Stagetips c18

Protein identification was performed by mass spectrometry (MS) analysis [25 ]. Selected protein bands, found exclusively in samples containing NPs, were manually excised from the SDS-PAGE gel. Spots were dissected to smaller pieces and de-stained with 1:1 volume ratio of 30 mM potassium ferricyanide and 100 mM sodium thiosulfate solution. Gel pieces were incubated in 200 mM NH₄HCO₃ solution for 20 min, washed two times with LC-MS Ultra CHROMASOLV^® water (Sigma-Aldrich, St. Luis, Missouri, USA), and dehydrated by covering with 100% acetonitrile. Reduction of proteins was performed with 10 mM dithiothreitol solution at 56°C for 45 min and alkylated by 55 mM iodoacetamide at room temperature for 30 min. Alkylation was stopped by addition of 25 mM NH₄HCO₃. Proteins were digested in gel with the MS grade modified trypsin (Sigma-Aldrich, Sigma-Aldrich, St. Luis, Missouri, USA) in 25 mM NH₄HCO₃ at 37°C overnight. Resulting peptides were extracted with 50% (v/v) acetonitrile / 5% (v/v) formic acid and concentrated in vacuum to 10 μL. Extracts were purified on StageTips C18 (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA) according to the manufacturer's instructions and analysed using an electrospray ionization (ESI) ion trap-MS (MSD Trap XCT Plus, Agilent, USA) as described in Leonardi et al. [41 (link)].

A total of 150 mg of trypsinized peptides per genotype were pooled and enriched for pTyrosine, as previously described⁵² . Lyophilized peptides were mixed in 1.4 ml immunoprecipitation buffer (IAP buffer 50 mM MOPS, pH 7.2, 10 mM sodium phosphate, 50 mM NaCl, pH 7.0). A stock solution of protein agarose G beads (Santa Cruz Biotechnology) 80 μL of slurry were conjugated with 300 μg of Phospho-Tyrosine Mouse monoclonal antibody (p-Tyr-100, Cell Signaling Technology). The beads-Anti-p-Tyr antibody conjugate was transferred to the peptide’s tube and incubated with gentle rotation for 2 h at 4 °C. The beads were washed and eluted with 55 μl and 45 μl of 0.15% trifluoroacetic acid (TFA), respectively. The two elution yields were pooled. The resulting peptide mixtures were purified by solid-phase extraction (stage tips C18, Thermo Scientific). The samples were dried by vacuum centrifugation. This approach was repeated three times with samples from all three groups run in parallel, 15 hearts per genotype in three technical replicates. The immunoprecipitation buffer flow-through was stored at −80 °C for subsequent TMT 9-plex. According to the manufacturer’s instructions, tryptic peptides were desalted and labeled with 9-plex isobaric TMT (Thermo Scientific). The labeling reaction was carried out for 1 h at room temperature, followed by quenching with 100 mM Tris.HCl (pH 8.0).

sBCMA was purified by immunoprecipitation and obtained by acidic elution. The eluate was then desalted and concentrated using StageTips, C18, microcolumns (Thermo Scientific, Bremen, Germany). Then two approaches were followed. (A) The material was digested in solution by trypsin or chymotrypsin and analysed using mass spectrometry (LTQ Orbitrap XL) as described before50 (link). (B) The immunoprecipitated material was separated via an SDS gel, silver-stained and the band corresponding to BCMA as detected using western blot analysis was excised and analysed using mass spectrometry (LTQ Orbitrap XL).