Identification of novel biomarkers for sepsis prognosis via urinary proteomic analysis using iTRAQ labeling and 2D-LC-MS/MS

Abstract

Objectives: Sepsis is the major cause of death for critically ill patients. Recent progress in proteomics permits a thorough characterization of the mechanisms associated with critical illness. The purpose of this study was to screen potential biomarkers for early prognostic assessment of patients with sepsis.

Methods: For the discovery stage, 30 sepsis patients with different prognoses were selected. Urinary proteins were identified using isobaric tags for relative and absolute quantitation (iTRAQ) coupled with LC-MS/MS. Mass spec instrument analysis were performed with Mascot software and the International Protein Index (IPI); bioinformatic analyses were used by the algorithm of set and the Gene Ontology (GO) Database. For the verification stage, the study involved another 54 sepsis-hospitalized patients, with equal numbers of patients in survivor and non-survivor groups based on 28-day survival. Differentially expressed proteins were verified by Western Blot.

Results: A total of 232 unique proteins were identified. Proteins that were differentially expressed were further analyzed based on the pathophysiology of sepsis and biomathematics. For sepsis prognosis, five proteins were significantly up-regulated: selenium binding protein-1, heparan sulfate proteoglycan-2, alpha-1-B glycoprotein, haptoglobin, and lipocalin; two proteins were significantly down-regulated: lysosome-associated membrane proteins-1 and dipeptidyl peptidase-4. Based on gene ontology clustering, these proteins were associated with the biological processes of lipid homeostasis, cartilage development, iron ion transport, and certain metabolic processes. Urinary LAMP-1 was down-regulated, consistent with the Western Blot validation.

Conclusion: This study provides the proteomic analysis of urine to identify prognostic biomarkers of sepsis. The seven identified proteins provide insight into the mechanism of sepsis. Low urinary LAMP-1 levels may be useful for early prognostic assessment of sepsis.

Trial registration: ClinicalTrial.gov NCT01493492.

Figure 1. Schematic of the experimental design based on iTRAQ labeling combined with 2-D LC-MS/MS analysis of SI, SP, and de.

SCX, strong cation exchange. SI: urine specimens from patients with SIRS. SP: urine specimens from sepsis patients, acquired within 24 h of admission to the ICU. de: urine specimens from sepsis patients, acquired within 48 h before death.

Figure 2. Proteins identified in the three sets (technical replicates).

Figure 3. The analytical process for candidate target protein identification.

SI: urine specimens from patients with SIRS. SP: urine specimens from sepsis patients, acquired within 24 h of admission to the ICU. de: urine specimens from sepsis patients, acquired within 48 h before death. de/SI: proteins differentially expressed in de relative to SI. de/SP: proteins differentially expressed in de relative to SP. SP/SI: proteins differentially expressed in SP relative to SI.

Figure 4. GO annotation of the final selected differentially expressed proteins.

These differentially expressed proteins were classified among three categories: cellular component (CC), molecular function (MF) and biological process (BP). According to the GO database, the top 10 components for CC, MF, BP of the selected differentially expressed proteins are shown along with their enrichment score, represented as a p-value.

Figure 5. Western blot validation of three candidate markers in individual sepsis patients with different prognoses.

(A) Relative protein expression of SBP-1. The survivor group and non-survivor groups were 0.938±0.347 and 0.945±0.602 (p>0.05), respectively. (B) Relative protein expression of LAMP-1. The survivor group and non-survivor groups were 0.752±0.246 and 0.617±0.166 (p<0.05), respectively. (C) Relative protein expression of HSPG-2. The survivor and non-survivor groups were 0.802±0.282 and 0.880±0.606 (p>0.05), respectively.