Data alignment, peak detection, and normalization were performed in Progenesis QI (Nonlinear Dynamics). The chromatographic region from 0.4 to 9.0 min was considered for peak detection. The reference sample for alignment was selected by Progenesis QI from the pooled quality control samples, and data were normalized to the bacterial pellet weights. The resulting features were filtered by analysis of variance (P ≤ 0.05). Student's t tests for two samples were performed by using a two-tailed distribution and equal variance. When possible, identifications were made against the METLIN database within a mass accuracy of 15 ppm (59 (link), 60 (link)). For lipid species not found in common lipidomics databases, in-house databases were generated with ChemDraw (DGDGs and lysyl-PGs; PerkinElmer) or LipidPioneer (MGDGs and CLs) (61 (link)). CCS values for lipid standard extracts were obtained by using the DriftScope v2.8 (Waters Corp.) chromatographic peak detection algorithm with lock mass correction.
Driftscope v2
Manufactured by Waters Corporation
Sourced in United Kingdom, United States
The DriftScope v2.1 is a lab equipment product from Waters Corporation. It is a device designed to measure and analyze the drift characteristics of various analytes in a sample. The core function of the DriftScope v2.1 is to provide precise and reliable data on the drift behavior of the analyzed substances.
Automatically generated - may contain errors
Lab products found in correlation
Masslynx v4, by Waters Corporation (9 mentions)
Masslynx 4, by Waters Corporation (2 mentions)
Chemdraw, by PerkinElmer (1 mentions)
Masshunter mass profiler 10, by Agilent Technologies (1 mentions)
Im ms browser 10, by Agilent Technologies (1 mentions)
Synapt hdms mass spectrometer, by Waters Corporation (1 mentions)
Masslynx 1, by Waters Corporation (1 mentions)
Synapt g2 si hdms instrument, by Waters Corporation (1 mentions)
Synapt hdms qtof mass spectrometer, by Waters Corporation (1 mentions)
Synapt g2 hdms, by Waters Corporation (1 mentions)
15 protocols using driftscope v2
1
Comprehensive Metabolomics Data Analysis Pipeline
2
Lipid profiling using LC-IM-MS
3
Single-field Calibration for DTIM-MS and TWIM-MS
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Single-field calibration for DTIM-MS was applied using Agilent IM-MS Browser 10.0. Single-field calibrated data was demultiplexed and pre-processed using PNNL Preprocessor 3.0 (2021.04.21) [27 ], and Agilent MassHunter Mass Profiler 10.0 was used for peak picking and alignment of triplicate measurements [14 ].
For TWIM-MS, DriftScope V.2.8 included in MassLynx 4.2 software (Waters) was used to determine the TWCCSN2 calibration functions, which were saved into corresponding measurement data files. Individual data files were investigated using DriftScope and MS-DIAL 4.60 [28 (link), 29 ] was used to batch-process TWIM-MS data. To this end, datafiles in raw format were converted to.ibf files using the built-in converter. Settings used for peak picking and alignment are provided in the Electronic Supplementary information. TWCCSN2 values were calculated from arrival times using the Enhanced Duty Cycle (EDC) coefficient to correct arrival times [30 (link)], and a detailed description of the applied calibration approach is presented in the Electronic Supplementary Information.
Finally, Microsoft® Office® (Excel® and PowerPoint®) and R (4.1.2) [31 ] together with RStudio (2021.9.1.372) [32 ] were used for data analysis, visualization and creation of final figures.
For TWIM-MS, DriftScope V.2.8 included in MassLynx 4.2 software (Waters) was used to determine the TWCCSN2 calibration functions, which were saved into corresponding measurement data files. Individual data files were investigated using DriftScope and MS-DIAL 4.60 [28 (link), 29 ] was used to batch-process TWIM-MS data. To this end, datafiles in raw format were converted to.ibf files using the built-in converter. Settings used for peak picking and alignment are provided in the Electronic Supplementary information. TWCCSN2 values were calculated from arrival times using the Enhanced Duty Cycle (EDC) coefficient to correct arrival times [30 (link)], and a detailed description of the applied calibration approach is presented in the Electronic Supplementary Information.
Finally, Microsoft® Office® (Excel® and PowerPoint®) and R (4.1.2) [31 ] together with RStudio (2021.9.1.372) [32 ] were used for data analysis, visualization and creation of final figures.
