In parallel to the above-described immunoblot, a band of 180 kDa was excised from a replicate gel and digested with 12.5 ng/μL of trypsin (Roche Molecular Biochemicals) in 25 mM ammonium bicarbonate (pH 8.5) overnight at 37°C43 (link). The supernatant was then collected and 1 μL was spotted onto a MALDI target plate and allowed to air-dry at RT. Then, 0.4 μL of α-cyano-4-hydroxy-cinnamic acid matrix (3 mg/mL) (Sigma) in 50% (v/v) ACN was added to the dried peptide digest spots and allowed to air-dry at RT. MALDI-TOF MS analysis (4800 Proteomics Analyzer MALDI-TOF/TOF mass spectrometer (Applied Biosystems) was performed by the Proteomics Service, University of Vigo. Mass spectra were calibrated with internal peptides from trypsin autodigestion. A database search was performed with MASCOT Daemon search engine 2.1 (Matrix Science) through the Global Protein Server version 3.6 (Applied Biosystems) against SwissProt 2012_07 (536789 sequences; 190518892 residues), as previously described43 (link).
4800 proteomics analyzer maldi tof tof mass spectrometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 4800 Proteomics Analyzer MALDI-TOF/TOF mass spectrometer is a laboratory instrument designed for protein analysis. It utilizes matrix-assisted laser desorption/ionization (MALDI) and tandem time-of-flight (TOF/TOF) technology to provide high-resolution mass spectrometry data for proteomics research.
Automatically generated - may contain errors
Lab products found in correlation
Maldi plate, by Thermo Fisher Scientific (2 mentions)
α cyano 4 hydroxycinnamic acid matrix, by Merck Group (1 mentions)
Trypsin, by Roche (1 mentions)
C18 ziptip, by Merck Group (1 mentions)
Decyder software, by GE Healthcare (1 mentions)
4800 proteomics analyzer, by Thermo Fisher Scientific (1 mentions)
4000 series explorer software, by Thermo Fisher Scientific (1 mentions)
Imagequant software, by GE Healthcare (1 mentions)
Ettan daltsix electrophoresis unit, by GE Healthcare (1 mentions)
Mascot, by Matrix Science (1 mentions)
9 protocols using 4800 proteomics analyzer maldi tof tof mass spectrometer
1
MALDI-TOF MS Identification of Protein
2
Tryptic Digestion of Live Bacterial Cells
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The efficiency of the tryptic digestion of the live bacterial cells was checked at the qualitative level by looking at the mass spectrum from MALDI-TOF MS. Aliquots of 1 μL of the cleaned peptides were mixed with 1 μL of matrix solution (α-cyanohydroxycinnamic acid at a concentration of 5 mg/mL in 70% ACN/ 0.1% trifluoroacetic acid) and spotted onto a MALDI plate using the dry-droplet method. A 4800 Proteomics Analyzer MALDI-TOF/TOF Mass Spectrometer (Applied Biosystems, Waltham, MA, USA) was used to acquire the mass spectra, in the m/z range of 800 to 4000, with an accelerating voltage of 20 kV in reflectron mode. The spectra were internally calibrated using peptides from trypsin autolysis ([M + H+] = 842.509, [M + H+] = 2211.104) with an m/z precision of ± 20 ppm.
3
Extraction and Mass Spectrometry Analysis of L. barbarum Root
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Dried cortex of L. barbarum root was purchased from a local distributor (Hung Soon Pte Ltd, Singapore)49 (link). The sample was extracted with the appropriate extraction solvent (water, 50% EtOH) in a 1:10 ratio (v/v) at room temperature. The extracts were then centrifuged at 3000 × g for 30 min to remove plant debris. ZipTip C18 (Millipore) was used for sample preparation of the crude extracts for mass spectrometry analysis. Mass spectra of the samples were obtained with a 4800 Proteomics Analyzer MALDI-TOF/TOF mass spectrometer (Applied Biosystems, California, USA). The MS spectra were acquired using reflector and/or linear mode, using up to 2500 shots and an accelerating voltage of 20 kV.
