Data‐independent acquisition‐based quantitative proteomic analysis reveals differences in host immune response of peripheral blood mononuclear cells to sepsis

摘要

Abstract This study was aimed to uncover proteins that are differentially expressed in sepsis. Data‐independent acquisition (DIA) was used for analysis to identify differentially expressed proteins in peripheral blood mononuclear cells (PBMCs) of patients. A total of 24 non‐septic intensive care unit (ICU) patients, 11 septic shock patients and 27 patients diagnosed with sepsis were recruited for the mass spectrometry (MS) discovery. PBMCs were isolated from routine blood samples and digested into peptides. A DIA workflow was developed using a quadrupole‐Orbitrap liquid chromatography LC‐MS system, and mass spectra peaks were extracted by Skyline software. Orthogonal partial least‐squares discriminant analysis (OPLS‐DA) and partial least‐squares discriminant analysis (PLS‐DA) were applied to distinguish the patient groups at the level of fragment ion and peptide. Differentially expressed proteins in the patient groups were verified by enzyme‐linked immunosorbent assay (ELISA). Receiver‐operating characteristic (ROC) curves were used to evaluate the protein expression. A total of 1062 fragment ions and 122 proteins were identified in the MS‐DIA analysis conducted by Skyline software. Using gene ontology clustering analysis, we discovered that 51 of the 122 identified proteins were associated with biological processes, including carbon metabolism, biosynthesis of antibiotics, platelet activation, bacterial invasion of epithelial cells and complement, and coagulation cascades. Among them, five proteins (high‐mobility group box1 [HMGB1], matrix metalloproteinase 8 [MMP8], neutrophil gelatinase‐associated lipocalin [NGAL], lactotransferrin [LTF] and grancalcin [GCA]) were identified by ELISA as closely related to the development of sepsis. The ROC curves displayed good sensitivity and specificity.