Inflated lungs were sectioned at 13 μm and thaw-mounted onto indium-tin oxide (ITO) coated glass slides, sections were desiccated for 20 mins., and stored under nitrogen at -80°C prior to use. Slides were prepared for MSI by coating with norharmane (NRM, 7 mg/mL in 2:1, v:v, chloroform:methanol) matrix30 (link),31 (link) using an HTX Technologies TM Sprayer (Charlotte, NC) with the following cycle settings: 30°C, 10 passes, 0.1 mL/min, 1200 mm/min, 2.5 mm spacing, 10 psi N2, CC pattern, 0 s dry time, at 40 mm nozzle height. Coated slides were dried with a gentle nitrogen stream prior to imaging. Three biological replicates (arbitrarily selected) were evaluated by MALDI-TOF at 50 μm pixel size in negative ion mode on a Bruker RapiFlex (Bremen, DE) (Figure S1 ). On-tissue MS/MS fragmentations were performed on a Bruker RapiFlex. The fourth biological replicates from each group were evaluated by DDA-imaging in negative ion mode at 80 μm pixel size as previously described3 (link) on a bespoke imaging instrument composed of a Spectroglyph dual MALDI/ESI ion source (Spectroglyph LLC, WA, USA) coupled to an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific GmbH, Bremen, Germany). Mass resolution for MSI (MS4 ) data was 240,000 @ m/z 400. In addition to MSI data DDA-imaging data also provided high mass accuracy measurements and MS/MS spectra to aid lipid identification.
Rapiflex
Manufactured by Bruker
Sourced in Germany
The Rapiflex is a high-performance liquid chromatography (HPLC) system designed for rapid and efficient separation and analysis of a wide range of chemical compounds. It features advanced technology and automation to deliver reliable, reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
Autoflex speed, by Bruker (3 mentions)
Sprayer, by HTX Technologies (2 mentions)
Fleximaging 5, by Bruker (2 mentions)
Grinded naoh pellets, by Merck Group (2 mentions)
Orbitrap elite, by Thermo Fisher Scientific (1 mentions)
Flexanalysis version 3, by Bruker (1 mentions)
Timstof flex, by Bruker (1 mentions)
Ultraflex 2, by Bruker (1 mentions)
Cryostat, by Thermo Fisher Scientific (1 mentions)
Catalog no 68100, by Thermo Fisher Scientific (1 mentions)
Spectramax m4, by Molecular Devices (1 mentions)
Scils 2019a, by Bruker (1 mentions)
Solarix xr fticr mass spectrometer, by Bruker (1 mentions)
Flexanalysis 3, by Bruker (1 mentions)
Lsm 710, by Zeiss (1 mentions)
Hitrap desalting column, by Cytiva (1 mentions)
Axio observer, by Zeiss (1 mentions)
Direct q 5 water purification system, by Merck Group (1 mentions)
Hitrap, by Cytiva (1 mentions)
Imageprep, by Bruker (1 mentions)
29 protocols using rapiflex
1
Mass Spectrometric Imaging of Lung Tissue
2
Synthesis of NIR-active Polymer Nanoparticles
3
MALDI-TOF Analysis of XyG Oligosaccharides
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A MALDI‐TOF mass spectrometer (Bruker Daltonik, Rapiflex) was used in positive linear mode with an accelerating voltage of 20,000 V to obtain the mass profiles of released XyG oligosaccharides.
4
Oligosaccharide Mass Profiling Protocol
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The oligosaccharides present in the prepared samples were analyzed by Oligosaccharide Mass Profiling as described (Günl et al., 2011 (link)). Briefly, the samples were spotted onto a dried spot of dihydroxy benzoic acid matrix (10 mg·mL−1) and analyzed by MALDI-TOF mass spectrometry (Bruker rapifleX instrument). The machine was set to linear, positive reflectron mode with an accelerating voltage of 20,000 V. The spectra from the samples were analyzed using flexanalysis software 4.0 (Bruker Daltonics).
5
High-Resolution MALDI-MSI Imaging
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High-speed MALDI-MSI
acquisition was performed at 10 μm spatial resolution by using
a MALDI-TOF instrument (rapifleX, Bruker Daltonics). The MALDI source
is equipped with a scanning Smartbeam three-dimensional (3D) laser
featuring a laser beam diameter of 5 μm. Spectra were acquired
by using custom laser settings with a resulting field size of 10 μm.
The measurements were performed with the laser operating at a frequency
of 10 kHz with 20 laser pulses per pixel. Acquisition and subsequent
processing were performed by using the instrument software FlexImaging
5.0 (Bruker Daltonics). Acquisition of high-mass-resolution MSI data
was performed by using an Orbitrap Elite mass spectrometer (Thermo
Fisher Scientific GmbH, Bremen, Germany) coupled to a reduced-pressure
ESI/MALDI ion source (Spectroglyph LLC, Kennewick, WA). Further details
on the ion source can be found in the literature.12 (link) The 349 nm MALDI laser (Spectra Physics, Mountain View,
CA) was operated at a repetition rate of 1000 Hz and pulse energy
of ∼1.5 μJ. The laser was focused to a spot size/step
size of ∼20 × 20 μm2, mass resolution
was chosen to be 120,000 (at m/z 400), and the total scan time was 1.05 s/scan and pixel.
acquisition was performed at 10 μm spatial resolution by using
a MALDI-TOF instrument (rapifleX, Bruker Daltonics). The MALDI source
is equipped with a scanning Smartbeam three-dimensional (3D) laser
featuring a laser beam diameter of 5 μm. Spectra were acquired
by using custom laser settings with a resulting field size of 10 μm.
