## 2 Material and Methods
### Serum Cytokines
#### 2.3.3 Data Processing  
The acquired raw data files were processed with the MaxQuant software v. 2.0.3.0 [11,12] using the internal search engine the Andromeda [13] and Uniprot [14] databases, restricted to specific protein sequences from Homo sapiens (Proteome UP000005640). To develop a comprehensive immune response profile, a selected number of cytokines was focused. This selection aimed to cover a broad spectrum of immune mediators, including proinflammatory and anti-inflammatory cytokines, chemokines, and growth factors particularly relevant to COVID-19 pathophysiology and immune responses, potentially enhancing the sensitivity for specific targets. Consequently, protein sequences were retrieved for pro-inflammatory cytokines (gene id) GM-CSF (_CSF2_), IFN-\(\alpha\) (_IFNA1_), IL-1\(\alpha\) (_IL1A_), IL-1\(\beta\) (_IL1B_), IL-6 (_IL6_), IL-15 (_IL-15_), and TNF-\(\alpha\) (_TNF_); anti-inflammatory cytokines (gene id) IL-1 RA (_IL1 RN_), IL-10 (_IL10_), and IL-13 (_IL13_); cytokines (gene id) IFN-\(\beta\) (_IFNB_), IFN-\(\gamma\) (_IFNG_), IL-2R (_IL2 RA_), IL-7 (_IL7_), and IL-8 (_CXCL8_); chemokines (gene id) eotaxin (_CCL1_), IP-10 (_CXCL10_), MIG (_CXCL9_), MIP-1\(\alpha\) (_CCL3_), MIP-1\(\beta\) (_CCL4_), and RANTES (_CCL5_); and growth factors (gene id) VEGF (_VEGF_), HGF (_HGF_), and EGF (_EGF_). PARK7 (Uniprot ID 099497), one of the proteins with lowest variability in abundance among different human cell types, was used as an internal control for data analysis due to its stable expression, low cell-type specificity, and consistent detection in a wide range of tissues [15,16]. This was crucial considering the diverse range of cytokines that were analyzed, as it ensures that internal control remained unaffected by biological variations, thereby supporting accurate and reproducible results. Furthermore, PARK7 exhibited an average log\({}_{2}\) (label-free quantification (LFQ)) across all samples of 14.56 \(\pm\) 0.07 (0.5% standard deviation), reflecting minimal variability and thereby supporting its reliability as a reference point. Methionine oxidation (+15.9949 Da), protein N-terminal acetylation (+42.0106 Da), as well as phosphorylation of serine, threonine, and tyrosine (+80.973607 Da), were defined as variable modifications, and cysteine carbamidomethylation (+57.0215 Da) was set as a fixed modification. A Q-TOF was used with default parameters (Bruker, Germany). Enzyme specificity was set to trypsin/P, allowing cleavages at the carboxyl side of arginine and lysine residues and before proline residues, with a maximum of two missed cleavages allowed. The false discovery rate for peptides and proteins was set to 1%. The minimum score for acceptance of MS/MS identification was set to 10 for unmodified peptides and 40 for modified peptides. A minimum of three razor or unique peptides was required for protein group identification, with a minimum sequence coverage of 25% and a minimum total score of 10. Match between runs was enabled with a 0.7 min match time window and a 20 min alignment window. Dependent peptides were enabled with a fold discovery rate of 1%. Normalized spectral protein LFQ intensities were calculated using the MaxLFQ algorithm [17], with a minimum ratio count of 1. MaxLFQ is a label-free intensity determination and normalization procedure that enables comparative proteomics studies to identify changes in protein expression levels across experimental conditions. This approach accounts for variability in sample preparation and fractionation; it relies only on common peptides between samples to avoid losing data due to missing intensities and assumes that most proteins remain unchanged between samples, eliminating the need for housekeeping proteins. As a result, MaxLFQ achieves high precision through global optimization and median-based calculations, handles incomplete datasets effectively, and accurately quantifies fold changes across several orders of magnitude [17]. MaxQuant output data were processed using Perseus v1.6.15.0 with default settings [18].