Non reduced peptide mapping using Lys C

Disulphide mapping analysis was performed on Waters ACQUITY UPLC coupled to Waters Synapt HDMS system. 100 µg of intact antibody was denatured using 6 M guanidine hydrochloride at 37 ºC for 30 min. 1 ml of the cooled Ethanol is added and stored in − 20 °C for 1 h for precipitation of the protein. The sample is centrifuged at 8000 rpm for 15 min and collected precipitate was treated with 50 µl of 2 M Urea, 2 mM CaCl₂, 0.2 M Tris HCl (pH 6.5) and 2.5 µg of Lys C enzyme (Roche sequencing grade modified; reconstituted with MilliQ water) in the ratio of 1: 20 (Lys C: antibody, w/w). The reaction mixture was incubated at 37 °C for 48 h. The digested sample was further analyzed LC MS. Standard operating conditions were used for LC MS as described below:

Mobile phase A: 100% acetonitrile.

Mobile phase B: 0.1% FA in water.

Column: C18, 2.1 × 100 mm, 1.7 µm, part no: 1860002352.

Flow rate: 0.3 ml/min.

Column temp: 40 °C.

Analyzer mode: sensitivity.Analyzer mode: sensitivity.

Cone voltage: 25 V.

Scan time: 1 s.

Mode: positive.

Mass range: 50–2500 m/z.

Trap collision: 4–30 V.