Disulfides, cleavage detection

A potential drawback for top-down analyses is that, as the molecular weight of a protein increases, it becomes less likely that the monoisotopic peak will be observed. For proteins > 15 kDa, the monoisotopic peak is 1% abundance and cannot be detected [131,132], It has been shown also that natural variation in C versus abundance can shift the most abundant isotopic peak of carbonic anhy-drase by 1 Da [131]. These phenomena place a limit on the accurate determination of protein molecular mass and, consequently, PTM assignment for example, is a 2 Da shift due to either abundance and deamination, or disulfide cleavage ... 

Another crosslinker, SAED (Chapter 5, Section 3.9), can be used in a similar fashion, but instead of transferring a radioactive label, it contains a fluorescent portion that is transferred to a binding molecule after cleavage. Similarly, sulfo-SBED routinely is used to study protein interaction. Cleavage of a disulfide bridge after capture of interacting proteins results in transfer of a biotin label to the unknown prey protein (Chapter 28, Section 3.1). The biotin modification then can be used to detect or isolate the unknown interactor for subsequent identification. 

Cleavage of the polypeptide chain prior to partial reduction is an efficient way of controlling the reaction. For example, co-agatoxin IVB was completely reduced with TCEP, but upon treatment with trypsin, partially reduced species were detected under the same conditions 48 In contrast to these all or none characteristics in reduction, the four disulfide bonds of echistatin (1) can be reduced in a stepwise manner, which even allowed the elucidation of its reductive unfolding pathway 52 ... 

The gas-liquid chromatography with mass spectrometric detection (GLC-MS) analysis of the electrolyzed solution has shown that thiophenol is the only reduction product and the S—S bond cleavage is quantitative. Such a mechanism of bond breaking was confirmed by electrochemical studies. In cyclic voltammograms, anodic and cathodic peak potentials were the same for thiophenol and diphenyl disulfides thus the same species were participating in these processes. Electrode reactions of diphenyl disulfide are given by the following equations [166] ... 

Once the protein s primary sequence has been determined, the location of disulfide bonds in the intact protein can be established by repeating a specific enzymatic cleavage on another sample of the same protein in which the disulfide bonds have not previously been cleaved. Separation of the resulting peptides shows the appearance of one new peptide and the disappearance of two other peptides, when compared with the enzymatic digestion product of the material whose disulfide bonds have first been chemically cleaved. In fact, these difference techniques are generally useful in the detection of sites of mutations in protein mole-... 

For the formation of the second disulfide between Cys and Cys, the method applying Tl(tfa)3 obtained yields of over 90% and only a few percent of disulfide isomer were observed. Iodine oxidation in methanol gave no bicyclic product, while in 95% AcOH cleavage of the Asp-Pro bond was detected. In the case of DMSO/ 1 M HCl the oxidation took 14 h and side reactions occurred (Tyr deletion and appearance of disulfide isomers). 

Detailed electrochemical studies and the detection of crossover disulfide products in mixed electrolyses support the first mechanism [178]. Cleavage of an a C-Si bond on oxidation of a-silyl sulfides to give phenylthio carbocations as shown in Eq. (34) M=SiMe3 is known [179-182] ... 

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