Oxidation is an underappreciated post-translational modification in the regulation of immune responses associated with changes in phosphorylation

Although macrophages are known to be affected by their redox status, oxidation is not yet a well-recognized post-translational modification (PTM) in regulating macrophages and immune cells in general. While it has been described that the redox status of single cysteines in specific proteins is relevant for macrophage functions, global oxidation information is scarce. Hence, we globally assessed the impact of oxidation on macrophage activation using untargeted proteomics and PTM-omics. We exposed THP-1 macrophages to lipopolysaccharide (LPS) for 4 h and 24 h and applied a sequential iodoTMT labeling approach to get information on overall oxidation as well as reversible oxidation of cysteines. Thus, we identified 10452 oxidation sites, which were integratively analyzed with 5057 proteins and 7148 phosphorylation sites to investigate their co-occurance with other omics layers. Based on this integrative analysis, we found significant upregulation of several immune-related pathways, e.g. toll-like receptor 4 (TLR4) signaling, for which 19 proteins, 7 phosphorylation sites, and 39 oxidation sites were significantly affected, highlighting the relevance of oxidations in TLR4-induced macrophage activation. Co-regulation of oxidation and phosphorylation was observed, as evidenced by multiply modified proteins related to inflammatory pathways. Additionally, we observed time-dependent effects, with differences in the dynamics of oxidation sites compared to proteins and phosphorylation sites. Overall, this study highlights the importance of oxidation in regulating inflammatory processes and provides a method that can be readily applied to study the cellular redoxome globally.


Co-isolation threshold 50
Average reporter S/N threshold 10

Scaling mode None
For Protein Roll-Up Use all peptides Maximum allowed fold change 100

Sample preparation
For the redoxome, the mixes of iodoTMT-labeled peptides were subjected to peptide clean-up on paramagnetic beads (paragraph 2.1.1.3).Only the volume had to be scaled up as described in the manuscript.

LC-MS data acquisition
To measure the redoxome samples, the same LC and MS parameters described for the proteome have been used (paragraph 2.1.2).

Table S7: Consensus workflow general parameters redoxome.
Compared to processing proteomics samples, the Peptide Isoform Grouper node has to be added to the workflow.The minimum number of peptide sequences per protein has to be decreased to 1 to keep all identified peptides.Notably, analysis is performed on the peptide level, not the protein level.

Peptide confidence at least High
Minimum number of peptide sequences 1 FDR (strict) 0.01

Apply strict parsimony principle True
Table S8: Consensus workflow quantification parameters redoxome.

Co-isolation threshold 50
Average reporter S/N threshold 10

Scaling mode None
For Protein Roll-Up Use all peptides Maximum allowed fold change 100

Sample preparation
For the phosphoproteome, the same paramagnetic bead approach described for the proteome (paragraph 2.1.1)has been applied.Only the elution after the peptide clean-up was done differently than for proteome and redoxome, not in two fractions but only with water.Afterwards, a two-step enrichment using a workflow based on the HighSelect™ TiO2 Phosphopeptide Enrichment Kit (Thermo Scientific, USA) and the High-Select™ Fe-NTA Phosphopeptide Enrichment Kit (Thermo Scientific, USA) was performed as described before (5).
Compared to processing proteomics samples, the Peptide Isoform Grouper node has to be added to the workflow.The minimum number of peptide sequences per protein has to be decreased to 1 to keep all identified peptides.Notably, analysis is performed on the peptide level and not on the protein level .

Figure S5 :
Figure S5: Overlap of affected oxidized proteins with proteins known to be modified on cysteine.....
Figure S1: Overlaps in proteome proteins and proteins found phosphorylated or oxidized.Shown are the overlaps of proteins found in the proteome and such for which phosphorylation sites (A) or oxidation sites (B) were identified.Since site intensities were normalized to changes on the protein level, only modified proteins with the protein intensity available in the proteome were investigated further.

Figure S2 :
Figure S2: Principal component analyses (PCAs) of reliably identified proteins and sites.PCAs were performed with reliably quantified proteins (proteome), phosphorylation sites (phosphoproteome), reversible oxidation sites (redoxome: reversible), overall oxidation sites (redoxome: overall), and the combination of both (redoxome: complete) to assess the data reproducibility after 4 h and 24 h LPS treatment.The very low explained variances in the first two principal components suggest a good reproducibility.

Figure S3 :
Figure S3: Assignment of identified proteins to cellular compartments.Shown are the compartments assigned to most phosphorylation sites, proteins, or oxidation sites quantified here.

Figure S5 :
Figure S5: Overlap of affected oxidized proteins with proteins known to be modified on cysteine.Proteins showing significantly (FDR≤0.05)altered overall (A) or reversible (B) oxidation after 4 h or 24 h, respectively, were compared to proteins known for cysteine modification in general or the presence of S-nitrosocysteine in particular.For this comparison, information available in the UniProtKB was used.

Figure S7 :
Figure S7: Top 5 significantly enriched Reactome pathways.Combination of the top 5 Reactome pathways, significantly enriched (FDR≤0.05)either in one of the investigated omics layers or based on the integrative pathway enrichment.The significance of enrichment is provided with asterisks: * -FDR≤0.05,** -FDR≤0.01,*** -FDR≤0.001.The color reflects the median Log2(FC) of the proteins/sites assigned to the pathway.The enrichment analysis was performed based on significantly (FDR≤0.05)altered proteins (proteome), phosphorylation sites (PP), oxidation sites in general (red: all), reversibly oxidized sites (red: rev), the combination of both types of oxidation sites (redoxome), or the combination of all three omics layers (integrative).