Comprehensive Lung Proteome Extraction

Lungs were perfused with PBS through the heart to remove blood. Then, ∼100 mg of total lung tissue (wet weight) was homogenized in 500 μl PBS (with protease inhibitor cocktail and EDTA) using an Ultra-turrax homogenizer. After centrifugation, the soluble proteins were collected and proteins were extracted from the insoluble pellet in three steps using buffers with increasing stringency [buffer 1: 150 mM NaCl, 50 mM Tris–HCl (pH 7.5), 5% glycerol, 1% IGEPAL® CA-630 (Sigma, #I8896), 1 mM MgCl₂, 1× protease inhibitors (+EDTA), 1% benzonase (Merck, #70746-3), 1× phosphatase inhibitors (Roche, #04906837001); buffer 2: 50 mM Tris–HCl (pH 7.5), 5% glycerol, 150 mM NaCl, fresh protease inhibitor tablet (+EDTA), 1.0% IGEPAL® CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 1% benzonase (Merck, #70746-3); and buffer 3: 50 mM Tris–HCl (pH 7.5), 5% glycerol, 500 mM NaCl, protease inhibitor tablet (+EDTA), 1.0% IGEPAL® CA-630, 2% sodium deoxycholate, 1% SDS, 1% benzonase (Merck, #70746-3)]. Insoluble pellets were resuspended in detergent-containing buffers and incubated for 20 min on ice (except for buffer 3, which was used at room temperature), followed by separation of soluble and insoluble material using centrifugation for 20 min at 16,000 g. The PBS from the tissue homogenate and the NP40-soluble fraction (buffer 1) was pooled, which together with the two fractions derived from ionic detergent extraction (buffers 2 and 3) resulted in a total of three soluble fractions and one insoluble pellet that were subjected to LC-MS/MS analysis. Soluble proteins were precipitated with 80% acetone and subjected to in-solution digestion using a modified published protocol (Kulak et al, 2014 (link)). In brief, protein reduction (10 mM TCEP) and alkylation (50 mM CAA) were performed at once in 6 M guanidinium hydrochloride (100 mM Tris–HCl pH 8.5) at 99°C for 15 min. Subsequent protein digestion was done in two steps. The first digestion was done at 37°C for 2 h with LysC (1:50 enzyme to protein ratio) in 10 mM Tris–HCl (pH 8.5) containing 2 M guanidinium hydrochloride (Gdm), 2.7 M urea, and 3% acetonitrile. The second digestion step was done using fresh LysC (1:50 enzyme to protein ratio) and trypsin (1:20 enzyme to protein ratio) in 600 mM Gdm, 800 mM urea, and 3% acetonitrile at 37°C overnight. For the insoluble protein pellet, which is strongly enriched for insoluble ECM proteins, we optimized the in-solution digestion protocol with additional steps involving extensive mechanical disintegration and ultrasonication-aided digestion. The insoluble material was cooked, reduced, and alkylated in 6 M Gdm for 15 min and then subjected to 200 strokes in a micro-Dounce device, which reduced the particle size of the insoluble protein meshwork. We then proceeded with the two-step digestion protocol described above, which was additionally aided by 15-min ultrasonication (Bioruptor, Diagenode) in the presence of the enzymes in both digestion steps.
For tissue proteome time course analysis, a similar sequential extraction procedure as described above was used. However, in these experiments, we employed slightly different buffers for extraction following a commercially available protein extraction kit (Compartment Protein Extraction Kit, Millipore). We collected three protein fractions for LC-MS analysis (two soluble fractions and one insoluble fraction). The first fraction measured was derived from proteins soluble in buffer M of the extraction kit [HEPES (pH 7.9), MgCl₂, KCl, EDTA, sucrose, glycerol, sodium deoxycholate, NP-40, sodium orthovanadate]; the second fraction was derived from proteins soluble in buffer CS [PIPES (pH 6.8), MgCl₂, NaCl, EDTA, sucrose, SDS, sodium orthovanadate], and finally, we also analyzed the proteins insoluble in buffer CS as described above for QDSP. To perform relative quantification of full proteomes in the various conditions, we summed up the peptide intensities of the three protein fractions in MaxQuant (Cox & Mann, 2008 (link)).
Peptides were purified using stage tips containing a poly-styrene-divinylbenzene copolymer modified with sulfonic acid groups (SDB-RPS) material (3M, St. Paul, MN, USA) as previously described (Kulak et al, 2014 (link)). For the QDSP experiments, we separated peptides in two fractions by sequentially eluting from the SDB-RPS stage tip material (buffer 1: 150 mM NH₄HCO₂, 60% acetonitrile, 0.5% FA; buffer 2: 5% ammonia and 80% acetonitrile).