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MudPIT analyses of the proteins associated with FBXW5 in HEK293T cells

ABSTRACT: Nutrient deprivation of the cell mobilizes an extensive amount of membrane to form and grow the autophagosome, allowing the progression of autophagy. By providing membranes and stimulating LC3 lipidation, Coat Protein Complex II (COPII) promotes autophagosome biogenesis. Here, we show that the F-box protein FBXW5 targets SEC23B, a component of COPII, for proteasomal degradation and that this event limits the autophagic flux in the presence of nutrients. In brief, TCA-precipitated protein eluted after Streptag-FLAG (SF)-FBXW5 affinity purification were urea-denatured, reduced, alkylated, and digested with endoproteinase LysC followed by trypsin. The resulting peptide mixtures were loaded onto tri-phasic microcapillary fused silica columns (100um i.d.), packed with C18 reverse phase (Aqua; Phenomenx), SCX (Luna; Phenomenex) and C18-RP, placed in-line with an Agilent 11000 quaternary pump, and analyzed by a 10-step MudPIT on linear ion traps. MS/MS datasets were searched using SEQUEST (v. 27.9) against a non-redundant human protein database (NCBI, 2010-11-22) containing 160 usual contaminants. To estimate false discover rates (FDRs), the amino acid sequence of each non-redundant protein was randomized. Peptide/spectrum matches were sorted and selected using DTASelect with the following criteria set: spectra/peptide matches were retained only if they had a DeltCn of at least 0.8, and minimum XCorr of 1.8 for singly, 2.0 for doubly, and 3.0 for triply charged spectra. Additionally, the peptides had to be minimum 7 amino acids in length and fully tryptic. MudPIT analysis revealed the presence of peptides corresponding to FBXW5, SKP1 and CUL1, as well as 15 unique peptides derived from the COPII coat subunit SEC23B.

INSTRUMENT(S): LTQ (Thermo Scientific instrument model)

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Anita Saraf

PROVIDER: MSV000083289 | MassIVE | Sat Dec 29 07:18:00 GMT 2018

SECONDARY ACCESSION(S): PXD012197

REPOSITORIES: MassIVE

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MudPIT analyses of the proteins associated with FBXW5 in HEK293T cells

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