Fig. 2: Workflow for global whole heart TMT phospho-tyrosine proteomics profiling.

a Ventricular tissues were rinsed with PBS and subjected to Heat Stabilization using a T1 Tissue Stabilizor to maximize phosphorylation preservation, then snap-frozen in liquid nitrogen and stored at −80 °C until further use. b Ntg, TgErbB2, and R403Q-αMyHC mice hearts (n = 5 per group) were processed in parallel to obtain whole heart lysates; 30 mg of protein lysates were used from each mouse heart for in-solution trypsin digestion, a total of 150 mg of trypsinized protein per genotype were C18 desalted and lyophilized at −20 °C for three days. c A total of 150 mg of tryptic peptides (pooled material from 5 hearts per group) were enriched for pTyrosine using immunoaffinity precipitation (Protein Agarose Beads + 300 micrograms of mAb Anti-pTyr) and desalted on C18 stage tips. d RP-HPLC ESI (electrospray ionization) MS/MS was performed on an LTQ Orbitrap Elite (Thermo Scientific) for a 120 min on a linear acetonitrile gradient (4−40%). Raw data were searched with Mascot 2.3, and label-free quantification with MS1 extracted ion chromatograms were performed using MaxQuant. This approach was repeated three times with samples from all three groups running parallel; 15 hearts per genotype in three technical replicates. e A fraction of the flow-through of pTyr enrichment was used for a 9-plexTMT; three replicates of each genotype (Ntg, TgErbB2, and R403Q-αMyHC) were pooled, fractionated in RPLC for full proteome quantification, f Data from pTyrosine and Full Proteome were processed in a similar way, pTyrosine signals were normalized to matched total protein expression to normalize data. Analyses are detailed in the text and supplemental data. g Shows the overlap of phosphorylated proteins found in the three groups, whereas h shows the overlapping tyrosine-phosphorylated peptides among the three groups.