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QXL-MS of insulin resistance in C2C12 myotubes

ABSTRACT: C2C12 myotubes were made insulin resistant with 0.5 mM palmitic acid for 24 h along with non-treated controls and then each group stimulated with or without 100 nM insulin for 30 min (n=4 each). Cells were then cross-linked with 2 mM of tPhoX for a further 30 min in the presence of insulin and the reaction quenched as previously described (Jiang et al., 2022). To increase the depth of analysis, each sample was subjected to a crude biochemical-based separation into four fractions (Wong et al., 2025) followed by digestion with trypsin/LysC. The 16 samples from each fraction were labelled with 16-plex Tandem Mass Tags (TMT) and pooled, followed by the depletion of phosphopeptides and enrichment of XL-peptides using immobilised metal-ion affinity chromatography (IMAC). Each of the four biochemical fractions underwent peptide-level size-exclusion chromatography (SEC) to partially fractionate larger XL-peptides from mono-/loop-linked peptides in 2-3 fractions which were further separated by high pH reversed-phase chromatography into 12 fractions and analysed by LC-MS/MS using data-dependent acquisition (DDA) resulting in the analysis of 132 individual runs. Data were analysed with pLink2 and searched against a skeletal muscle-specific mouse proteome database to limit the search space with filtering to 1% FDR followed by reporter-ion quantification and statistical analysis

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Benjamin Parker

LAB HEAD: Benjamin Parker

PROVIDER: PXD077371 | Pride | 2026-05-29

REPOSITORIES: Pride

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			Action	DRS
	240417_Eclipse_BP-8445_XL_P1_B1.raw	Raw
	240417_Eclipse_BP-8445_XL_P1_B10.raw	Raw
	240417_Eclipse_BP-8445_XL_P1_B11.raw	Raw
	240417_Eclipse_BP-8445_XL_P1_B12.raw	Raw
	240417_Eclipse_BP-8445_XL_P1_B2.raw	Raw

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