All of the immunoreactive proteins were selected and excised from 2D gels, which corresponded to the spots on the PVDF membranes and further in a gel digested with trypsin. MALDI-TOF-MS analysis was performed by using a MALDI-TOF/TOF instrument (5800 proteomics analyzer; Applied Biosystems) to identify proteins. Combined MS and MS/MS queries were conducted by using the Mascot search engine (Version 2.2; Matrix Science, Ltd.) on the database of UniProt M. bovis (downloaded on May 20, 2016; 1695 sequences) with the following parameter settings: Trypsin digestion, variable modification of oxidation (M), fixed modifications of carbamidomethyl (C), peptide mass tolerance for monoisotopic data of 100 ppm, one max missed cleavages, and MS/MS fragment tolerance of 0.4 Da. GPS Explorer protein with confidence index ≥ 95% (protein score C. I. %) was used for further manual validation.
5800 proteomics analyzer
Manufactured by Thermo Fisher Scientific
Sourced in United States, Australia
The 5800 Proteomics Analyzer is a mass spectrometry instrument designed for advanced protein analysis. It provides high-resolution, accurate mass measurements to enable detailed protein identification and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Peaks studio version 4.5 sp2, by Bioinformatics Solutions (2 mentions)
Mascot search engine, by Matrix Science (1 mentions)
Maldi plate, by Thermo Fisher Scientific (1 mentions)
Ziptip, by Merck Group (1 mentions)
C8 column, by Macherey-Nagel (1 mentions)
Famos autosampler, by Thermo Fisher Scientific (1 mentions)
Probot, by Thermo Fisher Scientific (1 mentions)
C18 pepmap trap column, by Thermo Fisher Scientific (1 mentions)
Biomap, by Novartis (1 mentions)
13 protocols using 5800 proteomics analyzer
1
Proteomic Identification of Mycobacterium bovis
2
MALDI-TOF Analysis of BoNT Substrates
3
Mass Spectrometric Detection of BoNT/FA Activity
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One nanogram of BoNT/FA was added to peptide substrate LSELDDRADALQAGASQFESSAAKLKRKYWWKNLK (SubB) in the presence of reaction buffer as described previously (24 (link)). Another aliquot of 1 ng of BoNT/FA was added to another peptide substrate TSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADAL (SubF) also in the presence of reaction buffer. Following a 4-h incubation at 37°C, a 2-µl aliquot of each reaction supernatant was mixed with 18 µl of matrix solution, and a 0.5-µl aliquot of this mixture was pipetted onto one spot of a MALDI plate as described previously (24 (link)). Mass spectra of each spot were obtained by scanning from 900 to 5500 m/z in MS-positive ion reflector mode on an Applied Biosystems 5800 proteomics analyzer (Framingham, MA). The instrument uses an Nd-YAG laser at 355 nm, and each spectrum is an average of 2,400 laser shots.
To positively identify BoNT/FA via its amino acid sequence, 1 µg of the toxin was added to 13 µl of 100 mM ammonium bicarbonate at pH 7.5 and 2 µl of trypsin at 0.5 mg/ml in water. Following mixing, the mixture was heated at 52°C for 10 min, followed by the addition of 1 µl of 10% of trifluoroacetic acid. Peptides from the toxin were identified by LC-MS/MS with database searching as described previously (20 (link)).
To positively identify BoNT/FA via its amino acid sequence, 1 µg of the toxin was added to 13 µl of 100 mM ammonium bicarbonate at pH 7.5 and 2 µl of trypsin at 0.5 mg/ml in water. Following mixing, the mixture was heated at 52°C for 10 min, followed by the addition of 1 µl of 10% of trifluoroacetic acid. Peptides from the toxin were identified by LC-MS/MS with database searching as described previously (20 (link)).
4
SALDI-MS Analysis of Amyloid-β1-42 Detection
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During the detection of Aβ1–42, GO played the role of matrix in SALDI-MS analysis. GO (1 mg) was dispersed in ultrapure water (1 mL) under sonication for 20 min. The above GO slurry (2 μL) was successively pipetted on the stainless steel target plate and dried for SALDI-MS analysis. Based on the reflection mode employing delayed extraction, the MS analysis was performed on 5800 Proteomics Analyzer (Applied Biosystems, Framingham, MA, USA) coupled with a pulsed Nd:YAG laser (1 kHz, 355 nm wavelength). In this study, the delay time, acceleration voltage, and repetition rate were optimized to 200 ns, 20 kV, and 200 Hz, respectively. For the SALDI-MS analysis, the number of laser shots was 200 per analysis.
