Dry lipid extracts were washed twice with 1 ml of methanol and then resuspended in 200 µl of a 2:1:0.25 chloroform-methanol-water solvent mixture. Aliquots of 1 µl each of norharmane matrix (10 mg/ml in 2:1 chloroform-methanol) and then analyte were spotted directly onto stainless steel target plates in duplicate. Mass spectra were recorded in negative-ion mode with a Bruker Microflex LRF MALDI-TOF mass spectrometer (Bruker Daltonics, Billerica, MA) operated in reflectron mode. The instrument was equipped with a 337-nm nitrogen laser, and analyses were performed at 39.5% global intensity. Typically, 900 laser shots were summed to acquire each spectrum and at least three mass spectra were acquired per spot. Electrospray tuning mix (Agilent, Palo Alto, CA) was used for mass calibration. Data were acquired and processed with flexControl and flexAnalysis version 3.4 (Bruker Daltonics Inc.). Mass spectra were smoothed by a Savitzky-Golay filter, and the baseline was corrected with TopHat. Molecular structures and m/z values of major ions identified in E. coli and K. pneumoniae lipid A mass spectra are presented in Fig. S1 . K. pneumoniae lipid A mass spectra not presented here are in Fig. S2 .
Microflex lrf maldi tof mass spectrometer
Manufactured by Bruker
Sourced in Germany, United States
The Microflex LRF MALDI-TOF mass spectrometer is a laboratory instrument designed for the analysis of biomolecules. It utilizes matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) technology to measure the masses of a wide range of analytes, including proteins, peptides, oligonucleotides, and other macromolecules.
Automatically generated - may contain errors
Lab products found in correlation
α cyano 4 hydroxycinnamic acid chca matrix, by Merck Group (4 mentions)
Msp 96 polished steel target plate, by Bruker (3 mentions)
Anti flag m2 magnetic bead, by Merck Group (3 mentions)
Sheep anti mouse igg magnetic dynabeads, by Thermo Fisher Scientific (3 mentions)
Flexcontrol software, by Bruker (3 mentions)
Flexanalysis software, by Bruker (2 mentions)
Electrospray tuning mix, by Agilent Technologies (1 mentions)
Flexanalysis version 3, by Bruker (1 mentions)
Flexcontrol, by Bruker (1 mentions)
Proteomics grade, by Roche (1 mentions)
Microtube, by Eppendorf (1 mentions)
Msp 96 target plate, by Bruker (1 mentions)
Dynabead, by Thermo Fisher Scientific (1 mentions)
Cary 50 uv vis spectrophotometer, by Agilent Technologies (1 mentions)
Tlc silica gel 60 f254, by Merck Group (1 mentions)
One shot bl21 star, by Thermo Fisher Scientific (1 mentions)
In gel trypsin, by Merck Group (1 mentions)
16 protocols using microflex lrf maldi tof mass spectrometer
1
MALDI-TOF Mass Spectrometry Analysis of Bacterial Lipid A
2
MALDI-TOF Analysis of Amyloid-Beta Peptides
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For matrix-assisted laser
desorption ionization time-of-flight (MALDI-TOF) mass spectrometry
analysis of Aβ peptides, a GSM
or inverse GSM compound was added to the CHO cells expressing one
of the APP mutant forms described. Secreted Aβ peptides from
conditioned media were analyzed as previously described52 (link),53 (link) with the following modifications. Briefly, the mutant peptides were
immunoprecipitated using Ab5 recognizing the Aβ1–16 epitope54 (link) and sheep anti-mouse IgG magnetic Dynabeads
(catalog no. 11201D, Life Technologies) and
eluted with 0.1% trifluoroacetic acid in water. Eluted samples were
mixed in a 2:1 ratio with saturated α-cyano-4-hydroxycinnamic
acid (CHCA) matrix (Sigma) in an acetonitrile/methanol mixture (60:40)
and loaded onto a CHCA-pretreated MSP 96 target plate-polished steel
(part no. 224989, Bruker, Billerica, MA). Samples were analyzed using
a Bruker Microflex LRF-MALDI-TOF mass spectrometer.
