Computational protocol: Comparative Proteomics of Milk Fat Globule Membrane Proteins from Transgenic Cloned Cattle

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Protocol publication

[…] LC-MS/MS was performed on a nano Acquity UPLC system (Waters Corporation) connected to an LTQ Orbitrap XL mass spectrometer (Thermo Electron Corp., Bremen, Germany) equipped with an online nano-electrospray ion source (Michrom Bioresources, Auburn, USA). The MS was operated in data-dependent mode to switch automatically between MS and MS/MS acquisition. Survey full-scan MS spectra (m/z 300–1800) were acquired in the Obitrap with one microscan and with a mass resolution of 60,000 at m/z 400, followed by MS/MS of the eight most-intense peptide ions in the LTQ analyzer. LC-MS/MS spectra were acquired using SEQUEST (v.28 revision 12, Thermo Electron Corp) against the International Protein Index bovine database version 3.54. A decoy database containing the reverse sequences was appended to the database. The search parameters were set as follows: partial trypsin cleavage with two missed cleavage sites was considered; the peptide mass tolerance was 1.4 Da; and the fragment ion tolerance was 1 Da. Trans Proteomic Pipeline software (revision 4.20; Institute of Systems Biology, Seattle, WA) was then used to identify proteins based on corresponding peptide sequences with ≥95% confidence. The peptide results were filtered by PeptideProphet with a p value>0.90 and a ProteinProphet probability = 0.95 , . […]

Pipeline specifications

Software tools Comet, TPP, PeptideProphet, ProteinProphet
Databases IPI
Application MS-based untargeted proteomics
Organisms Bos taurus, Homo sapiens
Chemicals Silver