Protein samples were loaded into BoltTM 4–12% Bis-Tris Plus gradient gel (Invitrogen), and the target protein gel band was excised and digested with trypsin as described40 (link). After cleanup steps using C18 ZipTips (Millipore), the samples were mixed with an equal amount of matrix solution containing 10 mg/ml R-cyano-4-hydroxycinnamic acid in 0.1% TFA/50% ACN and spotted onto a 384-well stainless steel MALDI target plate (Applied Biosystems, Foster City, CA). An ABI 4800 Proteomics Analyzer MALDI TOF/TOF mass spectrometer (Applied Biosystems) was used to analyze the samples. For protein ID, combined MS and MS/MS data were submitted via GPS Explorer (version 3.6, Applied Biosystems) to Mascot server (version 2.0, Matrix Science). The swissprot database (including 545536 sequences, 194023197 residues) was utilized for the search.
Abi 4800 proteomics analyzer maldi tof tof mass spectrometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI 4800 Proteomics Analyzer MALDI TOF/TOF mass spectrometer is a laboratory instrument designed for protein and peptide analysis. It utilizes matrix-assisted laser desorption/ionization (MALDI) and tandem time-of-flight (TOF/TOF) mass spectrometry technology to identify and characterize proteins and peptides within a sample.
Automatically generated - may contain errors
Lab products found in correlation
C18 ziptip, by Merck Group (1 mentions)
Bolt 4 12 bis tris plus gradient gel, by Thermo Fisher Scientific (1 mentions)
Maldi target plate, by Thermo Fisher Scientific (1 mentions)
Gps explorer, by Thermo Fisher Scientific (1 mentions)
Mascot server, by Matrix Science (1 mentions)
Gps explorer software version 3, by Thermo Fisher Scientific (1 mentions)
Mascot search engine, by Matrix Science (1 mentions)
3 protocols using abi 4800 proteomics analyzer maldi tof tof mass spectrometer
1
MALDI-TOF/TOF Protein Identification
2
Identifying Proteins via MALDI-TOF/TOF Mass Spectrometry
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Tryptic peptides were finally dissolved in MALDI matrix (7 mg/mL α-cyano-4-hydroxycinnamic acid in 0.1% TFA and 50% ACN), spotted onto 192-well stainless steel MALDI target plates, and analyzed using an ABI 4800 Proteomics Analyzer MALDI TOF/TOF mass spectrometer (Applied Biosystems, Carlsbad, CA, USA). The MS together with the MS/MS spectra were searched against the IPI Human database version 3.24 using GPS ExplorerTM Version 3.0 software and MASCOT database search algorithms (version 2.0). The following search criteria were used: trypsin specificity, cysteine carbamidomethylation (C), and methionine oxidation (M) as variable modifications, 1 trypsin miscleavage allowed, 100 ppm MS tolerance, and 0.25 Da MS/MS tolerance. All identified proteins had protein scores greater than 59 (P < 0.05) and individual ion scores greater than 21 with expected values (P < 0.05). All MS/MS spectra were further validated manually.
3
MALDI-TOF/TOF Mass Spectrometry Protein Identification
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The collected spots were destained with 50% acetonitrile/100 mM NH4HCO3. After 10 min, 2 μl of 25 ng/ml trypsin diluted in 50 mM NH4HCO3 were added to each gel piece and 30 μl of 50 mM NH4HCO3 were then added followed by incubation overnight at 37°C. The peptide mixtures from the gel pieces were extracted and dry-digested using a vacuum pump. Subsequently, 2 μl 50% acetonitrile/0.1% TFA and 0.5 μl matrix solution containing CHCA saturated in 50% acetonitrile/0.1% TFA were used to redissolve the powder. The samples were then analyzed using the ABI 4800 Proteomics Analyzer MALDI-TOF/TOF mass spectrometer (Applied Biosystems). For most mass spectrometers, the upper limit for m/z is between 650 and 800. MS/MS analyses were performed at collision energy of 2 KV with air. The Mascot search engine (version 2.1, Matrix Science) and the GPS Explorer™ software version 3.6.2 (Applied Biosystems) were used to explore the tandem mass spectra and peptide and protein. The Mascot searching engine was used to identify the protein.
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