Fig 3.

(A) Level of nonspecific cleavages as represented by the sum of XICs of the 52 NISTmAb semitryptic peptides and 6 nontryptic peptides relative to the sum of total identified peptides XICs as a function of trypsin resuspension time at room temperature (20°C). Dramatic differences were observed between the two conditions (i.e., 50 mM acetic acid and water); reconstitution in the acidic condition caused more nontryptic cleavages, the extent of which increased with longer reconstitution times. In contrast, the level of nonspecific cleavages remained consistent at approximately 1% with reconstitution of trypsin in water. (B) Evaluation of level of Trypsin-1 nonspecific cleavages under the recommended storage condition, with trypsin reconstituted in 50 mM acetic acid and stored at –80°C for different periods, versus the storage condition using water for reconstitution. The yield of nonspecific cleavages under the recommended storage condition (50 mM acetic acid) was significantly higher and increased with the length of the storage period. (C) Demographic profile of amino acids involved in the semitryptic and nontryptic cleavages, based on 220 semitryptic peptides and 34 nontryptic peptides involving five biotherapeutic samples in addition to NISTmAb.