Easy nanolc

Interacting proteins were identified and quantified by nano-LC-MS/MS (Liquid Chromatography tandem Mass Spectrometry), as previously described^{58 (link)}. Briefly, in-gel digestion was performed using trypsin and resulting peptides were C18-purified and analyzed on an EASY nanoLC (on a 25 cm C18 Pepmap column from Thermo) coupled to a Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific). Mass resolution was 70 000 and 17 500, for peptide and fragment scans, respectively. MaxQuant software version 1.5.2.8 was applied for protein identification (using 20 197 Homo sapiens sequences from Uniprot, August 2015), and label-free quantification by means of peptide peak areas^{60 (link)}. False discovery rate (FDR) was set to 0.01 for both protein and peptide identification. Proteins were considered significant only if the protein was present in all 3 replicates of LINC00958 pull-down and displayed an overall fold enrichment over controls greater than 3.

The protein expression levels obtained from the TMT analysis were confirmed by quantifying the expression levels of five selected proteins using Parallel Reaction Monitoring (PRM) analysis. Unique peptides from the target proteins were defined according to the TMT data. The proteins (50 µg) were prepared and digested following the TMT analysis protocol. The obtained peptide mixtures were analyzed by nano LC-PRM MS using easy nano-LC (Thermo Fisher Scientific) coupled to a Q Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometer (Thermo Fisher Scientific). The raw data were processed using Skyline 2.6, with the cut-off value set to 0.99. The five product ions with the highest signal intensities were allowed to enter each peptide segment for analysis. Each peptide segment was manually integrated, and the results were exported for data analysis.

For the metaproteomic analyses, filtration is used to separate extracellular peptides from the obtained (poly)peptides. The resulting smaller fractions are then desalted and analysed without proteolytic digestion via liquid chromatography (LC) and mass spectrometry (MS) on an EasyNano-LC coupled online to a QExactive-Plus mass spectrometer (ThermoScientific, Waltham). The identification of ribosomal peptides is done with an integrated catalogue of MG and MT data, while the non-ribosomal peptides are identified using different tools, that is, MyriMatch, DirecTag as well as CycloBranch.45 59 60 (link) The metaproteomic data also allow identification of extracellular (poly)peptides with possible pathogenic functions including protein misfolding and molecular mimicry.61 62 (link)

Eluted light and heavy proteins (64 µl) were diluted with 16 µl of 100 mM Tris buffer (pH 8.5) and reduced with 0.5 µl of 0.5 M Tris(2-carboxyethyl)phosphine hydrochloride (Thermo Fisher Scientific, Waltham, MA) for 20 min at 37°C, alkylated with 1.8 µl of 0.5 M chloroacetamide (Thermo Fisher Scientific) for 15 min at 37°C, and digested with 2 µl of 100 ng/µl lysyl endopeptidase (Lys-C, Wako Chemicals, Richmond, VA) for 4 hr at 37°C. Samples were diluted to a final concentration of 2 M urea by adding 240 µl of 100 mM Tris–HCl pH 8.5 and digested with 32 µl of 100 ng/µl trypsin (Thermo Fisher Scientific) and 3.2 µl 100 mM CaCl₂ for 18 hr at 37°C. After desalting with a Vivapure C18 micro spin column (Sartorius Stedim Biotech, Bohemia, NY), peptides were eluted with 50 µl of 75% acetonitrile and 0.2% formic acid twice. Solvent was removed using a SpeedVac. Dried samples were acidified by 0.2% formic acid and loaded onto an Easy Nano-LC connected to a hybrid LTQ-Orbitrap Classic (Thermo Fisher Scientific) as previously described (de Godoy et al., 2008 (link); Lee et al., 2011 (link)).