The measurements were performed with the laser operating at a frequency
of 10 kHz with 20 laser pulses per pixel. Acquisition and subsequent
processing were performed by using the instrument software FlexImaging
5.0 (Bruker Daltonics). Acquisition of high-mass-resolution MSI data
was performed by using an Orbitrap Elite mass spectrometer (Thermo
Fisher Scientific GmbH, Bremen, Germany) coupled to a reduced-pressure
ESI/MALDI ion source (Spectroglyph LLC, Kennewick, WA). Further details
on the ion source can be found in the literature.12 (link) The 349 nm MALDI laser (Spectra Physics, Mountain View,
CA) was operated at a repetition rate of 1000 Hz and pulse energy
of ∼1.5 μJ. The laser was focused to a spot size/step
size of ∼20 × 20 μm2, mass resolution
was chosen to be 120,000 (at m/z 400), and the total scan time was 1.05 s/scan and pixel.
6
Protein Mass Determination by MALDI-TOF
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Molecular masses of the proteins/peptides were determined by MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectroscopy on a Rapiflex (Bruker Daltonics, Bremen, Germany) mass spectrometer in positive linear mode and reflectron mode. Mass spectra were acquired in the mass ranges of m/z 500–3000 and 5000–25,000 for the reflectron and linear modes, respectively. Intensity and number of laser shots were adjusted to obtain an optimal signal-to-noise ratio; most data resulted from 5000 shots at a laser frequency of 5000 (linear)–10,000 (reflectron) Hz. External calibration was performed with Bruker peptide and protein standard kits. The samples were mixed 1:1 (v/v) with a solution of sinapinic acid (SA) at 10 mg/mL or a saturated solution of alpha-cyano-4-hydroxycinnamic acid (HCCA) for linear and reflectron modes, respectively. Peptide sequences were identified by manual analysis of fragment ions and subsequent comparison of predicted (ProtParam tool—Expasy for linear mode and sum of the monoisotopic mass of the amino-acids for the reflectron mode) and experimentally-obtained fragment patterns.
7
MALDI Imaging of Kidney Tissue
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Kidney organ sections were dewaxed, tryptically digested, and coated with matrix (α-cyano-4-hydroxycinnamic acid, Bruker Daltonics) for the MALDI TOF/TOF measurement with a sprayer for MALDI imaging (HTX TM-Sprayer: TMSP-M3, HTX Technologies). MALDI-IMS was performed using the Rapiflex (Bruker Daltonics). Experimental mass spectrometric data were compared with calculated peptide masses for each entry in the sequence database.
Additional experimental procedures and data processing are described in theSupplemental Methods .
Additional experimental procedures and data processing are described in the
8
MALDI-TOF MS Characterization of Polymers
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MALDI-TOF MS were recorded on a Bruker RapiFlex spectrometer operating at the following conditions: nitrogen laser (337 nm), accelerating potential (20 kV) in positive linear ion or reflection mode. In delayed extraction mode, the delay time was ∼300 ns, optimized based on the mass range of the polymer distributions. External calibration was performed using PEG polymer standards. Trans-2-[3-(4-tert-butylphenyl)-2-methyl-2- propenylidene]malononitrile (DCTB) was employed as the matrix. An analyte solution and matrix solution with a concentration of 10 g·L−1 in THF (1:4 v/v analyte-to-matrix solution) were mixed with 1 μL of potassium trifluoroacetate (10 g·L−1). An analyte solution (at 40 g·L−1 in THF) and matrix solution with a concentration of 10 g·L−1 in THF (1:4 v/v analyte-to-matrix solution) were mixed with 1 μL of potassium trifluoroacetate (10 g·L−1) (K+ ionization). A total of 1 μL of the resulting mixture was spotted on the MALDI plate for MS analysis. Data were analyzed and normalized using the FlexAnalysis version 3.0 software (Bruker, Billerica, MA, USA).
9
MALDI-TOF/TOF Venom Peptide Analysis
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Mass spectrometry experiments have been performed on a MALDI-TOF/TOF spectrometer (Rapiflex, Bruker Daltonics), externally calibrated from m/z 757 to 3150 while using PepMix Calibration Mixture II (Bruker). Dried venom fractions were dissolved in 200 µL of formic acid (0.1%), vortexed ten seconds, and then centrifuged. 1 µL of each solution was deposited on a MALDI plate together with 1 µL of saturated CHCA (80% ACN/20% FA 0.1%). The droplets were let to dry at room temperature to co-crystallize the matrix and the peptides. Laser shots at 38% irradiated the crystals, and 2000 spectra were accumulated to obtain a satisfying signal to noise ratio.
10
Permethylation of T. suis N- and O-glycans
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For permethylation, T. suis N- and O-glycans were dried in a glass tube and resuspended in 1 ml of a slurry of grinded NaOH pellets (Sigma-Aldrich) in DMSO followed by adding 500 µl iodomethane. The mixture was incubated for 20 min at room temperature under constant shaking and then the reaction was quenched using 200 µl dH2O. Subsequently, permethylated N- and O-glycans were extracted in chloroform by constant washing with dH2O and then applied to a pre-equilibrated C18 solid-phase extraction column. Salts and contaminants were removed using 15% (v/v) acetonitrile prior to elution of the permethylated glycans using a 50% (v/v) acetonitrile solution. MALDI-TOF MS was performed using an Autoflex Speed or a Rapiflex (Bruker Daltonics) instrument in positive reflection mode with 2,5-dihydroxybenzoic acid as matrix.
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