DOI: 10.6084/m9.figshare.24187128
title: Native lamin A/C brain proteome and novel partners in a mouse chronic inflammation model of human frailty

description: <b>Tandem Mass Tag (TMT) proteomics</b>Protein extracts (40 ug total at 1 ug/ul in 1% SDS) were reduced with 15 uL of 15 mM DTT for 1 hour at 56°C, alkylated by adding 15 uL 100 mM iodoacetamide and incubating in the dark for 30 min, and then TCA/Acetone precipitated. The protein pellet from each sample was digested overnight at 37°C by adding 100 uL Trypsin/LysC mixture (40 ug proteases in 1.2 mL of 100 mM triethylammonium bicarbonate (TEAB; Promega #V5071) or approximately 3.33 ug protease per sample. Individual samples (40 ug) were labeled with a unique isobaric mass tag reagent (TMT 10-plex, Thermo Scientific) according to manufacturer instructions. Both pairing and labeling order of TMT reagent and peptide sample were randomized. Briefly, the TMT reagents (0.8 ug vials) were allowed to come to room temperature before adding 41 uL anhydrous acetonitrile, then vortexed and centrifuged. The entire TMT reagent vial was added to the 100 ug peptide sample and reacted at room temperature for 1 hour. The reaction was quenched by adding hydroxylamine (8 uL) to a final concentration of 5%. All TMT-labeled samples were combined and vacuum centrifuged to dryness removing the entire liquid.<i>Basic Reverse Phase (bRP) fractionation</i>— Labeled peptide samples were fractionated by basic reverse phase (bRP) chromatography on Oasis HLB uElution plates (Waters). TMT labeled peptides (5%, approximately 20 ug) were bound to HLB resin in 10 mM triethylammonium bicarbonate (TEAB) buffer and step eluted with 0%, 5%, 10%, 25%, and 75% acetonitrile in 10 mM TEAB (0 and 5% fractions were combined). Fractions were dried by vacuum centrifugation.<i>Mass Spectrometry Analysis—</i><b> </b>The peptide fractions were resuspended in 20 uL 2% acetonitrile in 0.1% formic acid; approximately 0.5 ug (2 uL) was loaded onto a C18 trap (S-10 uM, 120Å, 75 um x 2 cm; YMC Co., LTD, Kyoto, Japan) and then separated on an in-house packed PicoFrit column (75 um x 200 mm, 15 um, +/-1 um tip, New Objective) with C18 phase (ReproSil-Pur C18-AQ, 3 um, 120Å, www.dr-maisch.com) using 2-90% acetonitrile gradient at 300 nL/min over 120 min on a EasyLC nanoLC 1000 (Thermo Scientific). Eluting peptides were sprayed at 2.0 kV directly into an Orbitrap Fusion Lumos (Thermo Scientific) mass spectrometer. Survey scans (full ms) were acquired from 360-1700 m/z with a cycle time of 3 sec. Precursor ions isolated in a 0.7 Da window and fragmented using HCD activation collision energy 39 and 15s dynamic exclusion, with a scan range of 116m/z-2000m/z. Precursor and fragment ions were analyzed at resolutions 120,000 and 30,000, respectively, with automatic gain control (AGC) target values at 4 x 10<sup>5</sup> with 50 ms maximum injection time (IT) and 1 x 10<sup>5</sup> with 118ms maximum IT, respectively.<i>Data analysis—</i> Isotopically resolved masses in precursor (MS) and fragmentation (MS/MS) spectra were extracted from raw MS data using spectrum selector with recalibration in Proteome Discoverer (PD) software (version 2.4.0.305, Thermo Scientific) and searched using Mascot (2.6.2; www.matrixscience.com) against a Mus musculus protein database (RefSeq2017_83, created 5/23/2019, containing 76,508 sequences. The following criteria were set for all database searches: (a) all species in database; (b) trypsin as the enzyme, (c) two missed cleavages allowed; (d) N-terminal TMT6plex and cysteine carbamidomethylation as fixed modifications; (e) lysine TMT6plex, methionine oxidation, serine, threonine and tyrosine phosphorylation, asparagine and glutamine deamidation, HexNAc on serine or threonine, as variable modifications; and (f) precursor and fragment ion tolerances were set to 5ppm and 0.03Da, respectively. Peptide identifications from Mascot searches were filtered at 5% False Discovery Rate (FDR) confidence threshold, based on a concatenated decoy database search, using the Proteome Discoverer. Proteome Discoverer uses only the peptide identifications with the highest Mascot score for the same peptide matched spectrum from the different extraction methods. The protein intensities were reported as S/N of each peptide and relative protein comparisons were calculated using the peptide grouping in Proteome Discoverer. Quan value correction factors were used (Lot TK271715) with a co-isolation threshold of 30. Peptide abundances were normalized against a custom sequence .FASTA file containing only prelamin A (XP_006501136.1 PREDICTED: prelamin-A/C isoform X1 [Mus musculus]) to ensure there was no experimental bias in protein quantification that depended on the total amount of lamin A/C immunoprecipitated from each sample.