1
2
3
4
5
|
<p><strong>Sample processing protocol:</strong> The proteomic data were generated with 3 batches of 16-plex and 2 batches of 11-plex TMT experiments. The rat brain samples from 31 HXB/BXH strains with replicates (i.e., male and female) were lysed, digested, and labeled with either 11 or 16 different TMT tags. The TMT-labeled peptides were pooled with an equal amount of each and fractionated into 42 fractions in a concatenated fashion on an RP-HPLC column (4.6 mm x 250 mm) under basic pH conditions. each fraction was run sequentially on a column (75 μm x 20 cm for the whole proteome, 50 μm x ∼30 cm for phosphoproteome, 1.9 μm C18 resin from Dr. Maisch GmbH, 65°C to reduce backpressure) interfaced with a Q Exactive HF Orbitrap or Fusion MS (Thermo Fisher). Peptides were eluted by a 2-3 hr gradient (buffer A: 0.2% formic acid, 5% DMSO; buffer B: buffer A plus 65% acetonitrile). MS settings included the MS1 scan (410-1600 m/z, 60,000 or 120,000 resolution, 1 × 106 AGC and 50 ms maximal ion time) and 20 data-dependent MS2 scans (fixed first mass of 120 m/z, 60,000 resolution, 1 × 105 AGC, 100-150 ms maximal ion time, HCD, 35%–38% normalized collision energy, ∼1.0 m/z isolation window). </p>
<p><strong>Data processing protocol:</strong> The MS/MS raw files are processed using the JUMP searching engine against the UniProt mouse database. Searches were performed using 8 ppm mass tolerance for precursor ions due to JUMP’s auto mass correction function and 15 ppm for fragment ions, allowing up to two missed trypsin cleavage sites. TMT tags on lysine residues and peptide N termini (+229.162932 Da) were used for static modifications and the dynamic modifications include oxidation of methionine residues (+15.99492 Da). The assigned peptides are filtered by minimal peptide length, maximum miscleavages, mass-to-charge accuracy and matching scores. The peptides are then divided into groups according to peptide length, trypticity, modification, miscleavage, and charge and then further filtered by matching scores to reduce protein or phosphopeptide FDR to below 1%. Proteins or phosphopeptides were quantified by summing reporter ion counts across all matched PSMs using our in-house software.</p>
<p><strong>Protein quantification</strong>: We first extracted the TMT reporter ion intensities of each PSM and corrected the raw intensities based on the isotopic distribution of each labeling reagent. We discarded PSMs with low intensities (i.e., the minimum intensity of 1,000 and the median intensity of 5,000). After normalizing abundance with the trimmed median intensity of all PSMs, we calculated the mean-centered intensities across samples (e.g., relative intensities between each sample and the mean) and summarized protein relative intensities by averaging related PSMs. Finally, we derived protein absolute intensities by multiplying the relative intensities by the grand mean of the three most highly abundant PSMs. We first used the internal standard to normalize 3 batches of 16-plex experiments and 2 batches of 11-plex experiments. We then used the LIMMA batch removal function to normalize all five batches of TMT experiments. </p>
|
