Cell culture

MC38, SK-MEL-37, and A549 cell lines were maintained in DMEM containing 10% FCS, 2 mM glutamine, 1 mM sodium-pyruvate, 100 units/mL penicillin, and 100 μg/mL streptomycin (control medium). The cell lines Jurkat, MEL-HO, D41-MEL, NCI-H226, and NCI-H1650 were cultured in RPMI complemented with 10% FCS, 2 mM glutamine, 1 mM sodium-pyruvate, 100 units/mL penicillin, and 100 μg/mL streptomycin (control medium). MC38 cells were kindly provided by H.-C. Probst (Institute for Immunology, University Medical Center, Johannes Gutenberg University Mainz), NCI-H226 (CRL-5826) and NCI-H1650 (CRL-5883) cells were obtained from ATCC, A549 (ACC 107) and MEL-HO (ACC 62) cells were obtained from the DSMZ-German Collection of Microorganisms and Cell Culture GmbH, D41-MEL cells were kindly provided by Catherine Wölfel (Department of Hematology and Oncology, University Medical Center, Johannes Gutenberg University Mainz), Jurkat cells were kindly provided by Ari Waisman (Institute for Molecular Medicine, University Medical Center, Johannes Gutenberg University Mainz), and SK-MEL-37 cells were obtained from Sigma-Aldrich (SCC262).

OT-I transgenic mice, purchased by Charles River Laboratories, were used to generate primary T cells. OT-I splenocytes were cultured in control medium: RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin, 50 μM β-mercaptoethanol, in the presence of IL-2 (supernatant of XL-63 cells used at a dilution of 1:250, kindly provided by H.-C. Probst (Institute for Immunology, University Medical Center, Johannes Gutenberg University Mainz)), and peptide (1 μg/mL ovalbumin 257–264) for 5 days. The culture in Met or AHA conditions was performed using the appropriate Stable Isotope Labeling by Amino acids in Cell culture (SILAC) medium without l-leucine, l-arginine, l-lysine, and l-methionine (SILAC DMEM or SILAC RPMI, both purchased by Athena Enzyme Systems) containing all the supplements used in the control medium and supplemented either with 0.8 mM l-leucine, 0.8 mM l-lysine, 0.4 mM l-arginine, and 0.2 mM l-methionine (SILAC medium + Met) or with 0.8 mM l-leucine, 0.8 mM l-lysine, 0.4 mM l-arginine, and 0.1 mM l-AHA (SILAC medium + AHA). All cells were maintained in a humidified incubator with 5% CO2 at 37 °C.

Cell growth and viability

Cell viability was performed using MC38 cells incubated either in control medium or in SILAC medium supplemented with Met or AHA. After 20 h of incubation, cells were fixed and stained using the fixable viability dye eFluor780 (eBioscience) before analyzing live and dead cells using the BD FACSCanto system and the DIVA software. For cell growth determination, 0.2 × 106 MC38 cells were seeded in 6-well plates and grown in control medium or in SILAC labeling medium supplemented with Met or AHA. After 18 and 24 h, cells were harvested and counted microscopically using trypan blue staining for dead cell exclusion. For each condition, three biological replicates were generated. For the Annexin V staining, 1 × 106 MC38, Jurkat, or primary OT-I cells incubated for 20 h in three different media (control medium, SILAC medium + Met, or SILAC medium + AHA) were harvested, washed in Annexin-binding buffer (10 mM Hepes, 140 mM NaCl, and 2.5 mM CaCl2), and stained with Annexin V–FITC (BD Biosciences) according to the manufacturer’s protocol for 15 min at 20 °C in the dark. Before analysis, TO-PRO-3 Iodide (Invitrogen) was added to a final concentration of 40 nM and samples were acquired using the Amnis ImageStream MK II and analyzed using the FlowJo software.

