Peptide-centric (bottom-up) de novo sequencing

Clones of interest were captured through fraction collection using the same chromatography setup used for LC-MS profiling. Samples were dried under vacuum and resuspended in a 50 mM ammonium bicarbonate buffer. To boost signal intensity, the fractions were pooled across the time points. Samples were equally split for digestion with four proteases.

For digestion with trypsin, chymotrypsin and thermolysin, a sodium deoxycholate (SDC) buffer was added to a total volume of 80 μL, 200 mM Tris pH 8.5, 10 mM tris(2-carboxyethyl)phosphine (TCEP), 2% (w/v) SDC final concentration. For pepsin digestion, a urea buffer was added to a total volume of 80 μL, 2M Urea, 10 mM TCEP. Samples were denatured for 10 min at 95 °C followed by reduction for 20 min at 37 °C. Next, iodoacetic acid was added to a final concentration of 40 mM and incubated in the dark for 45 min at room temperature for alkylation of free cysteines. Then for trypsin, chymotrypsin and thermolysin 50 mM ammonium bicarbonate buffer was added to a total volume of 100 μL. For pepsin 1 M HCl was added to a final concentration of 0.04 M. 0.1 μg of each protease was added and incubated for 4 hours at 37 °C. After digestion 2 μL HCOOH was added to precipitate the SDC. SDC was removed by centrifugation for 20 min at max speed (20,817 × g) after which the supernatant was moved to a new tube.

Samples were desalted by Oasis HLB (Waters Corporation, Millford, MA, USA) following a 5-step protocol. 1) Sorbent was wetted using 2x 200 μL CH₃CN, 2) followed by equilibration with 2x 200 μL water/10% HCOOH. 3) The sample was loaded and 4) washed with 2x 200 μL water/10% HCOOH. 5) Finally, the sample was eluted using 2x 50 μL water/50% CH₃CN /10% HCOOH and dried down by vacuum centrifuge. Prior to MS analysis samples were reconstituted in 2% HCOOH.

Data acquisition was performed on the Orbitrap Fusion Tribrid Mass Spectrometer (Thermo Scientific, San Jose, CA, USA) coupled to UHPLC 1290 system (Agilent Technologies, Santa Clara, CA, USA). Peptides were trapped (Dr. Maisch Reprosil C18, 3 μm, 2 cm × 100 μm) prior to separation (Agilent Poroshell EC-C18, 2.7 μm, 500 mm × 75 μm). Trapping was performed for 10 min in solvent A (0.1% HCOOH in Milli-Q), and the gradient was as follows: 0 – 13% solvent B (0.1% HCOOH in 80% CH3CN) over 5 min, 13 – 44% solvent B over 65 min, 44 – 100% solvent B over 4 min, and 100% B for 4 min (flow was split to achieve the final flowrate of approximately 200 nL/min). Mass spectrometry data was collected in a data-dependent fashion with survey scans ranging from 350-2,000 Th (resolution of 60,000 @ m/z 200), and up to 3 sec for precursor selection and fragmentation with either stepped higher-energy collisional dissociation (HCD) set to [25%, 35%, 50%] or electron transfer dissociation (ETD), used with charge-normalized settings and supplemental activation of 27%. The MS2 spectra were recorded at a resolution of 30,000 (@ m/z 200). The AGC targets for both MS and MS2 scans were set to standard within a maximum injection time of 50 and 250 ms, respectively.

Raw LC-MS/MS data were processed using PEAKS X software (Bioinformatics Solutions Inc., Waterloo, ON, Canada) for de novo sequencing of peptides. The following parameters were used for de novo sequencing: parent mass error tolerance – 12 ppm, fragment mass error tolerance – 0.02 Da, max number of variable PTMs per peptide – 3. Fixed modifications: Carboxymethyl; variable modifications: Oxidation (HW), Oxidation (M), Pyro-glu from E, Pyro-glu from Q, Carboxymethyl (KW, X@N-term), and Carbamylation. Resulting de novo peptide tables were exported as.csv files and used for filtering of the IMGT database and determination of the mature ^24.4 1 _47359.4 clone sequences.