At day 9, cells were stimulated for various time points with LPS (100ng/ml, rough, ultrapure E. coli K12 strain, Invitrogen) or MOCK (= no stimulation).
Growth protocol
GM-CSF-derived BMDCs were grown and selected in media containing medium- heavy L-arginine 13C6 (Arg6) and L-lysine 2H4 (Lys4) (Sigma). Just prior to LPS or MOCK stimulation, all cells were pooled, pelleted and re-suspended in media containing heavy L-arginine 13C6-15N4 (Arg10) and heavy L-lysine 13C6-15N2 (Lys8) (Sigma) and plated at 106 cells/ml on non-tissue culture treated Petri dishes. In parallel, GM-CSF derived BMDCs were grown in light L-arginine (Arg) and L-lysine (Lys) (Sigma) containing media. Concentrations for L-arginine and L-lysine were 42 mg/l and 40 mg/l, respectively. The cell culture media, RPMI-1640 deficient in L-arginine and L-lysine, was a custom media preparation from Caisson Laboratories (North Logan, UT) and dialyzed serum was obtained from SAFC-Sigma. We followed all standard SILAC media preparation and labeling steps as previously described (Ong and Mann, Nature Protocols, 2006).
Extracted molecule
protein
Extraction protocol
After stimulation (LPS or MOCK) and the appropriate time points, cells were washed twice with PBS and lysed for 30 min in ice-cold lysis urea buffer (8 M urea; 75 mM NaCl, 50 mM Tris HCl pH 8.0, 1 mM EDTA, 2 µg/mL aprotinin (Sigma, A6103), 10 µg/mL leupeptin (Roche, #11017101001), 1 mM PMSF (Sigma, 78830)). Lysates were centrifuged at 20,000g for 10 min, and protein concentrations of the clarified lysates were measured via BCA assay (Pierce). From this procedure, DC lysates produced ~100µg of protein per 1 million cells.
Label
Unlabeled
Label protocol
See growth protocol
Hybridization protocol
Light- standard and heavy/medium-labeled sample lysates were then combined in a 1:1 protein ratio (20µg each and therefore 40µg total). Protein disulfide bonds of the combined lysates were reduced for 45 min with 5 mM dithiothreitol (Thermo Scientific, 20291) and alkylated for 45 min with 10 mM iodoacetamide. Samples were then diluted 1:4 with 50 mM Tris HCl, pH 8.0, to reduce the urea concentration to <2 M. Lysates were digested overnight at room temperature with trypsin, or AspN, or LysN (depending on the experiment) in a 1:50 enzyme-to-substrate ratio (Promega, V511X) on a shaker. Peptide mixtures were acidified to a final volumetric concentration of 1% formic acid (Fluka, 56302) and centrifuged at 10,000g for 5 min to pellet urea that had precipitated out of solution. The peptide mixtures were fractionated by Strong Cation Exchange (SCX) using StageTips as previously described (8) with slight modifications. Briefly, one StageTip was prepared per sample by 3 SCX discs (3M, #2251) topped with 2 C18 discs (3M, #2215). The packed StageTips were first washed with 100µl methanol and then with 100 µl 80% acetonitrile and 0.5% acetic acid. Afterwards they were equilibrated by 100µl 0.5% acetic acid and the sample was loaded onto the discs. The sample was transeluted from the C18 discs to the SCX discs by applying 100µl 80% acetonitrile; 0.5% acetic acid, which was followed by 6 stepwise elutions and collections of the peptide mix from the SCX discs. The first fraction was eluted with 50µl 50mM NH4AcO; 20% MeCN (pH 4.1, adjusted with acetic acid), the second with 50µl 50mM NH4AcO; 20% MeCN (pH 5 4.8, adjusted with acetic acid), the third with 50µl 50mM NH4AcO; 20% MeCN (pH 6.2, adjusted with acetic acid), the fourth with 50µl 50mM NH4AcO; 20% MeCN (pH 7.2), the fifth with 50µl 50mM NH4HCO3; 20% MeCN (pH 8.5) and the sixth with 50µ l 0.1% NH4OH; 20% MeCN (pH 9.5). 200µl of 0.5% acetic acid was added to each of the 6 fractions and they were subsequently desalted on C18 StageTips as previously described (8) and evaporated to dryness in a vacuum concentrator. Peptides were reconstituted in 7µl 3% MeCN/0.1% formic acid (at an estimated concentration of 1µg/µl).
Scan protocol
LC-MS/MS
Data processing
All mass spectra were analyzed with MaxQuant software version 1.3.5 using the mouse UniProt database (March 2013). MS/MS searches for the proteome data sets were performed with the following parameters: Oxidation of methionine and protein Nterminal acetylation as variable modifications; carbamidomethylation as fixed modification. Trypsin/P was selected as the digestion enzyme for the pulsed-SILAC experiments and either LysN or AspN was selected as the digestion enzyme for the additional protein quantification experiments for time 0h, and a maximum of 3 labeled amino acids and 2 missed cleavages per peptide were allowed. The mass tolerance for precursor ions was set to 20 p.p.m. for the first search (used for nonlinear mass recalibration) and 6 p.p.m. for the main search. Fragment ion mass tolerance was set to 20 p.p.m. The IBAQ feature was enabled in order to estimate relative proteins levels. For identification we applied a maximum FDR of 1% separately on the protein and peptide level. We required 2 or more unique/razor peptides for protein identification and a ratio count of 2 or more for protein quantification per replicate measurement. We constructed “analysis groups” using the following rules: 1. All transcripts in the same transcript group are in the same analysis group, where transcript groups are defined by the UCSC Table Browser (query settings: assembly=”July 2007 (NCBI37/mm9)”, group=”Gene and Gene Predictions”, track=”UCSC Genes”, table=”knownIsoforms”; see the fields “clusterID” and “transcript”) and represent groups of transcripts (isoforms) that are derived from the same genic locus and cannot easily be resolved. 2. All proteins in the same MaxQuant protein group are in the same analysis group, where MaxQuant determines protein groups on the basis of the peptide library supplied. The groups represent the level of ambiguity MaxQuant can confidently resolve. 3. If a protein and a transcript are associated with one another, then they are in the same analysis group, where we associate (one or more) Uniprot protein ID with (one or more) UCSC transcript ID based on ftp://ftp.uniprot.org/pub/databases/uniprot/current_release/knowledgebase/idmapping/by_ organism/MOUSE_10090_idmapping.dat Expression estimates were based on MaxQuant’s un-normalized M/L and H/L ratios per protein group. For each protein and replicate, we scaled the ratio by the mean IBAQ L channel intensity observed for that protein across all conditions and time points (treating missing values as zeroes). Analysis groups were named according to the concatenation of all unique gene symbols associated with the UCSC transcripts in the analysis group. Analysis groups (henceforth referred to as genes for simplicity) were excluded if they were not MS2-identified in at least 6 of the 10 time points in all conditions (time courses for LPS and MOCK stimulation), channels, and replicates. After applying this filter, we also excluded genes that did not have positive RNA-Seq values for at least 6 out of the 10 time points in all conditions and channels. These filters were not applied to the AspN or LysN data. We renormalized such that at each condition/time point the RNA-Seq expression values added up to exactly 1,000,000. Meanwhile, we normalized the protein data such that at each condition/time point protein (M+H) IBAQ values added up to exactly 1,000,000 (missing values were treated as zeroes).