4
Glycan Fragment Drift Time Analysis
5
Mass Spectrometry Analysis of Outer Membrane Proteins
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All samples were buffer exchanged into 100 mM ammonium hydrogen carbonate (NH4HCO3), pH 7.8 immediately prior to ESI-IMS–MS analysis. For the DDM samples, the buffer also contained 0.02% DDM. ESI-IMS–MS experiments were conducted on a Synapt HDMS mass spectrometer (Waters Ltd., Wilmslow, Manchester, UK). OMPs were introduced into the gas-phase using a nano-ESI source and in-house manufactured gold-plated borosilicate capillaries. Capillary voltage, cone voltage, bias voltage and backing pressure were set at 1.7 kV, 70 V, 20 V, and 6 mbar, respectively. Collision voltages in the Trap (PagP and OmpT = 100–150 V; tOmpA = 50–100 V) and Transfer (50–100 V) regions prior to and immediately following the drift cell, respectively, were varied to optimise liberation of each OMP with minimal impact on its structure. The argon gas pressure in the Trap was 3.65 × 10−2 mbar. The IMS drift times allowed calculation of collision cross sections (CCSs) by calibration against drift times of ions of known CCS [48] , [49] (link). Theoretical CCS values of OMPs were predicted using a scaled Projected Superposition Algorithm (PSA) from the 3D structure coordinates in the Protein Data Bank [50] . Aqueous CsI was used for m/z calibration. Data were processed using MassLynx v4.1 and Driftscope v2.5 software (Waters Ltd., Wilmslow, Manchester, UK).
6
Neuropeptide and Protein Identification Using LESA-MS
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The LESA
extraction was controlled by a beta
version of the LESA Plus software (Advion, UK), and MassLynx 1.4 (Waters,
U.S.A.) was used for controlling the online washing step and the μLC
separation. Data were processed and visualized using Mass Lynx 1.4
(Waters, U.S.A.) and DriftScope v2.5 (Waters, U.S.A.). The identification
of the neuropeptides and proteins was performed using Progenesis QI
for proteomics v2.0.5556.29015 (Non Linear Dynamics, U.S.A.). For
this identification, a species-specific FASTA file was created, and
a nonspecific digest reagent was selected. The amount of missed cleavages
was set at three, and the post-translational modifications (PTMs)
allowed in this MSE search were N-acetylation, M-oxidation,
and C-carbamidomethylation.
extraction was controlled by a beta
version of the LESA Plus software (Advion, UK), and MassLynx 1.4 (Waters,
U.S.A.) was used for controlling the online washing step and the μLC
separation. Data were processed and visualized using Mass Lynx 1.4
(Waters, U.S.A.) and DriftScope v2.5 (Waters, U.S.A.). The identification
of the neuropeptides and proteins was performed using Progenesis QI
for proteomics v2.0.5556.29015 (Non Linear Dynamics, U.S.A.). For
this identification, a species-specific FASTA file was created, and
a nonspecific digest reagent was selected. The amount of missed cleavages
was set at three, and the post-translational modifications (PTMs)
allowed in this MSE search were N-acetylation, M-oxidation,
and C-carbamidomethylation.
7
Phosphorylated Peptide Characterization by IM-MS
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Phosphorylated peptide solutions were diluted in CH3CN:H2O 1:1 (v/v) to 1 pmol/μL and directly infused into a Synapt G2-Si HDMS instrument (Waters) through a nanospray source, at a flow rate of 0.5 μL/min. The capillary, cone voltage and source temperature were typically set to 2.7 kV, 40 V and 80 °C respectively. The IM traveling wave speed was set to 630 m/s and the wave height set at its maximum 40 V. The nitrogen drift gas flow was set at 90 mL/min for all experiments. Phosphopeptide CID was induced in the transfer cell using argon collision gas at collision energy (CE) of 30 V. For analysis of the dimer, the capillary voltage was set at 1.95 kV, while the wave speed was reduced to 311 m/s. Mass spectra were processed using MassLynx V4.1 and mobilograms using DriftScope v2.1 (both Waters, UK).
8
Ion Mobility Data Analysis Protocol
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Data were analyzed using Masslynx v4.1
(Waters Corporation) and Driftscope v2.1 (Waters Corporation). To
compare the data, the gross arrival time had the injection time (10
ms) subtracted to give drift-time. In the case of trapping experiments,
the time spent in the array (0–360 ms) was also subtracted.
