LC‐MS/MS spectra were acquired on a SYNAPT HDMS mass spectrometer (Waters, Milford, MA) with a nanoAcquity UPLC System interface (Waters). Each sample (8 μl) was loaded onto a Symmetry 300 C18 (180 μm × 20 mm) precolumn (Waters) and washed with 0.1% formic acid for 3 min at a flow rate of 5 μl/min. The precolumn was connected to a BEH130 C18 column (75 μm × 200 mm, 1.7 μm: Waters) equilibrated in 3% acetonitrile and 0.1% formic acid. Peptides were eluted directly onto a NanoEase Emitter (Waters) with a 30 min linear gradient of 3–60% acetonitrile. The capillary voltage was set to 3,000–3,500 V and the data‐dependent MS/MS acquisitions were performed on precursors with charge states of 2, 3 or 4 over a survey m/z range of 350–1,990.
Synapt hdms mass spectrometer
Manufactured by Waters Corporation
Sourced in United Kingdom
The Synapt HDMS mass spectrometer is a high-resolution, ion mobility-enabled mass spectrometer designed for advanced analytical applications. It utilizes hybrid quadrupole time-of-flight (Q-TOF) technology to provide accurate mass measurements and detailed structural information about complex molecules.
Automatically generated - may contain errors
Lab products found in correlation
Masslynx v4, by Waters Corporation (7 mentions)
Nanoacquity uplc system, by Waters Corporation (4 mentions)
Zeba spin desalting column, by Thermo Fisher Scientific (3 mentions)
Ltq orbitrap xl, by Thermo Fisher Scientific (2 mentions)
Masslynx 4, by Waters Corporation (2 mentions)
Avidin, by Merck Group (2 mentions)
Yeast alcohol dehydrogenase, by Merck Group (2 mentions)
β lactoglobulin a, by Merck Group (2 mentions)
Driftscope 2, by Waters Corporation (2 mentions)
Massign, by Waters Corporation (2 mentions)
Concanavilin a, by Merck Group (2 mentions)
Beh130 c18 column, by Waters Corporation (1 mentions)
Driftscope v2, by Waters Corporation (1 mentions)
Masslynx software, by Waters Corporation (1 mentions)
Micro bio spin 6, by Bio-Rad (1 mentions)
Porcine trypsin, by Promega (1 mentions)
Amicon filter, by Merck Group (1 mentions)
Nanouplc system, by Waters Corporation (1 mentions)
Total recovery vial, by Waters Corporation (1 mentions)
Polaron sc7620, by Quorum Technologies (1 mentions)
19 protocols using synapt hdms mass spectrometer
1
Nanoflow LC-MS/MS for Proteomic Analysis
2
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).
3
Mass Spectrometric Analysis of Chaperone-Substrate Complexes
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Samples of SurA and Skp were prepared for MS by buffer exchanging into 200 mM ammonium acetate at pH 10 using Zeba spin desalting columns (Thermo Scientific) immediately prior to analysis. Skp–tOmpA or SurA–tOmpA complexes were prepared by rapid dilution of denatured tOmpA [400 μM in 8 M urea, 50 mM glycine–NaOH (pH 9.5)] to a final concentration of 1 μM into a solution of Skp or SurA [1 μM in 50 mM glycine–NaOH (pH 9.5)]. The samples were subsequently buffer exchanged into 200 mM ammonium acetate at pH 10 using Zeba spin desalting columns.
Spectra were acquired using a Synapt HDMS mass spectrometer (Waters Corporation, UK) by means of nano-ESI using in-house prepared platinum/gold-plated borosilicate capillaries. Typical instrument parameters include the following: capillary voltage, 1.2 kV; cone voltage, 120 V; trap collision voltage, 10 V; transfer collision voltage, 10 V; trap DC bias, 20 V; and backing pressure, 4.5 mBar. Data were processed using MassLynx v4.1 and UniDec [69] (link).
Spectra were acquired using a Synapt HDMS mass spectrometer (Waters Corporation, UK) by means of nano-ESI using in-house prepared platinum/gold-plated borosilicate capillaries. Typical instrument parameters include the following: capillary voltage, 1.2 kV; cone voltage, 120 V; trap collision voltage, 10 V; transfer collision voltage, 10 V; trap DC bias, 20 V; and backing pressure, 4.5 mBar. Data were processed using MassLynx v4.1 and UniDec [69] (link).
