Scaffold 5

GR-AF1-DBD +/− phosphorylation samples were reduced, alkylated, digested with trypsin, and purified with C18 stage tips (Pierce, Dallas, TX, USA, #87781) [24 (link),25 (link)]. Peptides were separated by LC and analyzed on a QExactive HF Orbitrap (ThermoFisher, Waltham, MA, USA) mass spectrometer. Fully phosphorylated GR and singly phosphorylated GR were compared to unmodified GR-AF1-DBD using Scaffold 5.1.2 (Proteome Software Inc., Portland, OR, USA). Phospho-fragment frequencies were compared to the control to determine the level of phosphorylation at each site.

Mass spectrometry was performed using a commissioned analysis service provided by MS Bioworks (Ann Arbor, MI). Briefly, Myc- or HA-tagged proteins indicated were transiently expressed in HeLa cells, and immunoprecipitated with anti-Myc or anti-HA antibodies and magnetic beads. Proteins on the beads were eluted using 2 × sample buffer and submitted to MS Bioworks in freezing conditions. Samples were resolved in SDS-PAGE and then digested in the gel by trypsin before LC–MS/MS. Protein identification was performed by MASCOT software (Matrix Science) and analysed by Scaffold 5 (Proteome Software). Contaminants such as keratin, immunoglobulins, and trypsin were excluded from the analysis.

For peptide identification, MS/MS data were searched against the Uniprot database (UP000005640, Taxonomy Homo sapiens, 78,120 entries) using the Byonic algorithm (version 2.16.2 Protein Metrics Inc., Cupertino, CA, USA). A maximum mass deviation of 15 ppm was applied for precursor ions, whereas for product ions, max. 0.6 Da (linear ion trap data) and 30 ppm (orbitrap data) were allowed. Oxidation of Met, acetylation of protein N-termini, and phosphorylation of S, T and Y were set as variable modifications, and carbamidomethylation of cysteines modification of peptide N-termini as well as modifications of Lys by the TMT label were included as fixed modifications. A maximum of two missed cleavage sites were considered for peptides and allowed for semi-specific digestion. Automatic score cut for peptide output and protein FDR was left for downstream processing (as recommended). Individual results files were exported as mzIdentML files with associated MGF spectra files, and imported into Scaffold 5 (Proteome Software Inc., Portland, OR, USA). Proteins were accepted at 95% FDR threshold for quantification. Quantification was performed using the TMT reporter ion ratio derived from the FT HCD spectra.

10 ng of AGO2 protein from each RISC, as quantified by titration filter binding, was denatured with 4X LDS sample buffer (Invitrogen, NP0007) and run on an SDS-polyacrylamide gel (NuPAGE 4 to 12%, Bis-Tris [Invitrogen, NP0322]) in MOPS buffer (Invitrogen, NP0001), according to manufacturer’s instructions. Gels were stained with Imperial Blue Stain (Thermo Scientific, 24615). Bands corresponding to AGO2 were excised and cut into pieces, then stored in 50% (v./v.) methanol. Proteins were eluted, digested with trypsin, and run on the Thermo Scientific Orbitrap Elite mass spectrometer with a Waters NanoAcuity UPLC system. Alignment of peptides and identification of modifications were carried out using either the Proteome Discoverer (Thermo Fisher) or Mascot (Matrix Science) software, followed by Scaffold 5 (Proteome Software). The label-free total ion chromatogram (TIC) signal for each assigned peak was used for relative quantitative analyses. Fraction of phosphorylation at S387 was estimated based on quantification of the “SAS³⁸⁷FNTDPYVR” peptide observed.

To verify if the apparently N-terminal extended dominant TnI proteins present in catshark and Greenland shark as well as African lungfish corresponded with the predicted TNNI5 or TNNI3 sequences, protein identification with liquid chromatography-mass spectrometry (LC-MS) was performed with the University of Manchester Bio-MS Research Core Facility (RRID SCR_020987). 20 µg protein per lane was run on a 16% acrylamide Tris-glycine gel (Invitrogen XP00165BOX) which was then stained with SimplyBlue SafeStain (ThermoFisher LC6065). The band at the location corresponding with TnI identified by the immunoblot was excised and digested with elastase. The samples were analysed with LC-MS/MS using an UltiMate 3000 Rapid Separation LC (RSLC, Dionex Corporation, Sunnyvale, CA) coupled to an Orbitrap Exploris 480 (Thermo Fisher Scientific, Waltham, MA) mass spectrometer. Mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. The products were analyzed with Scaffold 5 (Proteome Software, Portland, OR, USA) and searched against an in-house database including the transcriptomics-predicted TNNI3 and TNNI5 sequences from each respective species.