Huh7 cells were infected with DENV at a multiplicity of infection (MOI) of ten. Two hours before cell harvest, cell media was spiked with 440μCi of [35S]-methionine and [35S]-cysteine (Easy tag EXPRESS35S; Perkin Elmer; Waltham, MA). Cells were lysed in lysis buffer (0.1% Triton; 150mM NaCl; 60mM MgCl2; 25mM Tris-HCl, pH 7.5; 1mM DTT, 1μM Microcystin (Cayman Chemical; Ann Arbor, MI), and 1× protease inhibitor tablet (Roche; Mannheim, Germany)), clarified by centrifugation, and incubated with a γ-phosphate ATP-affinity resin. The resin was washed 3× with low salt wash buffer (50mM Tris, pH 7.5; 150mM NaCl; and 60mM Mg Cl2) and bound proteins were removed from the resin by boiling in 5× SDS running buffer. Samples were subjected to SDS-PAGE and analyzed by silver stain. The gel was then dried and analyzed by autoradiogram for seven days. Indicated bands were excised and identified by matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS) on an ABSCIEX (Framingham, MA) TOF/TOF 5800 mass spectrometer.
Tof tof 5800 mass spectrometer
Manufactured by AB Sciex
Sourced in United States
The TOF/TOF 5800 mass spectrometer is a high-performance instrument designed for advanced proteomics and peptide analysis. It features a combination of time-of-flight (TOF) and tandem TOF (TOF/TOF) capabilities, providing accurate mass measurements and effective fragmentation of peptide ions for in-depth structural analysis.
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Lab products found in correlation
Protease inhibitor tablet, by Roche (1 mentions)
Easytag express 35s, by PerkinElmer (1 mentions)
M2 beads, by Merck Group (1 mentions)
Kromasil 100 3.5 c18 column, by Merck Group (1 mentions)
2 5 dihydroxybenzoic acid, by Merck Group (1 mentions)
Xylotetraose, by Megazyme (1 mentions)
Trypsin profile igd kit, by Merck Group (1 mentions)
Liquid dextrose diet, by Bio-Serv (1 mentions)
Proteinpilot software 4, by AB Sciex (1 mentions)
15 protocols using tof tof 5800 mass spectrometer
1
DENV-Induced Host Protein Interactome
2
Mass Spectrometric Analysis of Amyloid-Beta Peptides
3
Hydrazide-PEG Conjugation of m3-HwTx-IV
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Ligation of the hydrazide-PEG linker
with the aldehyde moiety of m3-HwTx-IV was performed with
100 mM sodium citrate (pH 4.5) using a peptide concentration of 1
mg/mL (130 μM) and a 2-fold molar excess of hydrazide-PEG linker
(260 μM). The reaction was allowed to proceed at –20
°C in the dark for 24 h. The product was confirmed by matrix-assisted
laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF
MS) on a TOF/TOF 5800 mass spectrometer (AB SCIEX, Framingham, MA)
and analytical RP-HPLC. For MALDI-TOF MS, the peptide sample was diluted
to approximately 1 pmol/μL in a MALDI solvent [0.05% TFA in
50% ACN (v/v) in H2O], mixed [1/1 (v/v)] with an α-cyano-4-hydroxycinnamic
acid matrix [5 mg/mL in 50% ACN (v/v) in H2O], and spotted
onto an Opti-TOF 384-well (123 mm × 81 mm) MALDI plate (AB Sciex).
For RP-HPLC, an analytical Kromasil 100-3.5-C18 column
(2.1 mm × 150 mm, 3.5 μm; Merck) with a flow rate of 0.2
mL/min was used with a linear gradient from 0% to 45% solvent B over
45 min, where solvent A was 0.05% TFA in H2O and solvent
B was 0.043% TFA in 90/10% (v/v) ACN/H2O. The UV absorbance
was monitored at 214 and 280 nm.
with the aldehyde moiety of m3-HwTx-IV was performed with
100 mM sodium citrate (pH 4.5) using a peptide concentration of 1
mg/mL (130 μM) and a 2-fold molar excess of hydrazide-PEG linker
(260 μM). The reaction was allowed to proceed at –20
°C in the dark for 24 h. The product was confirmed by matrix-assisted
laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF
MS) on a TOF/TOF 5800 mass spectrometer (AB SCIEX, Framingham, MA)
and analytical RP-HPLC. For MALDI-TOF MS, the peptide sample was diluted
to approximately 1 pmol/μL in a MALDI solvent [0.05% TFA in
50% ACN (v/v) in H2O], mixed [1/1 (v/v)] with an α-cyano-4-hydroxycinnamic
acid matrix [5 mg/mL in 50% ACN (v/v) in H2O], and spotted
onto an Opti-TOF 384-well (123 mm × 81 mm) MALDI plate (AB Sciex).
For RP-HPLC, an analytical Kromasil 100-3.5-C18 column
(2.1 mm × 150 mm, 3.5 μm; Merck) with a flow rate of 0.2
mL/min was used with a linear gradient from 0% to 45% solvent B over
45 min, where solvent A was 0.05% TFA in H2O and solvent
B was 0.043% TFA in 90/10% (v/v) ACN/H2O. The UV absorbance
was monitored at 214 and 280 nm.
