Spectra were acquired on a QToF II mass spectrometer (Waters) modified for high mass transmission (Sobott et al., 2002 (link)). Then, 2.5 μl of the solution was introduced into the mass spectrometer using a gold-coated capillary needle prepared in house (Hernández and Robinson, 2007 (link)). Spectra were acquired in the positive ion mode and MS conditions were kept constant while concentration effects were monitored. For instrument parameters, see the Supplemental Experimental Procedures . Spectra were processed with MassLynx V4.1 with minimal smoothing and analyzed using Massign (Morgner and Robinson, 2012 (link)) and Unidec (Marty et al., 2015 ) software.
Qtof 2 mass spectrometer
Manufactured by Waters Corporation
Sourced in United Kingdom
The QToF II mass spectrometer from Waters Corporation is a high-performance analytical instrument designed for precise mass measurements and structural elucidation of molecules. It combines a quadrupole mass analyzer and a time-of-flight (TOF) mass analyzer to provide accurate mass data and enhanced sensitivity.
Automatically generated - may contain errors
Lab products found in correlation
Masslynx v4, by Waters Corporation (2 mentions)
Masslynx 4, by Waters Corporation (1 mentions)
Micro bio spin 6 column, by Bio-Rad (1 mentions)
Vivaspin filter, by Sartorius (1 mentions)
Masslynx software, by Waters Corporation (1 mentions)
Superdex 200 10 300, by GE Healthcare (1 mentions)
Glu1 fibrinopeptide b, by Merck Group (1 mentions)
Massprep station, by Waters Corporation (1 mentions)
Trypsin gold, by Promega (1 mentions)
Amicon ultra 0.5 ml, by Merck Group (1 mentions)
Micromass, by Waters Corporation (1 mentions)
Ultimate 3000 nano hplc system, by Thermo Fisher Scientific (1 mentions)
Quattro ultima, by Waters Corporation (1 mentions)
16 protocols using qtof 2 mass spectrometer
1
Mass Spectrometry of Protein Complexes
2
Non-covalent Protein Complex Analysis by NanoESI-MS
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NanoESI-MS measurements were performed in the positive ion mode on a modified Q-ToF II mass spectrometer (Waters) [55] (link), using in-house prepared, gold-coated glass capillaries [47] (link). The MS parameters in order to preserve non-covalent interactions were as follows: capillary voltage, 1.5 kV; cone voltage, 80 V; extractor, 5 V; source backing pressure, 6–8 mbar; collision cell pressure, 5 psi. Collision cell energy was between 60 and 100 V. Measurements were taken for intact protein complexes prepared for SEC-MALS analysis and exchanged into 50 mM ammonium acetate (pH 7.8) using Biospin 6 microspin columns (Bio-Rad Laboratories). Data were processed using our in-house software Massign [50] (link). The results are representative of multiple independent experiments.
3
High-mass complex analysis via QToF MS
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All spectra shown here were obtained on a QToF II mass spectrometer (Waters, Manchester, UK) suitable for analysis of high-mass complexes [34 (link)]. For MS analysis, 2.5 μl of the solution containing the complex was introduced using a gold-plated capillary needle (Harvard Apparatus, Holliston, MA, USA) into the instrument. Spectra were acquired in the positive-ion mode, and all instrument conditions were kept constant where concentration effects were being monitored. Typical instrument parameters were as follows: capillary voltage 1.7–1.8 kV, cone voltage 100 V, extractor 5 V, collision voltage 100 V, backing pressure 9.65×10−3 mbar, analyser pressure 4×10−4 mbar and ToF 2.71×10−6 mbar. For MSMS experiments, collision voltages varied up to 200 V. All data shown were acquired and processed with MassLynx V4.1 (Waters, Manchester, UK), with minimal smoothing and no background subtraction. All recorded mass spectra were calibrated externally using caesium iodide prepared in water.
4
Peptide Identification by Tandem MS
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Analyses of samples were carried out by LC-ESI-tandem MS on a Q-TOFII mass spectrometer fitted with a nanoflow ESI (electrospray ionization) source (Waters Ltd, UK). Peptides were separated on a PepMap C18 reverse phase, 75 μmi.d., 15-cm column (LC Packings) and delivered on-line to the MS via a CapLC HPLC system. Sequence interpretation for individual peptides was performed using the PepSeq MASCOT tool of the MassLynx™ 4.0 software package (Waters).
