Exosomal Proteome Analysis of Human Plasma for the Diagnosis of Candidemia

Research Article

Austin Dent Sci. 2021; 6(1): 1035.

Exosomal Proteome Analysis of Human Plasma for the Diagnosis of Candidemia

Wang Z, Huang L and Tang J*

Department of Trauma-Emergency & Critical Care Medicine, Shanghai Fifth People’s Hospital, Fudan University, Shanghai, China

*Corresponding author: Jianguo Tang, Fifth People’s Hospital, Fudan University, No. 801, Heqing Road, Minxing District, Shanghai, 200240, China

Received: April 15, 2021; Accepted: May 07, 2021; Published: May 14, 2021

Abstract

Exosomes have been extensively studied as diagnostic biomarkers and for monitoring disease progression. This study examined the role of exosomes in the pathogenesis of candidemia. Exosomes in the serum of patients with candidemia were analyzed by liquid chromatography-mass spectrometry/ mass spectrometry. Proteins differentially expressed in patient (n=4) vs. control (n=4) sera were confirmed by Enzyme-Linked Immunosorbent Assay (ELISA). Protein expression was evaluated using receiver operating characteristic curve analysis. In the serum samples of the patients, 4,055 fragment ions and 418 proteins were detected, including 11 proteins whose expression levels differed significantly from the controls. Three of these proteins (cathepsin b, pentraxin 3, and the serine protease inhibitor A3N) were shown by ELISA to be closely related to the development of candidemia. Our results suggest a role for serum exosomes as molecular markers in the diagnosis and monitoring of candidemia.

Keywords: Candidemia; Candida albicans; Plasma exosomes; Biomarkers; Proteomics

Introduction

Candida albicans is present on the skin and mucosal surfaces; it is the most common opportunistic fungal pathogen in humans, and is responsible for up to 50% of all cases of invasive candidiasis [1]. Due to the lack of innovative prophylactic and therapeutic strategies against fungal pathogens, invasive candidiasis has high rates of morbidity and mortality [2]. While blood cultures have a very high positive predictive value, due to the small fungal load and difficulties in culturing fungal pathogens their overall positivity rate for the diagnosis of bloodstream infections is only 20% [3]. Identification of serum biomarkers would allow early diagnosis of candidemia.

Exosomes are lipid bilayer vesicles with a diameter of 30-200 nm that are produced by endocytosis and secreted by multiple cell types. They are present in the supernatants of cell cultures and body fluids, including plasma, saliva, breast milk, urine, blood, cerebrospinal fluid, bile, and lymph [4]. Their role as functional organelles in a variety of biological processes, including the immune response, is evidenced by their protein, lipid, and RNA contents [5]. For example, using Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/ MS), researchers have identified >100 phosphoproteins in plasma exosomes with significantly higher expression levels in breast cancer patients than healthy controls [6]. Another study demonstrated that the presence of the GPCI protein in exosomes can serve as a marker for early diagnosis of pancreatic cancer [7]. Similar roles for other exosome biomarkers (CD44, CD47, CXCR4, Del-1, HER2 and KDR) in the early diagnosis and prognosis of breast cancer have been reported [8].

In this study, LC-MS/MS was used for quantitative detection of fragment ions and peptides produced after the digestion of exosomes isolated from the serum of patients with candidemia. Our aim was to obtain a better understanding of the pathogenesis of candidemia based on comparison of exosome protein expression between patients with candidemia and healthy controls. A bioinformatics approach was then applied to analyze the cellular components, molecular functions, and potential signaling pathways of these proteins with respect to the pathogenesis of candidemia.

Materials and Methods

Patients and controls

Plasma samples for method optimization were contributed by ourselves, and by patients diagnosed with sepsis at The Fifth People’s Hospital of Shanghai (Shanghai, China), in accordance with the requirements of the Ethics Committee of the Fifth People’s Hospital of Shanghai. Candidemia was diagnosed by a clinician and confirmed by blood culture. Written informed consent was obtained from all patients. The plasma samples were stored in aliquots at -80oC prior to their use.

