Abstract:

Digestive stability analysis of exogenous protein is one of the important indexes of genetically modified organisms
(GMO) safety assessment. In the present study, we established a novel assessment assay for protein digestive stability based
on label-free quantification of mass spectrometry using bovine β-lacto globulin (BLG), bovine serum albumin (BSA) and
soybean trypsin inhibitor (STI) as standard proteins. Protein samples were treated with simulated gastric/intestinal fluids
(SGF/SIF), and then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The target bands were then
cut out and trypsinized. The extracted peptides were analyzed by nano liquid chromatography-electronic spray ion-mass
spectrum/mass spectrum and identified by Mascot software. By calculating the ratio between the matched peptide numbers
of the target protein before and after SGF/SIF treatment, the digestibility of the target protein was estimated. When the ratio
was lower than 0.50, the target protein was considered digestible. This newly developed assay was applied to Cry1Ab/1Ac,
the exogenous protein of the genetically modified rice ‘Huahui No. 1’. The results showed that the ratio was lower than 0.50
after digestion in SGF for 2 minutes, while it decreased to less than 0.50 after digestion in SIF for 15 seconds, indicating that it
is very liable to be digested. Our study provides a novel MS-based method for digestive stability analysis of target proteins from
genetically modified organisms, which is easy to operate with no need for specific antibody.

Key words: genetically modified organisms, digestive stability, mass spectrometry, exogenous protein Cry1Ab/1Ac, lable-free quantification