Computational protocol: New Insights to Clathrin and Adaptor Protein 2 for the Design and Development of Therapeutic Strategies

Similar protocols

Protocol publication

[…] LC-MS/MS analyses were performed on an EASY-nLC II system (Thermo Fisher Scientific, Waltham, MA, USA) connected to a TripleTOF 5600+ mass spectrometer (AB SCIEX, Framingham, MA, USA) equipped with a NanoSpray III source (AB SCIEX, Framingham, MA, USA) and operated under Analyst TF 1.6.0 control. Samples were injected and trapped on a C18 pre-column (5 μm, 2 cm × 100 μm I.D., packed in-house). Subsequently, the peptides were eluted to and separated on a 15 cm analytical column (75 μm I.D., packed in-house with RP ReproSil-Pur C18-AQ 3 μm resin (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) connected in-line to the mass spectrometer. Peptides were eluted at a flow rate of 250 nL/min using a 20 min gradient from 5% to 35% phase B (0.1% formic acid and 100% acetonitrile). An information dependent acquisition (IDA) experiment allowing post-acquisition area-based XIC quantification was used for all LC-MS/MS analyses. [...] MS files collected for identification of protein bands on SDS-PAGE were converted to Mascot generic format (MGF) using the AB SCIEX MS Data Converter beta 1.3 (AB SCIEX, Framingham, MA, USA), and the “proteinpilot MGF” parameters and searched against the Swiss-Prot Mus musculus database (2015_01) using Mascot 2.5 (Matrix Science, London, UK). Trypsin was employed as enzyme allowing one missed cleavage. Carbamidomethyl was entered as a fixed modification, and oxidation of methionine was entered as a variable modification. The mass tolerances of the precursor and product ions were 10 ppm and 0.2 Da, respectively, and the instrument setting was specified as ESI-QUAD-TOF. The significance threshold (p) was set at 0.01, the ion score expect cut-off at 0.005. Mascot results were parsed using MS Data Miner v. 1.3 []. Mascot Distiller 2.5.10 was used for the area-based XIC quantification and the mascot search was performed using the same settings as for protein identification above except that the default average (MD) quantitation protocol was selected, number of peptides used for quantitation was 3, matched rho was 0.8, XIC threshold was 0.3 and isolated precursor threshold was set at 0.7. The area-based XIC quantification was based on the average intensity of the three most abundant peptides per protein. […]

Pipeline specifications

Software tools Analyst TF, ProteinPilot, MS Data Miner, Mascot Distiller
Application MS-based untargeted proteomics
Organisms Mus musculus, Homo sapiens
Diseases Alzheimer Disease, Dementia