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Synapt g1 hdms instrument

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The Synapt G1 HDMS instrument is a high-resolution mass spectrometry system designed for analytical applications. It combines ion mobility separation with time-of-flight mass analysis to provide detailed structural information about molecules. The core function of the Synapt G1 HDMS is to analyze the composition and structure of complex samples with high sensitivity and resolution.

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Synapt HDMS (36 citations) Synapt G1 (24 citations) Synapt G1 HDMS (23 citations) Synapt HDMS instrument (5 citations)

7 protocols using synapt g1 hdms instrument

1

Native Mass Spectrometry of Protein Complexes

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MS measurements of native protein samples were collected on a Synapt G1 HDMS instrument (Waters Corporation) equipped with a radio frequency generator to isolate higher m/z species (up to 32k) in the quadrupole, and a temperature-controlled source chamber as previously described 44 . Instrument parameters were tuned to maximize signal intensity while preserving the solution state of the protein complexes. Data was collected in positive ion mode and typically takes 30 seconds to 60 seconds per sample. Instrument settings are as follows: source temperature of 25 ºC, capillary voltage of 1.7kV, sampling cone voltage of 100V, extractor cone voltage of 5V, trap collision energy of 20V, argon flow rate in the trap was set to 7 ml/min (5.6 × 10−2 mbar), and transfer collision energy set to 10V. The T-wave settings were for trap (300 ms−1/1.0V), IMS (300 ms−1/20V) and transfer (100 ms−1/10V), and trap DC bias (30V). Molar fractions were extracted from spectra analyzed using UniDec 45 . Titrations of AncSR1 and its variants were fit to the equation 2DP0=4P0+Kd−8P0Kd+Kd24P0 , using a custom Python script where D is the concentration of dimers, [P]0 is the total concentration of monomers, and Kd is the dissociation constant.
Hochberg G.K., Liu Y., Marklund E.G., Metzger B.P., Laganowsky A, & Thornton J.W. (2020). A hydrophobic ratchet entrenches molecular complexes. Nature, 588(7838), 503-508.
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2

Native Mass Spectrometry of Protein Complexes

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MS measurements of native protein samples were collected on a Synapt G1 HDMS instrument (Waters Corporation) equipped with a radio frequency generator to isolate higher m/z species (up to 32k) in the quadrupole, and a temperature-controlled source chamber as previously described 44 . Instrument parameters were tuned to maximize signal intensity while preserving the solution state of the protein complexes. Data was collected in positive ion mode and typically takes 30 seconds to 60 seconds per sample. Instrument settings are as follows: source temperature of 25 ºC, capillary voltage of 1.7kV, sampling cone voltage of 100V, extractor cone voltage of 5V, trap collision energy of 20V, argon flow rate in the trap was set to 7 ml/min (5.6 × 10−2 mbar), and transfer collision energy set to 10V. The T-wave settings were for trap (300 ms−1/1.0V), IMS (300 ms−1/20V) and transfer (100 ms−1/10V), and trap DC bias (30V). Molar fractions were extracted from spectra analyzed using UniDec 45 . Titrations of AncSR1 and its variants were fit to the equation 2DP0=4P0+Kd−8P0Kd+Kd24P0 , using a custom Python script where D is the concentration of dimers, [P]0 is the total concentration of monomers, and Kd is the dissociation constant.
Hochberg G.K., Liu Y., Marklund E.G., Metzger B.P., Laganowsky A, & Thornton J.W. (2020). A hydrophobic ratchet entrenches molecular complexes. Nature, 588(7838), 503-508.
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3

Mass Spectrometry Analysis of K2P4.1 Fusion Proteins

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Samples were loaded into gold-coated borosilicate glass capillaries prepared in-house and infused into the mass spectrometer using nanoelectrospray ionization with instrument settings tuned to minimize activation.27 (link) K2P4.1 fusion proteins were analyzed on a Synapt G1 HDMS instrument (Waters Corporation) with the capillary voltage set to 2.00 kV, sampling cone voltage at 180 V, extraction cone voltage at 10.0 V and argon flow rate at 7 mL/min. The T-wave settings for trap (300 ms and 2.0 V), IMS (300 ms and 20.0 V) and transfer (100 ms and 10 V). A backing pressure of 5.3 mbar, source temperature of 80 ˚C, trap bias of 32 V, and trap and transfer collision voltage set to 150V and 50 V, respectively. For collision-induced unfolding (CIU) experiments, all instrument settings were constant with the exception that the trap collision voltage varied from 60 to 240 V in 10 V steps. CIU plots were generated using the program Pulsar.54 (link) Impact55 (link) was used to calculate CCS for crystal structures of K2P4.1.
Schrecke S., Zhu Y., McCabe J., Bartz M., Packianathan C., Zhao M., Zhou M., Russell D, & Laganowsky A. (2020). Selective regulation of human TRAAK channels by biologically active phospholipids. Nature chemical biology, 17(1), 89-95.
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4

