Pierce high ph reverse phase peptide fractionation kit

Equal amounts of peptides from the five infected mice and the five control mice for each experiment were labeled with the TMT10plex™ Isobaric Mass Tagging kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Equal amounts of the 10 labeled samples were mixed and fractionated in eight samples using a Pierce™ High pH Reverse-Phase Peptide Fractionation Kit (Thermo Fisher Scientific). The peptide concentration in each fraction was measured and equal amounts of fractions, between 0.35 and 1 µg, were injected for each experiment. Two technical replicas were analyzed.

The hMSC 2D and 3D EVs were isolated using ExtraPEG and then extracted for proteins. Based on protein quantification result, up to 100‐μg proteins were isolated on an S‐trap micro column (Protifi, Farmingdale, NY, USA, K02‐micro). The isolated proteins (triplicate for each group) were alkylated and digested on column based on manufacturer's instructions. All the eluted peptides were fractionated by Pierce high pH reverse phase peptide fractionation kit (ThermoFisher Scientific, 84868) into five fractions for each sample. Then all the samples were vacuumed dried and submitted to the FSU Translational Science Laboratory. The samples were analysed on the Q Exactive HF Orbitrap LC‐MS/MS System (ThermoFisher Scietific) as previously described (Hurwitz & Meckes, 2017 (link); Hurwitz et al., 2018 (link)). Briefly, resulting raw files were searched with Proteome Discoverer 2.4 using SequestHT, Mascot and Amanda as search engines. Scaffold (version 5.0) was used to validate the protein and peptide identity. Peptide identity was accepted if Scaffold Local FDR algorithm demonstrated a probability greater that 99.0%. Likewise, protein identity was accepted if the probability level was greater than 99.0% and contained a minimum of two recognized peptides. GO annotation was carried out by g:Profiler.

TMT-labeled samples were reconstituted in 0.1% TFA and the pH is adjusted to 2 with 10%. The combined sample (20 µg) was separated into eight fractions by Pierce High pH Reverse-Phase peptide Fractionation kit (Thermo Scientific) with an extra wash before separation to remove extra labels. The eight fractions were dried almost to completion.

TMT label reagents were allowed to equilibrate to room temperature (RT) and anhydrous acetonitrile was added to each tube. 21.6 µL of the TMT label reagents were added to each 20 µg of samples and were incubated for 1 hr at RT. The reaction was quenched by adding 8 µL of 5% hydroxylamine to the labeled samples and were incubated for 30 minutes at RT. All TMT labeled samples were combined in equal amounts in a new tube (200 µg) and were run on Mass spec after fractionation.
TMT labeled samples were then reconstituted in 0.1% TFA and the pH adjusted to 2 with 10% TFA^{17 (link)}. The combined sample (200 µg) was separated into 8 fractions by Pierce High pH Reverse-Phase Peptide Fractionation kit (Thermo Scientific) with an extra wash before separation to remove extra labels^{17 (link)}. The eight fractions were dried almost to completion.