For selective enhancement of Aβ1–42, the Apt-GO slurry was centrifuged at 4,000 g for 15 min and the precipitate was redispersed in the binding buffer (20 mM Tris-HCl, 140 mM NaCl, and 2 mM MgCl2, pH = 7.5). Then, the Aβ1–42 standards and disrupted AD cells model solution were added. The mixture was incubated at 37°C for 30 min and centrifugated at 4,000 g for 15 min. Afterward, the precipitate was redispersed in 0.1% TFA solution for SALDI-TOF/MS analysis as described above.
For selective enhancement of Aβ1–42, the Apt-GO slurry was centrifuged at 4,000 g for 15 min and the precipitate was redispersed in the binding buffer (20 mM Tris-HCl, 140 mM NaCl, and 2 mM MgCl2, pH = 7.5). Then, the Aβ1–42 standards and disrupted AD cells model solution were added. The mixture was incubated at 37°C for 30 min and centrifugated at 4,000 g for 15 min. Afterward, the precipitate was redispersed in 0.1% TFA solution for SALDI-TOF/MS analysis as described above.
5
Peptide Identification and Synthesis for Antioxidant Activity
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The peptide with the highest antioxidant activity was picked out to identify its molecular mass and amino acid sequence. The sample was desalted using Zip Tips (Millipore) and analyzed by matrix-assisted laser desorption/ionization-time of flight (MALDI TOF)-TOF mass spectrometer using a 5800 Proteomics Analyzer (Applied Biosystems at Proteomics International Pty. Ltd., Nedlands, Western Australia). The amino acid sequence was determined by the de novo sequencing method. The PEAKS Studio version 4.5 SP2 (Bioinformatics Solutions Inc., Waterloo, ON, Canada) was used for analysis of the derived tandem mass spectrometry (MS/MS) spectra.
The identified peptide was synthesized using the solid-phase method (GL Biochem Shanghai Ltd., Shanghai, China) using the standard Fmoc (9-fluorenyl-methoxycarbonyl) chemistry. Crude synthetic peptides were subjected to RP-HPLC to purify the peptide using a semi-preparative C8 column (10 mm × 250 mm, Macherey–Nagel GmbH & Co.).
The identified peptide was synthesized using the solid-phase method (GL Biochem Shanghai Ltd., Shanghai, China) using the standard Fmoc (9-fluorenyl-methoxycarbonyl) chemistry. Crude synthetic peptides were subjected to RP-HPLC to purify the peptide using a semi-preparative C8 column (10 mm × 250 mm, Macherey–Nagel GmbH & Co.).
6
Protein Identification by MALDI-TOF/TOF
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Protein identification was carried out as previously described (Di et al. 2013 , 2015 ). Briefly, spots of interest were excised and trypsin digested trypsin, with 0.5 μL of extracted sample analyzed by matrix‐assisted laser desorption/ionization time‐of‐flight/time‐of‐flight (MALDI‐TOF/TOF) with a 5800 Proteomics Analyzer (Applied Biosystems, CA, USA). Gel extracts were pooled, dried down and resuspended in a gel matrix using 5 μL 50% (v/v) ACN, and 0.1% (v/v) trifluoroacetic acid (TFA). Next, 0.8 μL of sample was mixed with 0.3 μL of matrix solution (2 μg/μL R‐cyano‐4‐hydroxycinnamic acid) in 50% (v/v) ACN and 0.1% (v/v) TFA. The spot proteins were identified from the peptide mass fingerprints obtained following MALDI‐TOF/TOF using MASCOT with MS/MS spectra from selected peptides. MS/MS searches were conducted against the nrNCBI database with search parameters: enzyme was trypsin; allowance of one missed cleavage site; fixed modification was carbamidomethyl (cysteine); variable modification was oxidation of Met; monoisotopic mass values; protein mass unrestricted; ±200 ppm as peptide mass tolerance; and ±1 Da as fragment mass tolerance.
