Proteins were identified by using AB SCIEX MALDI TOF-TOF 5800 system (AB SCIEX, Foster City, CA, USA) equipped with a neodymium with laser wavelength 349 nm as described [51 ,52 (link)]. The laser can shot at a rate of up to 1000 Hz. CHCA was used as the matrix with TFA for an ionization auxiliary reagent. The spectrum was calibrated using the TOF/TOF calibration mixtures (AB SCIEX). All peptide mass fingerprint spectra were internally calibrated with trypsin autolysis peaks, and all known contaminants were excluded during this process. Peptide mass was used to database search.
Maldi tof tof 5800 system
Manufactured by AB Sciex
Sourced in United States
The MALDI-TOF/TOF 5800 system is a mass spectrometry instrument designed for protein identification and characterization. It utilizes matrix-assisted laser desorption/ionization (MALDI) technology combined with time-of-flight (TOF) mass analysis. The system is capable of performing both MS and MS/MS analyses to provide detailed information about the structure and composition of biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Proteinpilot, by AB Sciex (2 mentions)
Tof tof calibration mixture, by AB Sciex (1 mentions)
Bovine trypsin, by Roche (1 mentions)
Ultraflex 3 tof tof mass spectrometer, by Bruker (1 mentions)
Speedvac, by Thermo Fisher Scientific (1 mentions)
Uv 1601 spectrophotometer, by Shimadzu (1 mentions)
Unity plus spectrometer, by Agilent Technologies (1 mentions)
Mascot server, by Matrix Science (1 mentions)
Trypsin, by Roche (1 mentions)
Modified bovine trypsin, by Roche (1 mentions)
11 protocols using maldi tof tof 5800 system
1
Protein Identification via MALDI TOF-TOF
2
Proteomics Identification of Brassica napus Proteins
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DAPs were excised manually and digested in-gel with bovine trypsin (Roche, Cat. 11418025001) as described elsewhere49 (link). The digested peptides were mixed with α-cyano-4-hydroxycinnamic acid (CHCA) and analyzed by AB SCIEX MALDI TOF-TOF 5800 System (AB SCIEX, Foster City, CA, USA). B.napus genome sequences were downloaded from CoGe database (https://genomevolution.org/CoGe/ ). B.napus proteins sequences was released in August 2014 (101040 sequences, version 5.0). The local protein database was built using MASCOT containing 101040 protein sequences derived from the complete genome sequences of B.napus. MS peptides search the B.napus local database using ProteinPilot Software. When individual ions scores were higher than the threshold score (>62), proteins were considered as a confident identification or with an extensive homology (P < 0.05). At least 2 peptides matched the observed masses for an identification to be considered valid. The positive matches were BLASTP searched against the uniProt protein database for updated annotation and identification of homologous proteins18 (link).
3
Phosphopeptide Binding to Engineered Nanomaterials
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The phosphopeptide (LPSSPVpYEDAASFK) was dissolved at 1 μg/μL in water. 3 μL of this solution was mixed with 75 μL of each of the ENMs (La2O3, quartz and AP-MWCNTs) dispersed at 1 mg/mL. The blank control was water only. After the incubation at 37 °C for 6 h, the peptides were analyzed by MS, carried out by a MALDI-TOF/TOF 5800 System (AB SCIEX, Foster City, CA) equipped with a 1 kHz OptiBeam on-axis laser. 2,5-Dihydroxybenzoic acid solution (25 mg/mL, in 70% ACN-H2O containing 1% H3PO4) was used as the matrix to assist the ionization of peptides.
4
MALDI-TOF MS Analysis of Samples
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One microliter of the sample was deposited on the sample plate and dried, then 1 µL of 20 mg/mL DHB substrate (in 60% (v/v) acetonitrile and 0.1% (v/v) TFA aqueous solution) was introduced. MS spectra were acquired on an Ultraflex III TOF/TOF mass spectrometer (Bruker Daltonik GmbH, Germany) equipped with a 200-Hz smart-beam 1 laser at 355 nm or a MALDI-TOF/TOF 5800 System (AB SCIEX, Foster City, CA) quipped with a 1-kHz optibeam™ on-axis laser in the positive reflection mode, and controlled by the Flex Control 2.4 software and Data Explorer software 4.11. Flex Analysis software 3.3 and TOF/TOF Series Explorer software 4.1 were used to analyze MALDI-TOF MS data. The threshold for peak acceptance was a signal-to-noise ratio of 6.
