Synapt g1 hdms

Investigation of the payload conjugation site was performed by peptide mapping analysis using a slightly modified RapiGest SF Surfactant care and use protocol (Waters, Milford, MA). Briefly, 10 μg of each sample was mixed with RapiGest 0.1% final concentration and reduced for 30 min at 60 °C with 5 mM Dithiothreitol. Alkylation was performed with 15 mM iodoacetamide for 30 min at RT. Finally 1.5 μL of a 1 μg/μl trypsin solution were added and incubated overnight at 37 °C. After digestion, the sample was acidified by addition of 0.5% trifluoroacetic acid and analyzed by Nano-LC-MSe using a nanoAcquity UPLC coupled to a Synapt G1 HDMS (Waters, Milford, MA) operated in MSe mode. 0.1% formic acid in water was used as solvent A and 0.1% formic acid in acetonitrile as solvent B. Tryptic peptides were injected and trapped for 3 min on a Symmetry C18 pre-column (5 μm, 180 μm × 20 mm, Waters, Milford, MA) with a flow rate of 10 μl/min. Separation was performed using an UPLC 1.7 μm BEH130 column (C18, 75 μm × 100 mm, Waters) with a flow rate of 450 nl/min, starting with 2% B from 0 to 2 min followed by a linear gradient from 8–35% B for 67 min. The used MSe mode combines an alternating MS and MSe (full mass range fragmentation) function each second. Spectra were acquired from m/z 50 to 1600 and extracted ion chromatograms were analyzed manually.

In short, native MS was performed on a Synapt G1 HDMS instrument (Waters Corporation) equipped with a 32k RF generator or on a Thermo Scientific Exactive Plus extended mass range (EMR) with a rear-entry ion source (REIS)^{49 (link)}. IM data were processed using PULSAR^{55 (link)}. Mole fraction was determined from deconvolution of mass spectra with UniDec^{57 (link)}. Denatured MS was performed on the front end of the REIS-orbitrap. Tryptic digest analysis and protein sequencing was performed on Thermo Orbitrap Fusion. Further details can be found in Supplementary Methods.

Purified SOD1 samples with pre-conjugated ligand were buffer exchanged to remove free ligand and diluted to 5 µM in 100 mM NH₄OAc, 50% acetonitrile and 2.5% formic acid. Samples were incubated for 30 min at room temperature before being nano-electrosprayed. Spectra were externally calibrated using a solution of caesium iodide (10 mg/mL) in 50% n-propanol and masses were determined using Masslynx 4.1 software (Waters, UK).
Native MS analysis was performed using a SYNAPT G1 HDMS (Waters, UK) with parameters set according to previous work^{30 (link)}. Briefly, purified recombinant SOD1 protein was buffer exchanged into 200 mM NH₄OAc (pH 6.8) using centrifugal concentrators with a 10-kDa molecular weight cut-off. Buffer exchanged SOD1 was diluted to working concentrations in NH₄OAc, loaded into gold-coated borosilicate capillaries (made in-house), and subjected to nano-electrospray ionization (n = 1 electrosprays for 3 separate dilutions). All spectra were externally calibrated using 1 mg/mL caesium-iodide in 50% n-propanol (Supplementary Fig. 10), and were processed using Masslynx 4.1 software.

Native MS was performed on a Synapt G1 HDMS instrument (Waters corporation) with a 32 k RF generator. AmtB–GlnK samples were at a final concentration of 2 µM. Instrument parameters were tuned to maximize ion intensity and simultaneously preserve the native-like state of AmtB. The instrument was set to a capillary voltage of 1.7 kV, sampling cone voltage of 100 V, extractor cone voltage of 10 V, and argon flow rate at 7 mL min⁻¹ (5.2 × 10^–2 mbar). The T-wave settings for trap (300 ms⁻¹/2.0 V), IMS (300 ms⁻¹/20 V), and transfer (100 ms⁻¹/10 V), source temperature (110 °C) trap collision voltage (20 V) and transfer collision voltage (160 V) and trap bias (35 V) were also optimized.

For intact mass analysis of heavy and light chain, 20 μg of each sample was mixed with 1% mercaptoethanol final concentration and reduced for 30 min. at 37 °C. Reduced samples were loaded directly onto a CapLC-MS system (1100 series, Agilent Technologies) coupled to a Synapt G1 HDMS (Waters, Milford, MA) operated in MS positive ion mode. 0.1% trifluoroacetic acid in water was used as solvent A and 0.1% trifluoroacetic acid in 70% n-propanol as solvent B. The samples were separated via a Zorbax SB300 C8 column (3.5 μm, 150 × 0.3 mm, Agilent Technologies), tempered at 75 °C. The gradient started with 3% B and a flow rate of 10 μL/min followed by a linear gradient from 2 to 39 min from 10% B to 60% B. Protein signals were recorded with a DA-detector at 214 and 280 nm. MS spectra were acquired from m/z 500 to 3000. Finally, intact protein mass was calculated applying the MaxEnt1 deconvolution algorithm.

Purified SOD1 was destabilized via incubation in 20 mM DTT 5 mM EDTA in 1 × PBS (pH 7.4) at 37 °C for 2 h, following which samples were buffer exchanged into 200 mM ammonium acetate using gel-filtration chromatography (Superdex 75 10/300 GL, GE USA). Fractions off the column were immediately placed on ice, pooled, and diluted to a final concentration of 10 µM SOD1 monomer (measured via BCA assay). Mass spectrometry analysis was performed using a SYNAPT G1 HDMS (Waters, UK) with parameters set according to previous work^{32 (link)}. Briefly, SOD1 samples at 10 μM in 200 mM NH₄OAc were loaded into gold-coated borosilicate capillaries (made in-house) and subjected to nano-electrospray ionization. Results are representative of 3 separate unfolding experiments. All spectra were externally calibrated using 10 mg/ml caesium-iodide in 50% n-propanol, and were processed using Masslynx 4.1. For determination of the abundances of the observed conformations the area under the peak for each charge state was determined and the values plotted as a function of charge state. Following this, MATLAB R2014b (Version 8.4) was used to fit Gaussians to the plots and area under the peak was determined. Instrument parameters can be found in Appendix A.