4
Protein Identification by MALDI-TOF/TOF
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Spots present in only one of the conditions or displayed quantitative abundance changes of more than 1.5-fold were selected for identification by MALDI-TOF/TOF. Protein spots of interest were picked from the stained gel and were then washed and digested. The samples were mixed with a matrix solution CCA (α-cyano-4-hydroxycinnamic acid), spotted on a MALDI plate (Applied Biosystems, Foster City, CA, United States), and allowed to air-dry. To obtain a peptide mass fingerprint (PMF), lists of peak intensities and mass-to-charge (m/z) values were analyzed with a 4,800 Proteomics Analyzer MALDI-TOF/TOF Mass Spectrometer (Applied Biosystems, Foster City, CA, United States).
5
Quantitative Proteomic Analysis of SIRT2 Knockdown
6
Protein Identification by MALDI-TOF/TOF
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Spots that were present in only one of the conditions or displayed quantitative abundance changes of more than 2-fold were selected for identification by MALDI-TOF/TOF. Protein spots of interest were picked from the stained gel using the Investigator ProPic robotic workstation (Genomic Solution, Huntingdon, UK) and were then washed and digested. The samples were mixed with a matrix solution CCA (α-cyano-4-hydroxycinnamic acid), spotted on a MALDI plate (Applied Biosystems, Foster City, CA, USA) and allowed to air-dry. To obtain a peptide mass fingerprint (PMF), lists of peak intensities and mass-to-charge (m/z) values were analyzed with a 4800 Proteomics Analyzer MALDI-TOF/TOF Mass Spectrometer (Applied Biosystems).
7
Phosphorylation Site Identification by Mass Spectrometry
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In vitro kinase reactions after phosphorylating nNOS by a recombinant protein containing the active catalytic domain of PKD1 fused to GST (GST-PKD1-cat) were digested with trypsin and analyzed by HPLC followed by MALDI TOF/TOF and peptide fragmentation and de novo sequencing in the Proteomic Studies Unit (Unidad de Proteómica; Facultad de Farmacia Parque Científico de Madrid, Universidad Complutense de Madrid, Madrid, Spain) following standard procedures. MALDI-TOF MS analysis was performed in a 4800 Proteomics Analyzer MALDI-TOF/TOF mass spectrometer (Applied Biosystems, MDS Sciex, Toronto, Canada). The MALDI-TOF/TOF operated in positive reflector mode with an accelerating voltage of 20000 V. Selected peptides, were subjected to MS/MS sequencing analyzes using the 4800 Proteomics Analyzer (Applied Biosystems, Framingham, MA). Suitable precursors from the MS spectra were selected for MS/MS analysis with CID on (atmospheric gas was used) 1 Kv ion reflector mode and precursor mass Windows ± 4 Da. The plate model and default calibration were optimized for the MS/MS spectra processing. De novo sequencing from fragmentation spectra of peptides was performed using De novo tool software (Applied Biosystems), tentative sequences were manually checked and validated.
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In vitro kinase reactions after phosphorylating nNOS by a recombinant protein containing the active catalytic domain of PKD1 fused to GST (GST-PKD1-cat) were digested with trypsin and analyzed by HPLC followed by MALDI TOF/TOF and peptide fragmentation and de novo sequencing in the Proteomic Studies Unit (Unidad de Proteómica; Facultad de Farmacia Parque Científico de Madrid, Universidad Complutense de Madrid, Madrid, Spain) following standard procedures. MALDI-TOF MS analysis was performed in a 4800 Proteomics Analyzer MALDI-TOF/TOF mass spectrometer (Applied Biosystems, MDS Sciex, Toronto, Canada). The MALDI-TOF/TOF operated in positive reflector mode with an accelerating voltage of 20000 V. Selected peptides, were subjected to MS/MS sequencing analyzes using the 4800 Proteomics Analyzer (Applied Biosystems, Framingham, MA). Suitable precursors from the MS spectra were selected for MS/MS analysis with CID on (atmospheric gas was used) 1 Kv ion reflector mode and precursor mass Windows ± 4 Da. The plate model and default calibration were optimized for the MS/MS spectra processing. De novo sequencing from fragmentation spectra of peptides was performed using De novo tool software (Applied Biosystems), tentative sequences were manually checked and validated.
