Replicate preparative gels of 1000 μg of N2a/WT and N2a/APP cell proteins were prepared as for DIGE but without protein labeling. The gel was immersed overnight in dye (Coomassie blue solution containing 0.12% Coomassie brilliant blue G-250, 20% ethanol, 10% phosphoric acid, and 10% ammonium sulfate). Differential protein spots of interest identified by Decyder software analysis were manually excised from the stained gel. Gel pieces were destained and digested overnight at 37°C with 0.01 μg/μL trypsin (Promega Corp., WI, USA) as described by Robinson et al. [11 (link)]. The tryptic peptides were used for analysis by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) (SCIEX TOF/TOF™ 5800 System, AB SCIEX, Framingham, MA, USA).
Sciex tof tof 5800 system
Manufactured by AB Sciex
Sourced in United States
The SCIEX TOF/TOF™ 5800 System is a high-performance mass spectrometer designed for accurate and sensitive analysis of complex samples. It utilizes time-of-flight (TOF) and tandem time-of-flight (TOF/TOF) technologies to provide precise mass measurements and structural information about analytes.
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3 protocols using sciex tof tof 5800 system
1
Differential Proteome Analysis of N2a Cell Lines
2
MALDI-TOF-MS/MS-based Protein Identification
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Protein identification was carried out by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS, SCIEX TOF/TOF™ 5800 System, AB SCIEX, Framingham, MA, USA). Peptide extracts (0.6 μL) were crystallized with 10 mg/mL α-cyano-4-hydroxycinnamic acid (CHCA) in 0.1% TFA and 50% acetonitrile (ACN) directly onto the target, and dried at room temperature. The spectra were externally calibrated. Protein searching were conducted against the SwissProt Mus musculus database housed in MASCOT (Matrix Science, UK) with a mass measurement tolerance of 100 ppm in MS mode and 0.5 Da in MS/MS mode. Fixed carbamidomethyl modification was taken into account and up to two missed cleavages per peptide were allowed.
3
Co-IP Protein Identification via MS
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After co-immunoprecipitation, equal amounts of proteins were loaded in 4-12% SDS-PAGE gels and stained with Coomassie blue G250 (BioRad). After staining, the bands higher than 10 kDa were excised into individual fractions, excluding the stained IgG-H (52 kDa). These fractions were then further excised into small pieces and placed into a 1.5-ml tube. Sample preparation used for Q-Exactive mass spectrometry was performed according to the standard protocol [12] (link). After destaining and shrinking, the gel was treated with 20 mM DTT for protein reduction, followed by 50 mM iodoacetamide (IAA) treatment for alkylation. Protein digestions were performed with trypsin at 37 °C overnight and the digested proteins were then desalted for LC-MS/MS analysis (AB SCIEX TOF/TOF™ 5800 system, USA). Proteins were identi ed using Protein Pilot 4.0TM software (AB Sciex, USA) [13] (link).
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