Method：Mass spectrometry

Principle：
Contrast modification of the sample with IAA after reduction with DTT or incompletion reduction with TCEP with no treated sample, The samples are digested and then the peptides are analyzed by mass spectrometry to locate Disulfide Bonds.

Equipment：Finnigan LCQ Deca Xp / Finnigan LTQ

Samples Requirement：
1. Salt concentration: Volatile salt<20mM, nonvolatile salt<5mM
2. Protein amount: >10nmol; purity>90%

Example：



Fig1：MS/MS of peptide GSGGGCDLPQTHSLGNR.(Identified by digestion after reducted)
Fig2：MS/MS of peptide ISPFSCLK. (Identified by digestion after reducted)
Fig3：MS/MS of peptide FYTELYQQLNDLEACVIQEVGVEETPLMNVDSILAVR (Identified by digestion after reducted)
Fig4：MS/MS of peptide YSPCAWEVVR (Identified by digestion after reducted)
Fig5：Full scan of peptide of FYTELYQQLNDLEACVIQEVGVEETPLMNVDSILAVR和YSPCAWEVVR formed Disulfide Bonds

Result：
1. Not peptides found with Cys,with analysis of peptides mass spectrum without reduction after Trypsin digestion；These peptides（GSGGGCDLPQTHSLGNR；ISPFSCLK；YSPCAWEVVR；FYTELYQQLNDLEACVIQEVGVEETPLMNVDSILAVR）were found in mass spectrum if the sample was reduced before Trypsin digestion. Then we may speculate the sample has two pairs of Disulfide Bonds with no freed Cys.
2. In Fig 5, M/Z1817.8(+3)/1363.7(+4)/1091.6(+5) (corresponding to the peptide with disulfide bonds formed by YSPCAWEVVR and FYTELYQQLNDLEACVIQEVGVEETPLMNVDSILAVR)was found. Systematic analysis of Fig3，4，5, we can confirm the disulfide bonds location is Cys104-Cys144. For the same reason, we confirm the next disulfide bonds location is Cys104-Cys144.

Shanghai Applied Protein Technology Co. Ltd is a biotech company to provide customer service and product development in the proteomics filed.

The company is founded and owned by Research Center of Shanghai Institutes of Biological Sciences , Chinese Academy of Sciences.Our mission is to provide a total solution to our clients for their proteomics applications.

Through the cooperation with the proteomics center of SIBS we have developed innovate technologies and proteomics research platforms to provide the best service for our clients in the basic research, drug discovery & development, molecular diagnostics and other life sciences areas. We are the contributor to the life science.