For spinning, the extended cyclic motion (0–360 ms) and the
reinjection time (45 ms) were subtracted. CIU fingerprint plots were
created using Benthesikyme.52 (link) Population
fitting was performed using in-house software written in Python 2.7,
peak maxima were identified using the second derivative and manually
adjusted to ensure the same conformational populations were tracked
across the different experiments. During trapping and spinning experiments
the data were aligned according to the most intense peak to allow
the same centroid value for each Gaussian population for tracking.
In the case where a second conformational population became the maximum
peak, the two most intense peaks were used for alignment. Each conformational
family was approximated by a Gaussian distribution. The sum of all
distributions was optimized to produce the best fit to the experimental
data or trace. No restraint to the full width half-maximum value of
each Gaussian was imposed like in previous work.52 (link) The code is freely available athttps://github.com/ThalassinosLab/CIVU .
(Waters Corporation) and Driftscope v2.1 (Waters Corporation). To
compare the data, the gross arrival time had the injection time (10
ms) subtracted to give drift-time. In the case of trapping experiments,
the time spent in the array (0–360 ms) was also subtracted.
For spinning, the extended cyclic motion (0–360 ms) and the
reinjection time (45 ms) were subtracted. CIU fingerprint plots were
created using Benthesikyme.52 (link) Population
fitting was performed using in-house software written in Python 2.7,
peak maxima were identified using the second derivative and manually
adjusted to ensure the same conformational populations were tracked
across the different experiments. During trapping and spinning experiments
the data were aligned according to the most intense peak to allow
the same centroid value for each Gaussian population for tracking.
In the case where a second conformational population became the maximum
peak, the two most intense peaks were used for alignment. Each conformational
family was approximated by a Gaussian distribution. The sum of all
distributions was optimized to produce the best fit to the experimental
data or trace. No restraint to the full width half-maximum value of
each Gaussian was imposed like in previous work.52 (link) The code is freely available at
9
Conformational Analysis of α-LA by IM-MS
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The conformation of α-LA forms was investigated by IM-MS performed on a Synapt HDMS Q-TOF mass spectrometer (Waters Corporation, Manchester, U.K.) using a nano-electrospray ionisation source. Samples were prepared in 100 mM ammonium acetate (pH 7.0) to a final concentration of 25 μM. DTT α-LA was formed by the addition of DTT (2 mM) and RCM α-LA was incubated in the presence of β-CN (1 : 0.5 molar ratio; RCM α-LA : β-CN). Samples were loaded into platinum-coated borosilicate glass capillaries prepared in-house. Gentle source conditions were applied to minimise gas-phase structural changes prior to detection, with instrument parameters as follows: capillary voltage, 1.60 kV; sampling cone, 30 V; extraction cone, 1.5 V; trap/transfer collision energy, 10/15 V; trap gas, 5.5 l/h; backing gas, ∼4.5 mbar. The parameters for IM were as follows: IM cell wave height, 8 V; IM cell wave velocity, 350 m/s; transfer t-wave height, 8 V; transfer t-wave velocity, 250 m/s. Mass spectra and arrival time distributions (ATDs) were viewed using MassLynx (v4.1) and DriftScope (v2.1), respectively (Waters Corporation, Manchester, U.K.).
10
Ion Mobility Mass Spectrometry of Amyloid-β
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Experiments were performed using a hybrid Q-TOF mass spectrometer with IM capabilities—Synapt G2 HDMS (Waters Corp. Wilmslow, UK). Samples of Aβ 1–40 WT, SCR, NSCR at 100 μM concentration in 10 mM CH3COONH4, pH 7.4 (when necessary, pH was adjusted with ammonia) were infused directly (at 7 μL/min) to the ion source of a mass spectrometer, with a glass Hamilton syringe, through a stainless steel capillary. The mass signals were measured in the range 400–4000 m/z at the rate of 1 scan per second. The spectra analyzed were the average of 200 scans. The instrument was tuned to obtain the best possible signal and HDX efficiency, using the electrospray positive ion mode with a capillary voltage of 2.8 kV and a sample cone voltage of 37 V. The source and desolvation temperatures were maintained at 85 and 180°C, respectively. The mobility T-wave cell was operated at a pressure of 2.5 mbar of nitrogen, with a wave velocity of 300 m/s and amplitude (T-wave height) of 40 V. Data acquisition and processing were carried out with MassLynx V4.1 (Waters) and DriftScope V2.1 (Waters) software supplied with the instrument. Each analysis of drift time profiles in Aβ WT, SCR, NSCR was carried out under the same experimental conditions. All data were repeated for batch-to-batch replicates (n = 3 or more) to confirm the reproducibility of the results.
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