5
Chitosan-Assisted Oligosaccharide Purification
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Chitosan with a da of 48% (30 mg) was mixed with the recombinant PpChi protein (15 nmol) and incubated at 40 °C for
1 h. The oligosaccharides were eluted separately from carbon cartridges
using a 1–30% acetonitrile gradient. After drying in a centrifugal
evaporator (SpeedVac, Thermo Fisher Scientific, Waltham, MA), the
purified trisaccharide DDA and tetrasaccharide DDDA were weighed and
analyzed by HPAEC-PAD (Table S1 ) and MALDI-TOF
MS; MALDI CID MS/MS analysis was performed on a MALDI TOF/TOF 5800
system (AB Sciex, Framingham, MA).34 (link)
1 h. The oligosaccharides were eluted separately from carbon cartridges
using a 1–30% acetonitrile gradient. After drying in a centrifugal
evaporator (SpeedVac, Thermo Fisher Scientific, Waltham, MA), the
purified trisaccharide DDA and tetrasaccharide DDDA were weighed and
analyzed by HPAEC-PAD (
MS; MALDI CID MS/MS analysis was performed on a MALDI TOF/TOF 5800
system (AB Sciex, Framingham, MA).34 (link)
6
Analytical Techniques for Compound Characterization
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UV–visible spectra acquisition in the range 250–500 nm was performed using a UV1601 spectrophotometer (Shimadzu Italia S.r.l., Milan, Italy). 1H-NMR spectra were recorded with a Varian Unity Plus spectrometer (1H 400 MHz). Chemical shifts of 1H (δH) in parts per million were determined relative to DMSO-d6. A MALDI TOF/TOF 5800 system (AB SCIEX, Darmstadt, Germany) equipped with a neodymium-doped yttrium lithium fluoride (Nd:YLF) laser (345 nm), operating in linear positive ion mode, was used (typical mass accuracy was ≤100 ppm). At least 5000 laser shots were typically accumulated by a random rastering pattern, with a laser pulse rate of 400 Hz and a laser fluence of 1 mJ/cm2 in the MS mode. The delayed extraction (DE) time was set at 450 ns. Calibration in linear mode was carried out using a protein mixture composed of insulin beta chain (3497.0 Da), insulin (5735.0 Da) and cytochrome c (12361.1 Da).
7
Protein Identification via Mass Spectrometry
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Proteins spots were excised and digested with modified bovine trypsin (cat. no. 11418025001, Roche, Basel, Switzerland) as previously reported3 (link). Mass spectra of trypsin-digested peptide extracts were recorded on an AB SCIEX MALDI-TOF/TOF 5800 system (AB SCIEX, Framingham, MA, USA) with a laser wavelength of 349 nm. Unsing an in-house MASCOT server (Matrix Science, Boston, MA, USA), we searched for all spectra in a self-constructed database derived from the original Gh genome and expressed sequence tags36 (link)37 (link)38 (link) that included 77,051 protein sequences. All six protein spots were considered to be successfully identified onlyif peptide counts with 95% confidence >5 and peptide coverage >20%.
8
Protein Identification by MALDI-TOF/TOF
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Protein identification experiments were performed at Kocaeli University DEKARTproteomics laboratory using ABSCIEX MALDI-TOF/TOF 5800 system. In-gel tryptic digestion of proteins was performed using an in-gel digestion kit following the recommended protocol (Pierce, USA). The data obtained from MALDI-TOF/TOF were searched against the MASCOT database version 2.5 (Matrix Science) using a streamline software, ProteinPilot (ABSCIEX, USA), with the following criteria: National Center for Biotechnology Information non-redundant (NCBInr), species restriction to R. norvegicus, enzyme of trypsin, at least five independent matching peptides, at most one missed cleavage site, MS tolerance set to ±50 ppm and MS/MS tolerance set to ±0.4 Da, fixed modification being cells International carbamidomethyl (Cys) and variable modification being oxidation (Met), a peptide charge of 1+, and being monoisotopic. Only significant hits, as defined by the MASCOT probability analysis (p < 0.05), were accepted.
9
Proteomics Analysis of Zea mays Proteins
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The target protein spots of DEPs were manually excised from the 2-DE gels and digested in-gel with bovine Trypsin (Trypsin, Roche, Cat. 11418025001) as described previously23 (link). Digested proteins were then mixed with alpha-cyano-4-hydroxycinnamic acid (CHCA) matrix for determination of mass spectra and analyzed using an AB SCIEX MALDI TOF-TOF 5800 system (AB SCIEX, Shanghai, China) equipped with a neodymium with laser wavelength 349 nm. Mass spectra were obtained as described previously49 (link).
The raw MS and MS/MS spectra were combined and searched against the Zea mays amino acid sequence database (including 87,603 sequences), which was downloaded from Uniprot (http://www.uniprot.org ), using in-house MASCOT software for protein identification. The searched parameters were set as previously described22 (link). If peptides matched multiple proteins, the protein with the highest score was recorded in this study for bioinformatics analysis. For the unnamed proteins, BLAST search using NCBI (http: //www.ncbi.nlm) was performed to identify homologous proteins.
The raw MS and MS/MS spectra were combined and searched against the Zea mays amino acid sequence database (including 87,603 sequences), which was downloaded from Uniprot (
10
MALDI-TOF/TOF Protein Identification Workflow
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Protein identification experiments were performed at Kocaeli University DEKART proteomics laboratory (http://kabiproteomics.kocaeli.edu.tr/ ) by using ABSCIEX MALDI-TOF/TOF 5800 system. In-gel tryptic digestion of the proteins was performed by using an in-gel digestion kit following the recommended protocol of the manufacturer (Pierce, USA). Zip-Tip cleaning was performed for each digested sample following the recommended protocol (Millipore, USA) before deposition onto a MALDI plate. Peak data were analyzed with MASCOT by using a streamline software, Protein Pilot (ABSCIEX,USA). The parameters for searching were; enzyme of trypsin, 1 missed cleavage, fixed modifications of carbamidomethyl (C), variable modifications of oxidation (M), peptide mass tolerance: 50ppm, fragment mass tolerance: ±0.4 Da, peptide charge of 1+ and monoisotopic. Only significant hits, as defined by the MASCOT probability analysis (p < 0.05) were accepted. Classification of the proteins was performed by using a freely available classification system, PANTHER (http://www.pantherdb.org/ ) [18 (link)].
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