Chemicals were purchased from commercial sources (like Sigma‐Aldrich, TCI, AlfaAesar, ABCR and Fluka). Indolenine precursors and the protected DOTA chelator were synthesized according to published procedures. 1H, 13C and 71Ga NMR spectra in solution were measured with the spectrometers ECX 400 (400 MHz) and ECP 500 (500 MHz) from JEOL and Avance 500 (500 MHz) and Avance 700 (700 MHz) from Bruker. Mass spectra were measured on a 6210 ESI‐TOF and 6230 ESI‐TOF from Agilent. UV/VIS spectra were recorded using a PerkinElmer LAMBDA 950 UV/Vis/NIR spectrometer and fluorescence spectra recorded using a JASCO FP‐6500 spectrometer. NP automated column chromatography was done on a CombiFlash Rf (Teledyne ISCO) using prepacked silica columns (30 μm). Purification of compounds using size exclusion chromatography was done on a Sephadex column (NAP‐25, Sephadex G‐25 DNA) with water as eluent.
6230 esi tof
Manufactured by Agilent Technologies
The Agilent 6230 ESI-TOF is an electrospray ionization time-of-flight mass spectrometer. It is designed to provide accurate mass measurements and high-resolution mass analysis of a wide range of analytes.
Automatically generated - may contain errors
3 protocols using 6230 esi tof
1
Characterization of Ga-based Complexes
2
Verifying Stilbene-TTR Conjugation
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Before sample freezing, to verify that the major species in the (Stilbene)2-TTR sample is conjugated at both TTR binding sites, we carried out liquid chromatography followed by mass spectrometry (LC-MS, with LC equipment Agilent 1260 and mass spectrometer Agilent 6230 ESI-TOF) on a 3X diluted aliquot taken 3 hours after starting the reaction. For this, 5 µL of sample was injected in a reverse phase column (Agilent PLRP-S 2.1 x 50 mm 100A 5 µm) held at 60 °C and eluted with a water-to-acetonitrile gradient from 95% to 50% water, and a flow of 0.5 mL/min, 95% for 2 min then 50% water for 6 min.
To probe for the existence of A2 hydrolysis products, we carried out a time series of the A2+TTR reaction (same concentrations as used for sample preparation), injecting sample every 30 minutes for 5 hours. We monitored UV absorbance at 280 and 305 nm, as well as total ion count. We also analyzed samples containing only A2 (150µM A2 in TTR buffer + 10% DMSO) and only TTR (21µM TTR in TTR buffer). The baseline was removed using the ZhangFit method from the BaselineRemoval python package28 (link). The exponential labeling temporal curves were generated by normalizing the area under the curve dividing by the maximum, after baseline removal. See results inSupplementary Fig. 31 and Supplementary Text 4 .
To probe for the existence of A2 hydrolysis products, we carried out a time series of the A2+TTR reaction (same concentrations as used for sample preparation), injecting sample every 30 minutes for 5 hours. We monitored UV absorbance at 280 and 305 nm, as well as total ion count. We also analyzed samples containing only A2 (150µM A2 in TTR buffer + 10% DMSO) and only TTR (21µM TTR in TTR buffer). The baseline was removed using the ZhangFit method from the BaselineRemoval python package28 (link). The exponential labeling temporal curves were generated by normalizing the area under the curve dividing by the maximum, after baseline removal. See results in
3
High-throughput Protein Analysis via LC-MS
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The majority of the protein analysis was performed on an InnityLab II LC coupled to a single quadrupole MS (LC/MSD XT, Agilent Technologies) using an AdvanceBio RP-mAb Wide Pore Reversed-Phase column (2.1 × 50 mm, particle size 3.5 mm, Agilent Technologies) and a linear gradient of 0-95% B over 15 min (solvent A: H 2 O + 0.1% formic acid; solvent B: 80% isopropanol, 10% acetonitrile, 10% H 2 O + 0.1% formic acid; ow rate of 0.3 mL min -1 ). The protein samples were analyzed in positive mode as well as by UV absorbance at 210 and 280 nm. Deconvolution was performed with the Agilent OpenLAB CDS ChemStation LC/ MS soware using standard parameters and 2000 for the noise cut-off. For VAO and TOYE variants, analysis was performed on an Agilent 6230 ESI-TOF using the same column and eluent gradients. The calculated molecular weight of each protein was derived using Geneious Prime® 2021.2.2.
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