4
MALDI-TOF Mass Spectrometry Protocol
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MALDI-TOF analyses were performed on an AB Sciex TOF/TOF 5800 mass spectrometer using the reflectron mode29 (link),63 (link). Ionization was achieved by irradiation with an Nd:YAG laser (349 nm) operating with a pulse rate of 400 Hz. The laser intensity was set at 3500 and continuous stage motion was used with a velocity of 600 µm/s. Data was acquired in the negative ion mode. Typically, spectra from 2500 to 5000 laser shots were summed to obtain the final spectrum. 2,5-dihydroxybenzoic acid (Sigma) was used as the matrix at a concentration of 10 mg/mL in chloroform/methanol 9:1 or 1:1.
5
Proteomic Identification of Proteins
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Submitted protein gel bands were excised and in-gel digested with trypsin (0.6 μg), and the tryptic peptides were subjected to matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS) on an AB Sciex TOF/TOF 5800 mass spectrometer. Positive-mode time of flight was used to identify peptides, and individual peptides were sequenced by tandem mass spectrometry (MS/MS). All sequence and peptide fingerprint data were searched using the UniProt database and Mascot search engine.
6
MALDI-TOF MS Analysis of PEG Samples
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Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was carried out using an ABSCIEX TOF/TOF 5800 mass spectrometer at the Open Facility, Hokkaido University. A PEG sample (1.5 mg/mL, 10 µL) was mixed with a matrix (anthralin, 40 mg/mL, 25 µL) and ionization regent (silver trifluroroacetate, 40 mg/mL, 10 µL), and 0.4 µL of the mixture was drop cast on an Opti-TOF 384-Well Insert (123 × 81 mm) plate.
7
Glycan Profiling of Snail Samples
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Dried samples (n = 3, 3 biological replicates) were then dissolved in dimethylsulfoxide (DMSO) and methylated using NaOH/DMSO base and methyl iodide. The reaction was quenched using LC-MS grade water, and Per-O-methylated glycans were extracted with methylene chloride and dried under N2. Permethylated glycans were dissolved in methanol. Glycans were then mixed 1:1 with α-dihyroxybenzoic acid (DHB) matrix. MALDI-TOF-MS analysis was done in positive ion mode using an AB SCIEX TOF/TOF 5800 mass spectrometer on three samples. Glycans were identified according to previously established snail glycan assignments. Fetuin was processed identically as a glycoprotein control. 0.1 μg of xylotetraose (Megazyme) was added to each sample as a glycan control. Glycomics data were submitted to GlycoPost database under the accession number GPST000297.
8
Protein Identification by Mass Spectrometry
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A purified protein sample of IaaH was run on 12% SDS-PAGE under reducing conditions. The major band was excised and used for in-gel digestion of protein by trypsin. The Trypsin Profile IGD Kit (Sigma-Aldrich, Co., St. Louis, USA) was used for the preparation of MS/MS sample according to manufacturer’s protocol. These desalted tryptic digested peptides were then mixed with matrix solution consisting of 5 mg/ml of 4-cyanohydroxycinnamic acid (CHCA) in 50% acetonitrile and 0.1% trifluoroacetic acid. MS/MS spectra were acquired using a TOF/TOF™ 5800 mass spectrometer (AB Sciex LLC, CA, and USA). Mass spectral data was analysed using the MS/MS ion search of MASCOT program (http://www.matrixscience.com ).
9
CYP2E1 Knockout Mice Proteomic Study
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SV129 background CYP2E1 knockout mice were kindly provided by Dr. Frank J Gonzalez (Laboratory of Metabolism, National Cancer Institute, Bethesda, MD), and breeding colonies estabolished at Mount Sinai. SV129 wild type mice were purchased from Charles River Laboratory. Animal experiments were performed in Mount Sinai. Mice used in this study were all males at the age of 8 weeks, with body weight of 20–25 g, and fed with liquid dextrose diet (Bio-Serv, Frenchtown, NJ). When sacrificed, liver tissues were collected and rapidly excised into small fragments and washed with cold saline. With the use of the iTRAQ technique, a multifactorial comparative proteomic study between wild type and CYP2E1 knockout mice can be performed. MALDI plates were analyzed with a TOF/TOF 5800 mass spectrometer (AB Sciex). Functional annotation of protein was conducted using DAVID Bioinformatics Resources 6.7 (NIAID/NIH). For molecular pathway and network analysis of significantly changed proteins, the quantitative data were analyzed using Ingenuity Pathways Analysis (IPA).
10
MALDI-TOF/TOF Mass Spectrometry for Protein Identification
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MALDI plates were analyzed with a TOF/TOF 5800 mass spectrometer (AB Sciex). The instrument was calibrated using the 4700 mass calibration standard. MS spectra between m/z 800 and 4,000 were acquired for every spot using 1,000 laser shots in reflector mode. The 20 most intense ion signals per spot having a S/N>10 were selected as precursors for MS/MS acquisition using 2,000 laser shots. Peptide and protein identifications were performed with the ProteinPilot Software 4.5 (AB Sciex) using the Paragon algorithm. Combined data and spectra from all 24 OFFGEL fractions were searched against the UniProt mouse database (release-2010_11). The following search parameters were selected: iTRAQ 8-plex peptide label, cysteine alkylation, trypsin specificity, ID focus on biological modifications, and processing including quantitation and thorough ID. A protein with a confidence threshold of 95% (unused confidence threshold ProtScore>1.3) was reported and the corresponding False Discovery Rate (FDR) was less than 1%. In protein grouping, competitor threshold was set as 2.00 in ProtScore.
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