Mass Spectrometry Analysis of K2P4.1 Fusion Proteins

Cited 1 time
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Samples were loaded into gold-coated borosilicate glass capillaries prepared in-house and infused into the mass spectrometer using nanoelectrospray ionization with instrument settings tuned to minimize activation.27 (link) K2P4.1 fusion proteins were analyzed on a Synapt G1 HDMS instrument (Waters Corporation) with the capillary voltage set to 2.00 kV, sampling cone voltage at 180 V, extraction cone voltage at 10.0 V and argon flow rate at 7 mL/min. The T-wave settings for trap (300 ms and 2.0 V), IMS (300 ms and 20.0 V) and transfer (100 ms and 10 V). A backing pressure of 5.3 mbar, source temperature of 80 ˚C, trap bias of 32 V, and trap and transfer collision voltage set to 150V and 50 V, respectively. For collision-induced unfolding (CIU) experiments, all instrument settings were constant with the exception that the trap collision voltage varied from 60 to 240 V in 10 V steps. CIU plots were generated using the program Pulsar.54 (link) Impact55 (link) was used to calculate CCS for crystal structures of K2P4.1.
Schrecke S., Zhu Y., McCabe J., Bartz M., Packianathan C., Zhao M., Zhou M., Russell D, & Laganowsky A. (2020). Selective regulation of human TRAAK channels by biologically active phospholipids. Nature chemical biology, 17(1), 89-95.
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5

Mass Spectrometric Analysis of Nanojars

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Mass spectrometric analysis of the nanojars was performed with a Waters Synapt G1 HDMS instrument using electrospray ionization (ESI). 10−4–10−5 M solutions were prepared in N,N-dimethylformamide (DMF). Samples were infused by a syringe pump at 5 μL/min, and nitrogen was supplied as nebulizing gas at 500 L/h. The electrospray capillary voltage was set to –2.5 or +3.0 kV, respectively, with a desolvation temperature of 150 °C. The sampling and extraction cones were maintained at 40 V and 4.0 V, respectively, at 80 °C. The MS/MS conditions were the same, except the transfer collision energy was 5 V and the trap collision energies were 5, 30, 40, 50, 60 and 70 V.
Al Isawi W.A, & Mezei G. (2021). Doubling the Carbonate-Binding Capacity of Nanojars by the Formation of Expanded Nanojars. Molecules, 26(11), 3083.
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6

Native ESI-IM-MS Analysis of α-Synuclein

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Protein samples with a final concentration of 20 μM for native electrospray ionization-ion mobility-mass spectrometry (ESI-IM-MS) experiments were prepared in 50 mM ammonium acetate buffer (pH 6.8). Small molecule stocks in 100% DMSO were diluted into the buffer solution to achieve the required final concentrations (molar ratio of α-syn and small molecule: 1:1, 1:2.5 and 1:5). The percentage of DMSO in the solution was 2% (v/v) for all of the samples.
ESI-IM-MS analysis was performed on a Synapt G1 HDMS instrument (Waters Corp., Wilmslow, UK) with travelling-wave ion mobility (TWIMS). All the samples were analysed using positive ionization ESI with a spray capillary voltage of 1.8 kV using home-made borosilicate glass capillaries with filaments (Puller: the Flaming/Brown Micropipette Puller; Coater: Emitech Sputter Coater). The following instrumental parameters were used: sampling cone 50 V; source temperature 80 °C; backing pressure 3.0 mbar; trap collision energy 5 V; extraction cone 1 V; trap DC bias 22 V, transfer collision energy 4 V. Data were acquired over the m/z range of 500-4000. Data were processed by using MassLynx V4.1 and Driftscope V2.5 software supplied with the mass spectrometer.
Nahass G.R., Sun Y., Xu Y., Batchelor M., Reilly M., Benilova I., Kedia N., Spehar K., Sobott F., Sessions R.B., Caughey B., Radford S.E., Jat P., Collinge J, & Bieschke J. (2021). Brazilin Removes Toxic alpha-Synuclein and Seeding Competent Assemblies from Parkinson Brain by Altering Conformational Equilibrium. Journal of molecular biology, 433(8), 166878.
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7

High-Resolution Mass Spectrometry for Membrane Proteins

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MS was performed on a Q Exactive Ultra-High Mass Range (UHMR) from ThermoFisher Scientific, modified with a custom reverse entry ion source (REIS) and 1.5 m drift tube. A Synapt G1 HDMS instrument with a 32-k RF generator from Waters Corporation was also used for membrane and soluble protein measurements. Nanoelectrospray ionization was performed as previously described.19 (link) UHMR parameters applied for AmtB included 250 °C capillary temperature, 1.40 kV spray voltage, 200 maximum inject time, resolution set to 6250 without averaging, 50 eV in-source CID, 40 CE, trapping gas pressure of 5. Ion transfer was set to high m/z mode, with in-source desolvation voltage set to −180 V.
Lyu J., Liu Y., McCabe J.W., Schrecke S., Fang L., Russell D.H, & Laganowsky A. (2020). Discovery of Potent Charge-Reducing Molecules for Native Ion Mobility Mass Spectrometry Studies. Analytical chemistry, 92(16), 11242-11249.
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