7
MALDI-TOF MS Analysis of Babesia canis
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Subsequently, MALDI TOF MS analysis was carried out using the Applied Biosystems 5800 Proteomics Analyzer for peptide mass fingerprinting and MS/MS analysis. After MALDI analysis, MALDI MS/MS analysis was performed for the 10 most abundant ions of each sample. To identify proteins specifically belonging to the B. canis, strain Oliveri, a bioinformatics analysis from raw data was performed. For this purpose, the Mascot against the whole NCBI-nr and SwissProt protein databases was used, and for visualization, the Scaffold (Proteome Software, Inc. Portland, OR, USA) software was used.
The likelihood of protein match was determined using the expected values and the Mascot protein scores. Mascot search parameter values were established as 2 for missed cleavage of variable. MOWSE (Molecular Weight SEarch) scores greater than 83 were considered significant (P < 0.05).
Once the proteins were identified in B. canis strain Oliveri, they were characterized using multiple bioinformatic tools in order to determine patterns that could influence antibody production. Protein sequences were located and downloaded of the B. canis strain Oliveri genome (EMBL accession numbersHG803175.1 and HG803176.1 ) for further analyses.
The likelihood of protein match was determined using the expected values and the Mascot protein scores. Mascot search parameter values were established as 2 for missed cleavage of variable. MOWSE (Molecular Weight SEarch) scores greater than 83 were considered significant (P < 0.05).
Once the proteins were identified in B. canis strain Oliveri, they were characterized using multiple bioinformatic tools in order to determine patterns that could influence antibody production. Protein sequences were located and downloaded of the B. canis strain Oliveri genome (EMBL accession numbers
8
Peptide Identification by Nano-LC-MS/MS
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Peptides were redissolved in 20 µl 0.1% TFA and subjected to nano-liquid chromatography (nano-LC) tandem mass spectrometry (MS/MS). Peptides were delivered with a FAMOS autosampler at 30 µl/min to a Pepmap C18 trap column (5 mm × 300 µm i.d.; Dionex) and separated on an Alltima analytical capillary C18 column (150 mm × 100 µm i.d.) at 400 nl/min using the LCPacking Ultimate system. The peptides were separated using a linearly increasing concentration of acetonitrile from 5–35% in 80 min, from 35–45% in 7 min, from 45–90% in 2 min, and then held at 90% for an additional 11 min. The eluent was mixed with matrix (7 mg α-cyano-hydroxycinnaminic acid in 1 ml 70% acetonitrile, 0.1% trifluoroacetic acid, 10 mM ammonium monobasic phosphate) delivered at a flow rate of 1.5 µl/min and deposited offline to the Applied Biosystems metal target every 15 s for a total of 384 spots, using an automatic robot (Probot; Dionex). A 5800 proteomics analyzer (Applied Biosystems) was used for peptide analysis. CID was performed at 2 kV (the collision gas was nitrogen). MS/MS spectra were collected from 2,500 laser shots. The peptides with signal to noise ratio above 50 at the MS mode were selected for the MS/MS experiment; a maximum of 25 MS/MS were allowed per spot. The precursor mass window was 200 relative resolution (FWHM).
9
MALDI MS Analysis of Lipid Profiles
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All MALDI MS and MS/MS experiments were performed in positive ion reflection mode on a 5800 Proteomics Analyzer (Applied Biosystems, Framingham, MA, USA) equipped with a Nd:YAG laser (355 nm), a repetition rate of 400 Hz, and an acceleration voltage of 20 kV. Laser energy was set to 3500 and 3000 for DHB matrix and GO matrix, respectively. 1000 shots were accumulated for each spectrum. For tissue imaging, the laser step size was 60 μm. Assignment of detected lipids was based on the LIPID MAPS prediction tool (http://www.lipidmaps.org/tools/index.html ), the literatures and MS/MS experiments. During the database search, the [M + H]+, [M + Na]+ and [M + K]+ ions were considered, with a mass tolerance of ±0.1 Da. Two-dimensional ion density maps were created using the image reconstruction software (BioMap, Novartis, Basel, Switzerland).
10
MALDI-TOF Enzymatic Activity Assay
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