AHA labeling and enrichment of newly synthesized proteins

Secretome analysis was performed using the click chemistry–based approach similar to the method developed by Eichelbaum et al. [18]. Cells were grown in their appropriate control medium to 70% confluency in two T75 flasks (Greiner Bio-One CELLSTAR) for each biological replicate. After removing the medium, cells were washed twice with warm PBS and 7 mL of starvation medium (appropriate SILAC medium with the required supplements as described for the control media of the different cells used, including 0.8 mM l-leucine, but without Met or AHA, l-lysine, and l-arginine) was added. After 30 min of starvation, the medium was removed and cells were cultured either in AHA labeling medium (SILAC medium supplemented with 0.8 mM l-leucine, 0.8 mM l-lysine, 0.4 mM l-arginine, and 0.1 mM l-AHA) or in Met medium (same SILAC medium supplemented with 0.2 mM l-methionine instead of l-AHA). After 20 h labeling, the supernatants were collected, centrifuged for 5 min at 1000 g and 4 °C to remove remaining cells, and concentrated using Amicon Ultra-15 tubes (molecular mass cutoff 3000 Da, Millipore) to 0.25 mL. For all the cell lines and for the primary OT-I cells, three biological replicates were generated. Newly synthesized, AHA-containing proteins were isolated from the concentrated supernatants by performing click chemistry–based enrichment using the Jena Bioscience Click Chemistry Capture Kit according to the manufacturer’s protocol. For both conditions (AHA and Met), supernatants were washed thoroughly to remove unspecifically bound proteins. For subsequent proteomic analysis by mass spectrometry, proteins bound to the beads were reduced, alkylated, and digested with trypsin. For digestion, beads were suspended in 50 µL digestion buffer (50 mM Tris, pH 8, 2 mM CaCl2, and 0.1% RapiGest), and 0.5 μg trypsin was added and incubated overnight at 37 °C. The peptide solution was collected and the resin was washed with 50 μL 50 mM ammonium bicarbonate (NH4HCO3). Both solutions were combined and kept frozen until sample preparation for mass spectrometry analysis.

RNAseq—sample preparation and biostatistics

After collecting supernatants for secretome analysis, pellets from the same cells grown in both conditions (Met and AHA) were washed twice with PBS and subjected to transcriptome analysis. RNA was purified with the RNeasy Plus Micro Kit according to the manufacturer’s protocol (Qiagen). RNA was quantified with a Qubit 2.0 fluorometer (Invitrogen) and the quality was assessed on a Bioanalyzer 2100 (Agilent) using an RNA 6000 Pico chip (Agilent). Samples with an RNA integrity number (RIN) of ≥ 8 were used for library preparation. Barcoded mRNA-seq cDNA libraries were prepared from 150 ng of total RNA using NEBNext® Poly(A) mRNA Magnetic Isolation Module and NEBNext® Ultra™ II RNA Library Prep Kit for Illumina® according to the manual with a final amplification of 12 PCR cycles. Quantity was assessed using Invitrogen’s Qubit HS assay kit and library size was determined using Agilent’s 2100 Bioanalyzer HS DNA assay. Sequencing was performed on Illumina’s NovaSeq 6000 at Novogene (Cambridge, UK). Raw sequencing reads (approx. 30 mio 150 PE reads per sample) were preprocessed according to the Illumina standard protocol. Sequence reads were trimmed for adapter sequences and further processed using Qiagen’s software CLC Genomics Workbenchv20.0 with CLC’s default settings for RNAseq analysis. Reads were aligned toGRCm38 (file version GRCm38.p6) or GRCh38 (file version nGRCh38.104) genome dependent on cell line with the following settings: mismatch cost = 2; insertion cost = 3; deletion cost = 3; length fraction = 0.8; similarity fraction = 0.8. Detailed tables with expression values TPM, RPKM, total, and unique gene reads for each sample are deposited under the GEO accession number GSE211231. PCA plots were generated using CLC’s tool “PCA for RNA-Seq” with the following filtering and normalization settings: (a) “log CPM” (counts per million) values are calculated for each gene. The CPM calculation uses the effective library sizes as calculated by the TMM normalization. After this, a Z-score normalization is performed across samples for each gene: the counts for each gene are mean centered, and scaled to unit variance. Genes or transcripts with zero expression across all samples or invalid values (NaN or + / − infinity) are removed.

For differential expression analysis, in order to filter out non- or low-expressed genes, genes with TPM mean expression ≥ 4 in any group (Met or AHA treated) were used for subsequent analysis. For statistical analysis, CLC’s count based “Empirical analysis of Differential Gene Expression” implementing the “Exact Test” for two-group comparisons developed by Robinson and Smyth [41] was applied for each Met- versus AHA-treated cell line. For the generation of pie charts showing up- and down-regulated genes upon AHA treatment, the gene list of Met vs AHA comparism of each cell line was filtered on absolute fold change ≥ 2 and p values less than 0.05, which were considered statistically significant in this study.

Mass spectrometry—sample preparation

Secretome samples were digested “on-bead” during secretome protocol and subsequently desalted, using a Sep-Pak tC18 µElution Plate (Waters Corporation, Milford, MA) and a vacuum manifold. Purified peptides were lyophilized and reconstituted in 20 µL 0.1% (v/v) formic acid (FA) for LC–MS analysis. Proteome samples were digested according to filter-aided sample preparation (FASP) as described previously [42]. In brief, cell pellets were solved in 500 µL lysis buffer, containing 8 M urea, and disrupted in 15 cycles at 30 s sonification, followed by 30 s break in a Bioruptor (Diagenode, Liège, Belgium). Protein concentration was determined via Pierce 660 nm protein assay (Thermo Fisher Scientific), according to the manufacturer’s protocol and 20 µg protein was reduced using dithiothreitol (DTT), followed by alkylation with iodoacetamide (IAA). DTT was added again, to quench excess IAA. Buffer was exchanged by washing the membrane three times with 50 mM NH4HCO3 prior to digestion overnight at 37 °C using trypsin at an enzyme-to-protein ratio of 1:50 (w/w). After digestion, peptides were eluted via centrifugation, followed by washing the membrane once again with 50 mM NH4HCO3. Next, the samples were lyophilized and finally the purified peptides were reconstituted in 20 µL 0.1% (v/v) FA for LC–MS analysis.

