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

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.

Development of Prediction Models for New Integrated Models and a Bioscore System to Identify Bacterial Infections in Systemic Lupus Erythematosus

“…Previous studies have reported that the PLS-DA/OPLS-DA model was used to process multiple protein indicators to effectively predict bacterial infections (Kuusela et al, 2017;Tong et al, 2019). In our study, the PLS-DA/OPLS-DA models showed clear identification ability for bacterial infections in SLE.…”

Low-Density Granulocyte Contamination From Peripheral Blood Mononuclear Cells of Patients With Sepsis and How to Remove It – A Technical Report

“…This is an explanation for our observation, that the number of low-density granulocytes co-purifying with the PBMC fraction varies highly between patients. Although some of the studies depicting the problem of contaminating, co-purifying low-density granulocytes have been published a decade ago, it is still common practice today to use density gradient centrifugation for PBMC isolation from patients with sepsis (21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33). This goes along with a lack of reported information on the isolation purity and identification of the contained cell types.…”

Human peripheral blood mononuclear cells: A review of recent proteomic applications

“…Tong et al. studied PBMCs proteome to uncover biomarkers associated with sepsis and septic shock [18]. Non‐septic intensive care unit (ICU) patients, septic shock patients, and patients with sepsis were enrolled.…”