Endoproteinase glu c

Reduction and alkylation of the cystine bonds was carried out as previously described [28 (link)]. Sigma proteomics sequencing grade trypsin and endoproteinase Glu-C were used to digest the reduced and alkylated venom samples and the enzymes were activated in 40 mM NH₄HCO₃ buffer. A ratio of 1:100 (w/w) of enzyme to venom peptides was used. The digestion was carried out overnight at 37 °C and enhanced in a microwave apparatus for 4 min on the lowest power setting.

Sodium acetate (NaOAC), sodium bicarbonate (NaHCO₃), formaldehyde-d2, sodium cyanoborohydride (NaBH₃CN), ammonium hydroxide, dl-dithiothreitol (DTT), iodoacetamide (IAM), ammonium bicarbonate (NH₄HCO₃), guanidine hydrochloride (Gd-HCl), hydrochloric acid (HCl), sequence-grade chymotrypsin, α-lactalbumin type I, and endoproteinase Glu-C were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sequence-grade PNGase F were purchased from Promega (Madison, WI, USA). LC–MS grade acetonitrile (ACN), methanol (MeOH), formic acid (FA), dimethylformamide (DMF) were purchased from Thermo Fisher Scientific Inc (Walthan, MA, USA). Water (ddH₂O) was deionized to 18 MΩ by a Milli-Q system. Amicon centrifugal filters (10 or 30 kDa MWCO) were obtained from Millipore (Billerica, MA, USA). Human IgG (Trastuzumab) was from commercially available antibody drug product Her-IgG as the solution form (22 mg/mL). 2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)acetaldehyde (AD-PEG-N₃) and 2,5-Dioxopyrrolidin-1-yl 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)acetate (NHS-PEG-N₃) linkers were synthesized in house and their structures were confirmed by NMR and MS: ¹H-NMR (400 MHz): δ 9.71 (s, 1H), 4.14 (s, 2H), 3.71–3.64 (m, 10H), 3.37 (t, J = 5.0 Hz, 2H) for NHS-PEG-N₃; and ¹H-NMR (300 MHz): δ 4.53 (s, 2H), 3.82–3.79 (m, 2H), 3.74–3.66 (m, 8H), 3.39 (t, J = 5.0 Hz, 2H), 2.34(s, 4H) for AD-PEG-N₃.

Sequencing-grade modified trypsin was purchased from Promega (Madison, WI, USA), endoproteinase Glu-C from Sigma-Aldrich (St. Louis, MO, USA), Tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) and NHS-Activated Agarose Slurry from ThermoFisher Scientific (Rockford, IL, USA), S-methyl methanethiosulfonate (MMTS) from TCI (Tokyo Chemical Industry, Tokyo, Japan), and C18 MacroSpin Column from NestGroup (Southborough, MA, USA).

8701-BC cells were grown for 48 h, medium was collected, and proteins quantified with Bradford reagent. Amounts of 2.5, 5 and 10 µg of total proteins were loaded in SDS PAGE. After the incubation of the gel with a Blue Coomassie solution, the most evident band at 55 kDa was excised from the gel and analyzed by MALDI-TOF mass spectrometry [74 (link)].
Briefly, the spot from the gel was destained, reduced, alkylated, and submitted to hydrolysis with endoproteinase Glu-C (SIGMA-Aldrich, Milan, Italy). Peptide mixtures were analyzed by MALDI-TOF mass spectrometry using a Voyager DE-PRO mass spectrometer (Applied Biosystems, Monza, Italy). Samples were freeze-dried and then dissolved in 10 μL of 0.2% trifluoroacetic acid; 1 μL was mixed with 1 μL of a solution of alpha-cyano-4-hydroxycinammic acid, 10 mg/mL in acetonitrile, 0.2% trifluoroacetic acid 7:3 (v:v), and the mixture was applied onto the metallic sample plate and air dried. Mass calibration was performed using a peptide standard mixture provided by the manufacturer. All mass values are reported as monoisotopic masses, and raw data were analyzed using software provided by the manufacturer.