Mass spectrometry—data acquisition

Nanoscale liquid chromatography (nanoLC) of tryptic peptides was performed on an Ultimate 3000 RSLCnano LC system (Thermo Fisher Scientific) equipped with a PEPMAP100 C18 5 µm 0.3 × 5 mm trap (Thermo Fisher Scientific) and an HSS-T3 C18 1.8 μm 75 μm × 250 mm analytical reversed-phase column (Waters Corporation). Mobile phase A was 0.1% (v/v) FA and 3% (v/v) dimethyl sulfoxide (DMSO) in water. Mobile phase B was 0.1% (v/v) FA and 3% (v/v) DMSO in acetonitrile. Peptides were separated running a gradient from 2 to 35% at a flow rate of 300 nL/min at 55 °C over 40 min. Together with wash- and column re-equilibration steps, the total analysis time was 60 min. Eluting peptides underwent mass spectrometric analysis on an Orbitrap Exploris 480 (Thermo Fisher Scientific) in a data-dependent acquisition (DDA) mode targeting the 10 most abundant peptides for fragmentation (Top10). Spray voltage was at 1.8 kV, the funnel RF level at 40, and heated capillary temperature at 275 °C. Full MS resolution was set to 120,000 at m/z 200 and full MS automated gain control (AGC) target to 300% with a maximum injection time of 50 ms. Mass range was set to m/z 350–1500. The limit of isolated peptide precursors for MS2 scans was set to an ion target of 1 × 105 (AGC target value of 100%) with maximum injection times of 25 ms. Fragment ion spectra were acquired at a resolution of 15,000 at m/z 200. Intensity threshold was kept at 1E4. Isolation window width of the quadrupole was set to m/z 1.6 and normalized collision energy was fixed at 30%. All data were acquired in profile mode using positive polarity.

Mass spectrometry—data processing

Acquired raw data were processed in MaxQuant Version 2.0.3.0 [43] with database search performed in the integrated search engine Andromeda. For human cell lines, the UniProt human proteome database (UniProtKB release 2020–3-2_2-0–11, 20,365 entries), including 172 common contaminants, was used and for MC38 and OT-I cell line the UniProt mouse proteome database (UniProtKB release 2020–3-2_2-0–11, 17,033 entries), including 172 common contaminants. Trypsin was specified as enzyme for digestion and a maximum of two missed cleavages per peptide was allowed. Fixed modification was set for carbamidomethyl cysteine and variable modification was set for oxidized methionine. False discovery rate assessment for peptide and protein identification was done using the target-decoy strategy by searching a reverse database and was set to 0.01 for database search in MaxQuant. TOP3 quantification [44] was used to infer protein level quantities.

Mass spectrometry—statistical analysis

Obtained LFQ intensities were statistically analyzed in Microsoft Excel (2019) by performing two-tailed, unpaired t-tests across all technical and biological replicates and subsequent Benjamini–Hochberg correction [45]. The log2 ratio was calculated for each cell line by subtracting the log2 of the average LFQ intensities across all technical and biological replicates in the AHA group from the log2 of the average LFQ intensities across all technical and biological replicates in the methionine group. For entries with no detection in either group (Met or AHA), values were imputed by dividing the minimum intensity available in the corresponding dataset by 2. To define the secretome dataset, we applied a filter to retain only proteins with a log2 ratio ≤  − 1 between Met and AHA conditions after click chemistry–based enrichment. In the proteome data, proteins with a log2 ratio ≤  − 1 or ≥ 1 were considered to be differentially expressed, in AHA or Met condition, respectively. To align secretome, proteome, and transcriptome data, the entry lists of each dataset were used to detect uniquely or commonly present entries (proteins or genes), using R version 4.0.4 (2021–02-15).

Bioinformatics

Differentially expressed genes between both Met and AHA conditions were subjected to a GO analysis using the Enrichr web application (https://maayanlab.cloud/Enrichr/) [24,25,26]. The enrichment results in the category biological process were visualized in tables ranking the enriched gene set library terms according to their combined score. Before regulated proteins between Met and AHA treatments were analyzed using the Enrichr software, the UniProt proteins IDs were converted to gene IDs using the SYNGO tool (https://www.syngoportal.org/convert) [46]. GO enrichment results of regulated proteins in the category biological process were listed in tables, allowing visualization of GO terms with the highest combined scores.