Levels of CL and MLCL were detected by adapting a previously described technique using MALDI-TOF mass spectrometry [27 (link)]. Lipids were extracted from isolated mitochondria by adding 200 μg of mitochondrial protein to a mixture of methanol and chloroform and then vortexed for 45 seconds. 0.88% KCl was then added to the sample mixture, and samples were centrifuged at 800 x g for 10 minutes to separate the aqueous and organic phases. The organic phase was removed and subsequently dried under a steady stream of N2. The dried lipids were subsequently reconstituted in chloroform. 10mg/ml 9-aminoacridine in 60:40 isopropanol: acetonitrile was used as the matrix. The matrix was added to the reconstituted samples in a 1:1 ratio. Matrix by itself was added to an MTP AnchorChip 384 well plate, and the sample matrix mixture was then added onto a layer of dried matrix. After drying, an additional layer of matrix was added to the wells. The sample plate was then analyzed using a Bruker Autoflex Speed MALDITOF system in negative ionization reflectron mode with 5,000 spectra collected per sample.
Autoflex speed malditof system
Manufactured by Bruker
Sourced in Germany
The Autoflex Speed MALDI-TOF system is a mass spectrometry instrument designed for the analysis of a wide range of molecules, including proteins, peptides, and small molecules. It utilizes matrix-assisted laser desorption/ionization (MALDI) technology to generate ions from the sample, which are then detected and analyzed by a time-of-flight (TOF) mass analyzer.
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Lab products found in correlation
4 protocols using autoflex speed malditof system
1
Mitochondrial Lipid Profiling by MALDI-TOF
2
MALDI-TOF Mass Spectrometry of Macromolecules
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Mass spectra were acquired with an Autoflex
Speed MALDI-TOF system (Bruker Daltonics). Samples were spotted using
the dried droplet technique with sinapic acid as a matrix on MTP 384
ground steel target plates (Bruker Daltonics). The mass spectrometer
was operated in a linear positive mode. Mass spectra were acquired
over an m/z range from 30,000 to
210,000, and data were analyzed with FlexAnalysis software provided
with the instrument.
Speed MALDI-TOF system (Bruker Daltonics). Samples were spotted using
the dried droplet technique with sinapic acid as a matrix on MTP 384
ground steel target plates (Bruker Daltonics). The mass spectrometer
was operated in a linear positive mode. Mass spectra were acquired
over an m/z range from 30,000 to
210,000, and data were analyzed with FlexAnalysis software provided
with the instrument.
3
Identification of SpRDH Enzyme by MALDI-TOF/TOF
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Purified SpRDH enzyme was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF/TOF) undertaken at Genomine (Pohang, South Korea). The trypsin-digested protein samples of SpRDH were analyzed using an Autoflex Speed MALDI-TOF system (Bruker) with LIFTTM ion optics. Mass spectrum data were acquired using the instrument’s default calibration without applying internal or external calibration (S1A Fig ). Peaks in the spectrum were selected for inclusion in a Mascot search by using Flexanalysis Biotool Mascot software. The MS/MS ion searches against those in the NCBI protein database were performed by using the Mascot program (S1B Fig ). Similar searches were performed by applying the NCBI BLAST tool. Amino acid sequence alignments of SpRDH and other RDHs were performed by using Clustal Omega [24 (link)].
4
MALDI-TOF Mass Spectrometry Protocol
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The MALDI-TOF mass spectra were measured in linear positive detection mode by an autoflex® speed MALDI-TOF system (Bruker Daltonics GmbH, Bremen, Germany) equipped with a smartbeam™ II (modified Nd:YAG) laser having a wavelength of 355nm. Sinapidic acid and α-cyano-4-hydroxycinnamic acid (HCCA) (both by Sigma Aldrich, Damstadt, Germany) were used as matrix (both 10 g L−1) dissolved in Methanol and mixed at a ratio of 1:1 (v/v). 1 µL of a 0.5 g L−1 methanolic sodium trifluoroacetate solution was added per 100 µL matrix solution. The sample was prepared in deionized water at a concentration of 2 g L−1, mixed with the matrix solution in a ratio of 1:1 (v/v) and spotted on the MALDI plate via the dried droplet method [61 ]. BSA of different batches was used for calibration and for verification. The samples were measured with an acceleration voltage of 19.5 kV, a laser attenuation of 30%, a laser repetition rate of 1 kHz and a detector gain of 70× (3.446 kV). Each mass spectrum was recorded by accumulation of 8000 shots.
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