4
Analyzing PPARγ-LBD Interactions by MS
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The concentration of PPARγ-LBD was fixed at 1 mM, and an excess amount of TPT or TBT was added. After dilution with buffer (20 mM Tris and 150 mM NaCl pH 7.5) to 10 μM, sample solutions of PPARγ-LBD, PPARγ-LBD/TPT, and PPARγ-LBD/TBT were subjected to a buffer exchange with 150 mM ammonium acetate, pH 7.5, by passing them through mini gel filtration columns (BioRad) prior to analysis. All samples were analyzed by use of nanoflow electrospray using in-house capillaries prepared as described previously25 (link). Samples were loaded into capillaries, and spectra were recorded on a modified Synapt HDMS mass spectrometer (Waters), which provides the molecular mass of a protein complex formed through a non-covalent interaction26 (link). All mass spectra were calibrated against cesium iodide and analyzed by Mass Lynx software (Waters). Typical conditions included 2–3 μL of aqueous protein solution, capillary voltage of 1.1–1.7 kV, cone voltage of 190 V, and trap and transfer collision energy voltages of 30 and 10 V, respectively. The source pressure was maintained at 3 × 10−2 mbar.
5
Ion Mobility Mass Spectrometry of Proteins
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Lyophilised proteins (5 μM) were dissolved in 50 mM ammonium acetate buffer, pH 6.9 and desalted using Micro BioSpin 6 (Bio-Rad, Hemel Hempstead, UK) columns. ESI-IMS–MS experiments were performed using a Synapt HDMS mass spectrometer (Waters Ltd., Wilmslow, Manchester, UK). Sample introduction was achieved by nano-ESI using in-house prepared gold-plated borosilicate capillaries. Typically, a capillary voltage of 1.4 kV was applied, the cone voltage was set to 40 V, and a backing pressure of 4.5 mBar was used. IMS separation was achieved using a wave height of 5 V, and a wave velocity of 250 ms−1. Drift times were calibrated using experimentally determined CCSs of native proteins and applying a procedure described in detail elsewhere [30] , [31] , [32] (link), [33] (link). CCSs were calculated from coordinates deposited in the Protein Data Bank using a scaled projection approximation (PSA) [34] . 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
Monocyte Proteome Profiling of HTLV-1 Infection
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Eight samples from each group (AC, HAM/TSP and uninfected) were pooled before protein digestion. Following four times buffer exchange to 50 mM NH4HCO3 using 3-kDa cutoff Amicon filter (Millipore), the protein extract supernatant samples were quantified. Monocyte lysate supernatant (2 µg/μL) in 50 mM NH4HCO3 was mixed to RapiGEST 0.2% (w/v) (Waters) and incubated at 80 °C for 15 minutes.Samples were reduced by the addition of dithiothreitol (100 mM) at 60 °C for 30 minutes, followed by incubation with iodoacetamide solution (300 mM) at room temperature (RT) for 30 minutes. Digestion was performed with porcine trypsin (Promega, 1:100) at 37 °C overnight. To precipitate and remove the surfactant RapiGEST and stop digestion, 5% TFA (v/v) solution was added. Samples were vortexed, incubated for 90 minutes at 37 °C, and then centrifuged at 6 °C for 30 minutes. Supernatants were dried down in vacuum centrifuge24 (link), and pellets were resuspended in anion exchange column loading buffer (5 mM NH4HCO2, 5% CH3CN (v/v) pH 3.2). Aliquots of each sample were transferred to Waters Total Recovery vials and injected onto a nanoUPLC system coupled to Synapt HDMS mass spectrometer (Waters).
7
Native Mass Spectrometry of Proteins
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All nanoESI-TWIMS-MS protein measurements were carried out using a Synapt HDMS mass spectrometer (Waters Corp., Wilmslow, UK). Samples were introduced to the mass spectrometer using in-house pulled borosilicate capillaries (Sutter Instrument Co., Novato, CA, USA) coated with palladium using a sputter coater (Polaron SC7620; Quorum Technologies Ltd., Kent, UK). All protein samples were analyzed in positive ESI mode. The m/z scale was calibrated using 10 mg/mL aqueous caesium iodide (CsI) clusters across the acquisition range (typically m/z 500–15,000).
