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OBJECTIVE To establish a high resolution mass spectrometry method for characterization of recombinant human follicle-stimulating hormone disulfide bonds. METHODS For pre-treatment, follicle-stimulating hormone(FSH) was diluted to 0.5 mg·mL-1 with diluent, subtilin, trypsin, and PNGase F were added into the stock solution respectively, and 1 μL 10% formic acid was finally added to stop the reaction. For LC-MS analysis, ACQUITY UPLC Peptide BEH C18(2.1 mm×100 mm, 1.7 μm) column was used, the mobile phase was 0.1% formic acid/aqueous solution(A)-0.1% formic acid/acetonitrile solution(B), and gradient elution was performed. The mass spectrum acquisition mode was ddMS2, the ion source was ESI+, and the scanning range was m/z 250-2 000. RESULTS The 6-p-disulfide bond of β-subunit was successfully identified, with the values β as followes: C3=β:C51. β:C17=β:C66; β:C20=β:C104; β:C28=β:C82; β:C32=β:C84; β:C87=β:C94. The 5-p-disulfide bond of α-subunit was successfully identified, α:C7=α:C31; α:C10=α:C60; α:C28=α:C82; α:C32=α:C84; α:C59=α:C87. CONCLUSION The localization method of disulfide bond of recombinant human follicle stimulating hormone(hFSH) is established, which provides a new idea for the quality control of disulfide bond connection.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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