Journal: Journal of the American Chemical Society

Article Title: Time-Resolved Native Mass Spectrometry for Direct Measurement of Biomolecular Kinetics

doi: 10.1021/jacs.5c21842

Figure Lengend Snippet: Capturing an array of mixing times using theta emitters. (A) Mass spectrum of 0.8 μM green fluorescent protein (GFP) binding 2 μM Nb15 (15) and 4 μM NbAS (AS) at equilibrium. (B) Extracted mass chromatogram of apo, Nb15, and NbAS-bound GFP over three ESI power cycles acquired in one data acquisition. (C–E) Mass spectra 0.8 μM GFP binding 2 μM Nb15 and 4 μM NbAS in a theta emitter shortly after ESI voltage was turned on (C), and after (D) 1 or (E) 1.5 min of sustained voltage. The concentrations listed account for a 2-fold dilution within the Taylor cone during rapid mixing. Deconvoluted spectra are shown in Figure S9 .

Article Snippet: Briefly, codon-optimized genes for anti-alpha synuclein (NbAS), anti-GFP (Nb15), and anti-ALFA (NbALFA) were obtained from Twist Biosciences.

Techniques: Binding Assay

Journal: Journal of the American Chemical Society

Article Title: Time-Resolved Native Mass Spectrometry for Direct Measurement of Biomolecular Kinetics

doi: 10.1021/jacs.5c21842

Figure Lengend Snippet: Capturing kinetics of protein–protein interactions using theta emitters. (A, B) Biolayer interferometry (BLI) sensorgrams showing GFP binding to biotinylated (A) NbAS and (B) Nb15, with corresponding global fits (solid lines) based on a 1:1 binding model. (C) Crystal structure of Nb15 bound to GFP ( Table S1 and Figure S14 ). (D) Time-resolved native MS kinetic curves showing GFP binding to Nb15, timed using NbAS. Data from five bursts are plotted in distinct colors, fit to a 1:1 global kinetic model in which each burst is assigned its own mixing factor (α). The colored traces represent individual bursts, and solid lines denote the corresponding model fits, with each α value labeled in the matching color. (E) Kinetic rate constants and equilibrium dissociation constants ( K D ) for the GFP–Nb15 interaction timed with GFP-NbALFA and SOS–NbALFA, and for the GFP–NbAS interaction timed with GFP-NbALFA, as determined by BLI and time-resolved native mass spectrometry. A table of kinetic values for each system is provided in Table S2 .

Article Snippet: Briefly, codon-optimized genes for anti-alpha synuclein (NbAS), anti-GFP (Nb15), and anti-ALFA (NbALFA) were obtained from Twist Biosciences.

Techniques: Protein-Protein interactions, Binding Assay, Labeling, Mass Spectrometry