Proteomics Analysis of Protein Complexes

Proteomics was performed as previously described (45 (link)), with a few modifications. A single gel slice for each pulldown was subjected to in-gel tryptic digestion using a ProGest automated digestion unit (Digilab UK). The resulting peptides were fractionated using a Dionex Ultimate 3000 nanoHPLC system in line with an LTQ-Orbitrap Velos mass spectrometer controlled by Xcalibur 2.1 software (Thermo Scientific) operated in data-dependent acquisition mode. The raw data files were processed and quantified using Proteome Discoverer software v1.2 (Thermo Scientific) and searched against the UniProt Human database (122604 sequences) using the SEQUEST (Ver. 28 Rev. 13) algorithm. The reverse database search option was enabled, and all peptide data were filtered to satisfy a false discovery rate of 5%.

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Battram A.M., Durrant T.N., Agbani E.O., Heesom K.J., Paul D.S., Piatt R., Poole A.W., Cullen P.J., Bergmeier W., Moore S.F, & Hers I. (2016). The Phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) Binder Rasa3 Regulates Phosphoinositide 3-kinase (PI3K)-dependent Integrin αIIbβ3 Outside-in Signaling. The Journal of Biological Chemistry, 292(5), 1691-1704.

Publication 2016

LTQ-Orbitrap Velos Xcalibur 2.1 Proteome Discoverer software v1.2

Digestion Human Peptide Progest Proteome Tryptic

Corresponding Organization : University of Bristol

Other organizations : University of North Carolina at Chapel Hill

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Variable analysis

independent variables

Proteomics was performed

dependent variables

Identified and quantified proteins
Protein abundance

control variables

Gel slice for each pulldown
In-gel tryptic digestion using a ProGest automated digestion unit
Dionex Ultimate 3000 nanoHPLC system in line with an LTQ-Orbitrap Velos mass spectrometer
Xcalibur 2.1 software (Thermo Scientific) operated in data-dependent acquisition mode
Proteome Discoverer software v1.2 (Thermo Scientific)
UniProt Human database (122604 sequences)
SEQUEST (Ver. 28 Rev. 13) algorithm
Reverse database search option
False discovery rate of 5%

controls

Reverse database search option

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