8
Saliva Proteome Analysis in MCI and AD
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Saliva samples from four male subjects from each group (MCI, AD, and elderly nondemented controls) were pooled by mixing equal amounts. Fifty micrograms of each pool were loaded on a SDS-PAGE gel. ImageQuant software (GE Healthcare) was used for quantity determination.
Protein bands matching the corresponding molecular weight were excised from the gel and distained. In-gel digestion was carried out sequentially with trypsin and endopeptidase Asp-N for 16 h each. To validate the presence of lactoferrin in human saliva, this protein was identified by MALDI-TOF/TOF mass spectrometer 4800 Proteomics Analyzer (Applied Biosystems, Framingham, MA) and 4000 Series ExplorerTM software (Applied Biosystems).
Protein bands matching the corresponding molecular weight were excised from the gel and distained. In-gel digestion was carried out sequentially with trypsin and endopeptidase Asp-N for 16 h each. To validate the presence of lactoferrin in human saliva, this protein was identified by MALDI-TOF/TOF mass spectrometer 4800 Proteomics Analyzer (Applied Biosystems, Framingham, MA) and 4000 Series ExplorerTM software (Applied Biosystems).
9
Proteomic Analysis of Stem Tissues
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Isoelectric focusing (IEF)
was carried out on Immobiline DryStrip gel (IPG) strips (17 cm, nonlinear,
GE Healthcare, Piscataway, NJ, U.S.A.). Prior to the second dimension
of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE),
the focused IPG strips were equilibrated in equilibration buffers
for 15 min. The equilibrated IPG strips were transferred onto 12.5%
SDS acrylamide gels by use of an Ettan DALT Six Electrophoresis Unit
(GE Healthcare, Piscataway, NJ, U.S.A.). Protein spots were visualized
by staining with Coomassie Brilliant Blue dye (CBB-G250).23 Gels were scanned, and protein spot profiles
were analyzed by PDQuestTM7.0 (Bio-Rad Laboratory). Protein spots
were considered differentially expressed if the intensity changed
more than one and a half times in different stems. All the 2-DE experiments
were repeated three times. MS/MS analysis was conducted using a MALDI-TOF/TOF
mass spectrometer 4800 Proteomics Analyzer (Applied Biosystems, Framingham,
MA, U.S.A.). MS spectra were acquired in positive reflector mode.
Combined MS and MS/MS spectra were submitted to MASCOT (V2.2, Matrix
Science, London, U.K.) by GPS Explore software (V3.6, Applied Biosystems,
Framingham, MA, U.S.A.), and the intracellular localization of the
identified protein was analyzed through WOLF PSORT (http://wolfpsort.seq.cbrc.jp ), SwissPort, NCBI, and so on.
was carried out on Immobiline DryStrip gel (IPG) strips (17 cm, nonlinear,
GE Healthcare, Piscataway, NJ, U.S.A.). Prior to the second dimension
of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE),
the focused IPG strips were equilibrated in equilibration buffers
for 15 min. The equilibrated IPG strips were transferred onto 12.5%
SDS acrylamide gels by use of an Ettan DALT Six Electrophoresis Unit
(GE Healthcare, Piscataway, NJ, U.S.A.). Protein spots were visualized
by staining with Coomassie Brilliant Blue dye (CBB-G250).23 Gels were scanned, and protein spot profiles
were analyzed by PDQuestTM7.0 (Bio-Rad Laboratory). Protein spots
were considered differentially expressed if the intensity changed
more than one and a half times in different stems. All the 2-DE experiments
were repeated three times. MS/MS analysis was conducted using a MALDI-TOF/TOF
mass spectrometer 4800 Proteomics Analyzer (Applied Biosystems, Framingham,
MA, U.S.A.). MS spectra were acquired in positive reflector mode.
Combined MS and MS/MS spectra were submitted to MASCOT (V2.2, Matrix
Science, London, U.K.) by GPS Explore software (V3.6, Applied Biosystems,
Framingham, MA, U.S.A.), and the intracellular localization of the
identified protein was analyzed through WOLF PSORT (
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