each other by peptide bonds. Moreover, the protein produced by the ribosome may undergo covalent modifications, called post-translational modifications, after its incorporation of amino acids. Over 200 such modifications have been detected already [13,14], the most important being glycosylation, the formation of disulfide bridges, phosphorylation, sulfation, hydroxylation, carboxylation and acetylation of the N-terminal acid [15]. The most frequent are listed in Table 8.1 and a more comprehensive database of mass changes due to post-translational modifications of peptides and proteins is available on the Internet [16]. 

Another example of the use of mass spectrometry to delect toxins is to identify ricin, a highly toxic protein that inhibits cell protein synthesis. Ricin is produced from the seeds of Ricinus communis plants (known conunonly as castor beans) [73]. Structurally, ricin is made of A- and B-chains linked by a disulfide bridge. The toxicity of ricin is due primarily to the A-chain, which acts as an RNA A-glycosidase, which leads to ribosome incapacitation and ultimately to cell death. Ricin was identified from crude castor bean extracts using LC-MS/MS. The extract was denatured, reduced, and alkylated prior to trypsin digestion. Ricin identification was based on the detection of marker peptides in the digest. These markers include T5, T7, Til, T12, and T13 from the A-chain and T3, T5, T14, T19, and T20 from the B-chain. MS/MS can provide the amount and sequence of each marker for irrefutable evidence. For quick screening of ricin in crude extracts, MALDI-MS can be used to provide the molecular mass profile of the marker peptides. 

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