5
Peptide Analysis by LC-MS/MS
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Samples were analysed by LC–ESI-MSMS on a Q-TOFII mass spectrometer fitted with a lockspray nano-ESI (electrospray ionization) source (Waters Ltd.). Peptides were captured and desalted on a C18 cartridge and delivered on-line via a C18 column to the MS via a CapLC HPLC system. The mass spectrometer was operated with a capillary voltage of 3000 V in positive ion mode, using argon as the collision gas. Tandem MS data were acquired using an automated data-dependent switching between MS and MS/MS scanning based upon ion intensity, mass and charge state (data directed analysis (DDA™)). Charge state recognition was used to select doubly, triply and quadruply charged precursor peptide ions for fragmentation. One precursor mass at a time was chosen for tandem MS acquisition. Collision energy was automatically selected based on charge and mass of each precursor and varied from 15 to 55 eV.
6
Preparation and Mass Spectrometry Analysis of cATPase
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Prior to MS analysis cATPase aliquots in n-dodecyl-β-D-maltopyranoside-containing buffer were exchanged against 200 mM ammonium acetate using Micro Bio-spin 6 columns (Bio Rad). Mass spectra were acquired in replicates on a Q-ToF II mass spectrometer (Waters) modified for high masses28 (link) using gold-coated glass capillaries46 (link). Optimized instrument parameters were as follows: capillary voltage 1.7 kV, cone voltage 190 V, extractor 5 V, source backing pressure 7–10 mbar and a collision cell pressure of 10 psi. Collision cell energy was 150–200 V. Spectra were processed using MassLynx v4.1 and spectra were analysed using Massign47 (link). Representative mass spectra are shown for each experiment.
7
High-Mass Transmission Native Mass Spectrometry
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Approximately 10 μM of the purified complex was analyzed in 200 mM ammonium acetate (pH 7.0). Spectra were acquired on a Q-ToF II mass spectrometer (Waters) modified for high mass transmission (Sobott et al., 2002 (link)) and proteins were electrosprayed using gold coated glass capillaries prepared in-house (Hernández and Robinson, 2007 (link)). Typical MS conditions were capillary voltage 1.6–1.7 kV, cone voltage 50–100 V, extractor 5 V, collision voltage 20–80 V, and backing pressure ∼1.0 × 10−2 mbar. Spectra were calibrated externally using Cesium iodide (100 mg/ml). MS spectra were processed and analyzed using Masslynx version 4 (Waters).
8
Mass Spectrometry Protocol for Positive Ion Analysis
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Mass spectra were acquired on a QToF II mass spectrometer (Waters, Milford, MA) using positive ion mode. Instrumental control and data acquisition were conducted by using MassLynx 4.1 software. Voltage of microchannel plate (MCP) detector, sample cone voltage and source temperature were set at 2.1 kV, 30 V and 40 o C, respectively.
High voltage applied to the samples was typically set at 3.5 kV, a voltage obtained after optimization. Mass spectra were acquired in an m/z range of 100 -1000 Da with an interscan time of 0.1 s and a scan time of 1 s. Typically, mass spectra were generated by accumulating data from the first minute. Before DI-MS analysis, sodium iodide was used for m/z calibration of the instrument.
High voltage applied to the samples was typically set at 3.5 kV, a voltage obtained after optimization. Mass spectra were acquired in an m/z range of 100 -1000 Da with an interscan time of 0.1 s and a scan time of 1 s. Typically, mass spectra were generated by accumulating data from the first minute. Before DI-MS analysis, sodium iodide was used for m/z calibration of the instrument.
9
Native mass spectrometry analysis
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Proteins were analyzed on a Q-ToF 2 mass spectrometer (Waters Corporation) modified for transmission of high-molecular-mass species [68] (link) using native MS following protocols previously described [69] . Samples were nanoelectrosprayed at concentrations of 10 μM, in 200 mM ammonium acetate (pH 6.9), after retrieval from size exclusion chromatography (Superdex 200 10/300; GE Healthcare) or, in the case of the dissolved crystals, after desalting using 3-kDa MWCO Vivaspin filters (Sartorius). Data were calibrated externally and analyzed using Masslynx software (Waters Corporation).
10
Mass Spectrometric Analysis of Lipid-Modified Peptide
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