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I am learning protein sequencing and it seems that diagonal electrophoresis is a common method in the identification of disulphide bonds (S-S) exist between polypeptides in a protein.

Questions:

  1. After horizontal electrophoresis, we need to cleave the S-S bond using performic acid. According to Wiki,

Its strong oxidizing properties are used for cleaving disulfide bonds in protein mapping.

However, from what I have learnt in SDS-PAGE, 2-mercaptoethanol OR dithiothreitol are commonly used for cleaving S-S bonds, may I ask why do we need to use formic acid specifically here?

  1. the first electrophoresis is done horizontally and the second is done vertically (bottom to top). May I ask whether we can do it vertically then horizontally instead? It seems to me that it should be OK since there doesn't exist any disruption to the proteins but I would like to confirm.

Thank you.

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    $\begingroup$ You probably don’t need to oxidize the disulfide bonds rather than reduce them. Performic acid has the advantage that the oxidation products can’t spontaneously reform a disulfide bond whereas the reduction products from a thiolate reductant can. This may not be a significant issue in the actual application. Oxidation by performic acid also causes a significant mass change for the cysteine residues which can be useful for downstream mass spec. These oxidation products are also negatively charged which can increase mobility in PAGE and possibly aid separation. $\endgroup$
    – canadianer
    Jun 4, 2021 at 17:44
  • $\begingroup$ @canadianer thanks for pointing out my mistake :) So can I say: performic acid oxidize the sulfur in cysteine residue, forming cystenic acid i.e. disulphide bond is cleaved. Moreover, this is the cause of 'significant mass change' (-SH --> -SOOH), which can be utilized by MS for easy determination of S-S bond (where B-ME and DTT cannot achieve this)? Thanks $\endgroup$
    – Questions
    Jun 5, 2021 at 2:47
  • $\begingroup$ ref: researchgate.net/profile/Tanea-Reed/publication/51828850/figure/… $\endgroup$
    – Questions
    Jun 5, 2021 at 2:47
  • $\begingroup$ This article states that complete oxidation of the cystine to cysteic acid occurs (SO3-), which is a molecular mass change of 48 u. With BME or DTT reduction, you are left with a thiol or thiolate. $\endgroup$
    – canadianer
    Jun 5, 2021 at 11:12
  • $\begingroup$ Now I understand thank you very much $\endgroup$
    – Questions
    Jun 5, 2021 at 13:09

1 Answer 1

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You probably don’t need to oxidize the disulfide bonds rather than reduce them. Performic acid has some potential advantages:

  • The oxidation products can’t spontaneously reform a disulfide bond whereas the reduction products from a thiolate reductant can.
  • Oxidation by performic acid causes a significant mass change for the cysteine residues which can be useful for downstream mass spec. This article states that complete oxidation of the cystine to cysteic acid occurs (-SO3-), which is a molecular mass change of 48 u. With BME or DTT reduction, you are left with a thiol or thiolate.
  • These oxidation products are also negatively charged which can increase mobility in PAGE and possibly aid separation.
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