Serum exosome isolation and enrichment

Four 1mL aliquots of the plasma samples were quickly thawed, and then centrifuged for 30min at 3,000 × g and 4oC to remove cellular debris and impurities. The supernatants were diluted to 7mL with precooled 1× phosphate-buffered saline (PBS; Sangon Biotech, China), filtered (0.22mm; Millipore, USA), and then subjected to two rounds of ultracentrifugation for extraction and washing, respectively. Ultracentrifugation was carried out at 150,000 × g and 4oC using a SW41 rotor (Beckman Coulter, USA). After the first round of ultracentrifugation, the supernatant was removed and the pellet was resuspended in 1mL of 1× PBS for the second round. The washed, pelleted exosomes were resuspended in 200mL of 1× PBS.

Nanometer particle size analysis of exosomes

The enriched exosomes were suspended in 100μL of PBS. A 10μL aliquot was then diluted to 1mL with ultrapure water and injected into a S300 nanometer particle size analyzer (NanoSight, UK) to determine the size distribution and concentration of the particles according to the standard procedure. The measurement was performed in triplicate. NanoSight NTA (V3.2) software was used to analyze the results.

Proteolysis

FASP enzymolysis [9] was carried out as follows: 1) Exosome proteins were precipitated in five volumes of an organic solution composed of 50% ethanol, 50% acetone, and 0.1% acetic acid, and the obtained precipitate was dissolved in 6mol guanidine hydrochloride/L. After overnight freezing at -20oC, 2μL of the reducing agent DLdithiothreitol was added and the mixture was incubated for 1h at 60oC, followed by the addition of 10μL of the alkylating agent indole- 3-acetic acid and a 40min incubation in the dark at 4oC. The resulting protein solution was transferred to a 10kDa filter tube and centrifuged for 20min at 12,000 × g and 4oC to concentrate the proteins. Trypsin (Promega, USA) was then added at an enzyme:protein ratio of 1:20. After enzymatic hydrolysis, the peptides were desalted using a C18 cartridge, freeze-dried, and then reconstituted in 40μL of dissolution buffer [9].

LC-MS/MS data acquisition

Dried peptides were resuspended in 0.1% formic acid to a final concentration of 0.5mg/mL; 2mL of each sample was then analyzed in an Easy Nano-UPLC 1000 coupled with a QE Plus mass spectrometer (Thermo Scientific, USA). The peptides were eluted using a gradient of buffer B (0.1% TFA in acetonitrile), as follows: 0-65 min, 3-35% B; 65-68 min, 35-80% B; and 68-75 min, 80% B at a flow rate of 300nL/ min. The MS data files were acquired in data-dependent mode. The resolution of the full-scan MS spectra (m/z 300-1,500) was 70,000 (at m/z 200), with an Automatic Gain Control (AGC) of 2 × 105, a maximum injection time of 100ms, and a precursor charge of 2-6. The 20 most abundant precursors identified in the full scan were selected with an isolation window of 2.0m/z and fragmented by higher energy collision-induced dissociation at a normalized energy of 35%. The AGC was 1 × 104, the maximum injection time was 35ms, and the dynamic exclusion time was 60s.

Protein identification and quantitative analysis

The original files (raw files) were searched using MaxQuant software (version 1.5.5.1; www.maxquant.org). Protein identification and the results of the quantitative analysis were obtained using the following search criteria: missed trypsin cleavage site to allow two loci, oxidative and acetylated modifications as variables, quality of the precursor and fragment ion error of 6 ppm [10-6], 0.5-Da protein identification of standards: peptides based on at least two characteristics, p <0.05, a False Positive Rate (FDR) of <1%, and determination of the relative abundance of the proteins based on the values of at least two peptides. A significant difference between patients and controls was defined as a p value <0.01. A >2-fold change was considered to reflect significant up-regulation, and a <0.5-fold change significant down-regulation.

Enzyme-linked immunosorbent assay (ELISA)

Protein markers identified in the serum exosome proteomics analysis as potentially associated with candidemia were confirmed by ELISA, which was performed using commercially available kits (Biosh, China) in accordance with the manufacturer’s instructions.

Results

Enrichment and separation of serum exosomes

The exosomes isolated from serum were 100-150 nm (Figure 1A and B) in size, consistent with their known morphological characteristics. The size range was further confirmed by transmission electron microscopy, which also showed the bilayer goblet structure of the exosomes (Figure 1C). The efficiency of the exosome preparation method was assessed by immunoblot analysis of a known exosome marker, CD63 (Figure 1D). Together, these results demonstrated successful enrichment of serum exosomes and their suitability for use in the experiments.