Procise protein sequencing system

Purified mAb Guy’s 13 samples were separated by SDS-PAGE on 4–15% gels (Bio-Rad), blotted on PVDF membrane, and stained with Coomassie suspension G250. The N-terminal sequencing of mAb degradation fragments was performed by M. Weldon (University of Cambridge) on a Procise Protein Sequencing System (Applied Biosystems, Foster City, CA, USA).

The hydrolytic activity for matrix metalloproteinase was assayed by using synthetic fluorescence quenching substrates [22 (link),23 (link)]. Substrates were prepared as 10 mM stock solutions in dimethyl sulfoxide. Assay was carried out by incubating 5 μL of substrate stock solution with 10 μL of venom fraction in 50 mM Tris-HCl buffer (pH 7.5) containing 0.15 M NaCl, 10 mM CaCl₂, 0.05% Brij 35, and 0.02% NaN₃. The reaction was stopped by the addition of 0.9 mL of 3% acetic acid, and the intensity of fluorescence was measured at λ_ex of 325 nm and λ_em of 393 nm. The amount of substrate hydrolyzed was calculated from the standard curve of the reference compound, MOCAc–Pro–Leu–Gly.
The effect of venom on human prothrombin, fibrinogen, collagen, and laminin were measured by monitoring the time course degradation of these proteins on SDS polyacrylamide gel. Oxidized insulin B chain was used for the determination of enzyme specificity. Briefly, the hydrolyzed peptides were fractionated by a reversed-phase HPLC column (Develosil 300 ODS-7), and the fragments were identified by MALDI-TOF MS spectrum using Autoflex speed (Bruker Daltonics). The N-terminal amino acid sequence of each fragment was also detected by a Procise protein sequencing system (Applied Biosystems, Foster City, CA, USA).

The N-termini of the heavy chain, light chain, and Protac-digested heavy chain were analyzed by Edman degradation [37 ]. Purified proteins were subjected to reducing 12% SDS-PAGE and electrotransfered overnight at 30 V in CAPS buffer (Sigma-Aldrich, Oakville, ON) onto Immobilon-P^SQ PVDF membrane (Millipore, Billerica, MA). The membrane was incubated for 10 mins in 0.2% (w/v) Ponceau S (Sigma-Aldrich, Oakville, ON) in 1% (v/v) acetic acid and washed three times in Milli-Q water. It was then stored in a moist chamber and submitted for sequencing (The Advanced Protein Technology Centre, Hospital for Sick Children, Toronto, ON). Stained protein bands were excised and analyzed by the Procise Protein Sequencing System (Applied Biosystems, Foster City, CA) as previously described [38 ].