desorption ionization time-of-flight (MALDI-TOF) mass spectrometry
analysis of Aβ peptides, a GSM
or inverse GSM compound was added to the CHO cells expressing one
of the APP mutant forms described. Secreted Aβ peptides from
conditioned media were analyzed as previously described52 (link),53 (link) with the following modifications. Briefly, the mutant peptides were
immunoprecipitated using Ab5 recognizing the Aβ1–16 epitope54 (link) and sheep anti-mouse IgG magnetic Dynabeads
(catalog no. 11201D, Life Technologies) and
eluted with 0.1% trifluoroacetic acid in water. Eluted samples were
mixed in a 2:1 ratio with saturated α-cyano-4-hydroxycinnamic
acid (CHCA) matrix (Sigma) in an acetonitrile/methanol mixture (60:40)
and loaded onto a CHCA-pretreated MSP 96 target plate-polished steel
(part no. 224989, Bruker, Billerica, MA). Samples were analyzed using
a Bruker Microflex LRF-MALDI-TOF mass spectrometer.
3
Immunoprecipitation and Mass Spectrometry Analysis of Amyloid-Beta Peptides
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Immunoprecipitation and mass spectrometry (IP-MS) of Aβ and Aβ-like peptides in cell-free assay or conditioned media were performed as previously described [7 (link), 15 (link), 24 (link), 28 (link)–30 (link)]. Briefly, the peptides were immunoprecipitated using anti Aβ Ab5 antibody bound to sheep anti-mouse IgG magnetic Dynabeads (Life Technologies) and eluted with 0.1% trifluoroacetic acid (TFA) in water. COOH-terminal fragments (CTFs) were immunoprecipitated with anti-FLAG M2 magnetic beads (Sigma). Eluted samples were mixed 2:1 with saturated α-cyano-4-hydroxycinnamic acid (CHCA) matrix (Sigma) in a mixture of acetonitrile (60%) and methanol (40%) and loaded onto a CHCA pretreated MSP 96 grounded steel target (Bruker, Billerica, MA). Spectra were collected and processed with a Bruker Microflex LRF-MALDI-TOF mass spectrometer using the flexControl and flexAnalysis software.
4
Synthesis and Characterization of Fluorescent Peptides
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All fluorescent peptides and scrambled (SCR) peptide were purchased from GL Biochem (Shanghai, China) and were synthesized using standard Fmoc solid-phase synthesis to give peptides with a C-terminal amide. The scrambled peptide sequence EFTEVYEFDFKYDAPD is based on BK1.1. They were all deemed to be >90% pure by HPLC analysis and verified by LC–MS. The peptides were dissolved in DMSO, and the concentration was determined using NMR with TSP as an internal standard (57 (link)). All peptides were analyzed using a Bruker Microflex LRF MALDI-TOF mass spectrometer.
5
MALDI-TOF Mass Spectrometry-Based Protein Identification
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Protein spots were excised, transferred in 1.5 mL Eppendorf microtubes, and subjected to trypsin digestion (Proteomics grade, ROCHE, Milan, Italy). Afterward, 2 µL of sample was mixed with an equal volume of α-cyano-4-hydroxycinnamic acid-based matrix (C8982 Sigma Life Science, St. Louis, MO, USA) saturated in 50% acetonitrile. Finally, 0.8 µL aliquots of this mixture were released on the metal target plate of a Microflex® LRF MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) in reflector mode. Protein identification was carried out by searching the NCBI and/or Mascot protein database. The parameters used for the search of a protein database with PMF (peptide mass fingerprinting) were as follows: enzyme, trypsin; species, Homo sapiens; pI range, ±1; Mr range, ±20%; missed cleavage sites allowed, 1; minimum peptide hits, 4; mass tolerance, ±100 ppm; modifications, cysteine treated with iodoacetamide to carboxamidomethyl and methionine in the oxidized form.
6
Immunoprecipitation and Mass Spectrometry of Amyloid-Beta Peptides
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Immunoprecipitation and mass spectrometry of Aβ and Aβ‐like peptides in cell‐free assay or conditioned media were performed as previously described (Ran et al, 2014 ). Briefly, the peptides were immunoprecipitated using anti‐Aβ Ab5 antibody bound to sheep anti‐mouse IgG magnetic Dynabeads (Life Technologies) and eluted with 0.1% trifluoroacetic acid (TFA) in water. COOH‐terminal fragments (CTFs) were immunoprecipitated with anti‐FLAG M2 magnetic beads (Sigma). Eluted samples were mixed 2:1 with saturated α‐cyano‐4‐hydroxycinnamic acid (CHCA) matrix (Sigma) in a mixture of acetonitrile (60%) and methanol (40%) and loaded onto a CHCA‐pretreated MSP 96 target plate (Bruker, Billerica, MA). Samples were analyzed using a Bruker Microflex LRF‐MALDI‐TOF mass spectrometer.