Protein samples were dialyzed into 150 mM aqueous ammonium acetate before being infused into the Synapt HDMS instrument. nESI-MS and nESI-TWIMS-MS experiments were conducted under the following settings: capillary voltage, 1.5 kV; sample cone, 30 V; extraction cone, 4 V; source temperature, 60–80 °C; backing pressure, 3.0–5.0 mBar; trap voltage, 10–40 V; trap/transfer gas flow, 1.5 mL/min; IMS nitrogen gas flow, 20 mL/min, IMS wave height (ramped), 5–30 V; and traveling wave speed, 300 ms.
All data were processed and analyzed with the MassLynx v4.1 and Driftscope software, supplied with the mass spectrometer.
Protein samples were dialyzed into 150 mM aqueous ammonium acetate before being infused into the Synapt HDMS instrument. nESI-MS and nESI-TWIMS-MS experiments were conducted under the following settings: capillary voltage, 1.5 kV; sample cone, 30 V; extraction cone, 4 V; source temperature, 60–80 °C; backing pressure, 3.0–5.0 mBar; trap voltage, 10–40 V; trap/transfer gas flow, 1.5 mL/min; IMS nitrogen gas flow, 20 mL/min, IMS wave height (ramped), 5–30 V; and traveling wave speed, 300 ms.
All data were processed and analyzed with the MassLynx v4.1 and Driftscope software, supplied with the mass spectrometer.
8
Protein Identification by LC-MS/MS
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The protein spots most differentially expressed were manually excised and submitted for protein identification by LC-MS/MS, performed exactly as described elsewhere [18 (link)]. Briefly, selected SDS-PAGE spots were in-gel digested using trypsin (Roche, Basel, Switzerland) and the peptide mixture was analysed using a SYNAPT HDMS mass spectrometer (Waters, Milford, MA) interfaced with a nanoAcquity UPLC System (Waters). Obtained spectra were processed using VEMS [19 (link)] and searched for against the NCBI non-redundant (nr) database restricted to Fungi (version 20150309) using the online MASCOT server (Matrix Science Ltd., London; http://www.matrixscience.com ). Even though the L. prolificans genome has been released it is not yet available publicly [20 (link)], so protein identification was performed by comparison with orthologous proteins from other fungi whose genomes were already available in the NCBInr database.
9
Quantitative Proteomic Analysis of Ischemic Brain
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Following 30 min of ischemia, CN‐105 or vehicle was administered at 15 min after reperfusion. At 30 min after reperfusion, mice were killed, brains were removed and dissected in the midsagittal plane. Each hemisphere was flash‐frozen separately in liquid nitrogen and stored at −80°C. The injured right hemispheres were used for analysis. Peptides were prepared from brain samples for liquid chromatography–tandem mass spectrometry (LC‐MS/MS) analysis (Data S2 ). Peptide digests obtained from each of the samples were analyzed in a label‐free quantitative fashion using a nanoAcquity UPLC system coupled to a Synapt HDMS mass spectrometer (Waters Corp, Milford, MA) for unenriched peptide analyses and an LTQ Orbitrap XL (Thermo Fisher Scientific, Waltham, MA) for phosphopeptide analyses. Robust peak detection and label‐free alignment of individual peptides across all sample injections was performed using the commercial package Rosetta Elucidator v3.3 (Rosetta Biosoftware, Inc., Seattle, WA) with PeakTeller algorithm.
10
Native Mass Spectrometry of Protein Complexes
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Spectra were recorded on a Synapt HDMS mass spectrometer (Waters) modified for studying high masses. MtbCoaBC and MsmCoaBC were exchanged into NH4OAc (500 mM, pH 7.0) solution using Micro Bio-Spin 6 chromatography columns (Bio-Rad). A sample volume of 2.5 μL was injected into a borosilicate emitter (Thermo Scientific) for sampling. Instrument conditions were optimised to enhance ion desolvation while minimising dissociation of macromolecular complexes. Typical conditions were capillary voltage 1.8–2.0 kV, sample cone voltage 100 V, extractor cone voltage 1 V, trap collision voltage 60 V, transfer collision voltage 60 V, source temperature 20 °C, backing pressure 5 mbar, trap pressure 3–4 × 10−2 mbar, IMS (N2) pressure 5–6 × 10−1 mbar and TOF pressure 7–8 × 10−7 mbar. Spectra were calibrated externally using caesium iodide. Data acquisition and processing were performed using MassLynx 4.1 (Waters).
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