Proteomics and phosphoproteomics datasets of a muscle-specific STIM1 loss-of-function mouse model

Abstract

STIM1 is an ER/SR transmembrane protein that interacts with ORAI1 to activate store operated Ca²⁺ entry (SOCE) upon ER/SR depletion of calcium. Normally highly expressed in skeletal muscle, STIM1 deficiency causes significant changes to mitochondrial ultrastructure that do not occur with loss of ORAI1 or other components of SOCE. The datasets in this article are from large-scale proteomics and phosphoproteomics experiments in an inducible mouse model of skeletal muscle-specific STIM1 knock out (KO). These data reveal statistically significant changes in the relative abundance of specific proteins and sites of protein phosphorylation in STIM1 KO gastrocnemius. Protein samples from five biological replicates of each condition (+/- STIM1) were enzymatically digested, the resulting peptides labeled with tandem mass tag (TMT) reagents, mixed, and fractionated. Phosphopeptides were enriched and a small amount of each input retained for protein abundance analysis. All phosphopeptide and input fractions were analyzed by nano LC-MS/MS on a Q Exactive Plus Orbitrap mass spectrometer, searched with Proteome Discoverer software, and processed with in-house R-scripts for data normalization and statistical analysis. Article published in Molecular Metabolism [1].

Keywords: Calcium homeostasis; Isobaric tags; Mass spectrometry; PTM normalization; Protein abundance; Protein phosphorylation; R script.

Fig. 1

Proteomics workflow and key data metrics. Mice with skeletal muscle-specific loss of STIM1 (iStim^−/−, KO) and control animals (STIM1^fl/fl) were sacrificed after an overnight fast and gastrocnemius muscles were collected. Proteins were extracted from five biological replicate samples, enzymatically digested, the resulting peptides labeled with unique tandem mass tag (TMT) reagents. Samples were mixed and fractionated by high-pH reversed phase (HPRP) chromatography. Phosphopeptides were enriched by immobilized metal affinity chromatography (IMAC) after a small amount of the input material retained from each fraction for assessment of protein abundance. All phosphopeptide and input fractions were analyzed by nano LC-MS/MS on a Q Exactive Plus (QE+) Orbitrap mass spectrometer, searched with Proteome Discoverer software, and processed with in-house R-scripts for normalization of quantitative values and analysis of statistical significance. For each measurement type, the numbers quantified and statistically significant are indicated. Article published in Molecular Metabolism.

Fig. 2

Sample loading normalization with custom R scripts. Log₂ intensity of each TMT channel for all protein relative abundance measurements before (A-B) and after (C-D) normalization for any subtle (and unintended) deviations in equal peptide input. The data are displayed as both density plots (A, C) and box-and-whisker plots (B, D). Phosphopeptide quantitative values (not shown) were corrected with the same loading normalization factors.

Fig. 3

Correlation between phosphopeptide abundance and relative occupancy. Scatter plot of phosphopeptide relative abundance on the vertical axis and the phosphopeptide relative occupancy on the y axis for the same protein phosphorylation sites (both measurements expressed as log₂ KO/WT). Red dots represent phosphosites that only exhibited a statistically significant (10% FDR) change when considering phosphopeptide abundance, whereas blue indicates sites only changing by phosphopeptide relative occupancy, and site changing by both metrics are shown in purple. Sites that were not significant (N.S) are shown in grey (also slightly transparent to better visualize statically significant sites behind them). Phosphopeptides for which a corresponding protein was not measured are not shown.