7
Quantifying Secreted Amyloid-Beta Profiles
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Conditioned media from WT-APP and 3xK-APP cells were harvested and used to analyze secreted Aβ profiles using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry analysis. Secreted Aβ peptides from conditioned media were analyzed as previously described with the following modifications [37] (link), [38] (link), [39] (link). Briefly, the Aβ peptides were immunoprecipitated using Ab5 recognizing the Aβ1-16 epitope [40] (link) and sheep anti-mouse IgG magnetic Dynabeads (Life Technologies, catalog no. 11201D) and eluted with 0.1% trifluoroacetic acid (TFA) in water. Eluted samples were mixed 2∶1 with saturated α-cyano-4-hydroxycinnamic acid (CHCA) matrix (Sigma) in mixture of acetonitrile (60%) and methanol (40%) and loaded onto a CHCA pretreated MSP 96 target plate-polished steel (Bruker, Billerica, MA, USA - Part No. 224989). Samples were analyzed using a Bruker Microflex LRF-MALDI-TOF mass spectrometer. The theoretical average molecular weights of Aβ and AICD fragments were calculated using ExPASy Compute pI/Mw tool.
8
Quantitative Analysis of Aβ and AICD
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Conditioned media from the CHO-2B7 cells and the samples prepared from in vitro γ-secretase studies were used to analyze Aβ and AICD profiles using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry analysis. The secreted Aβ peptides were analyzed as previously described with the following modifications [2 (link), 75 (link), 76 (link)]. Briefly, the Aβ peptides were immunoprecipitated using Ab5 recognizing the Aβ1-16 epitope [77 (link)] and sheep anti-mouse IgG magnetic Dynabeads (Life Technologies, catalog no. 11201D) and the AICD fragments were captured using anti-Flag M2 magnetic beads (Sigma). The samples were washed and eluted with 10 μM solution of 0.1 % trifluoroacetic acid (TFA) in water. Eluted samples were mixed 2:1 with saturated α-cyano-4-hydroxycinnamic acid (CHCA) matrix (Sigma) in acetonitrile: methanol (60:40 %) and loaded onto a CHCA pretreated MSP 96 target plate-polished steel (Bruker, Billerica, MA, USA - Part No.224989). Samples were analyzed using a Bruker Microflex LRF-MALDI-TOF mass spectrometer.
9
MALDI-TOF Mass Spectrometry Protocol
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MALDI-TOF analysis was performed on a Microflex LRF MALDI-TOF mass spectrometer (Bruker, Bremen, Germany) equipped with a pulsed 337-nm nitrogen laser using SA (20 mg/mL in 50% acetonitrile [ACN]/50% water/0.1% TFA v/v/v), HCCA (10 mg/mL in 30% ACN/70% water/0.1% TFA v/v/v), and DHB (10 mg/mL in 30% ACN/70% water/0.1% TFA v/v/v) matrix. One microliter of sample was deposited on a Microflex Anchorchip plate (Bruker, Bremen, Germany), dried under ambient condition, and then overlayered with 1 μL of matrix. When the matrix was dried, MALDI-TOF MS analysis was performed under linear positive mode. The optimized parameters were as follows: 70% laser intensity, laser attenuator with 35% offset and 40% range, accumulation of 500 laser shots, 10.3× detector gain, and 150 ns delayed extraction time. External mass calibration was performed using cytochrome c (2 mg/mL) and myoglobin (2 mg/mL).
10
Purification and Characterization of 1-Phenyloctane
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All commercially obtained chemicals were used without further purification unless stated otherwise. The purity of 1-phenyloctane, the solvent in which the additional currents were observed, was confirmed by 1H NMR spectroscopy; moreover, the currents were observed in multiple batches of this solvent, of two different suppliers (ACROS Organics or Sigma-Aldrich), independent whether it was vacuum-distilled prior to use or used as received. For TLC analysis, TLC silicagel 60 F254 (Merck, Burlington, MA, USA) and for column chromatography silica gel 0.035–0.070 mm, (Acros, Branchburg, N.J., USA) was used. MALDI-TOF mass spectra were measured in reflective mode with dithranol as a matrix on a Bruker Microflex LRF MALDI-TOF mass spectrometer (Bruker, Billerica, MA, USA). UV–vis spectra were recorded on a Varian Cary 50 UV–vis spectrophotometer.
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