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A proteomics-based method for identifying antigens within immune complexes

ABSTRACT: A novel approach to recover and identify immune complexes (ICs) was developed using size exclusion chromatography (SEC) and affinity chromatography on immunoglobulin binding columns (HiTrap Protein G). The purification process was monitored by 1D SDS-PAGE, protein staining, Western blotting and, finally, liquid chromatography tandem mass spectrometry (LC MS/MS) was used to identify the recovered antigens. This approach was applied to serum with artificially created immune complexes (ICs) comprising vaccine antigen (influenza) and antibody, which led to recovery and identification of influenza peptides within the recovered ICs. This approach was compared with the established method for IC detection and recovery, polyethylene glycol (PEG) precipitation, followed by LC MS/MS. Both approaches successfully enabled capture, recovery and characterization of immunoglobulins and influenza antigen(s) in complex with the immunoglobulins. However, PEG precipitation has the advantage of simplicity and is more suited for large scale studies.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human) Betainfluenzavirus Alphainfluenzavirus

TISSUE(S): Blood

SUBMITTER: Andrew McArdle

LAB HEAD: Michael Levin

PROVIDER: PXD021575 | Pride | 2021-01-04

REPOSITORIES: Pride

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A proteomics-based method for identifying antigens within immune complexes.

Menikou Stephanie S McArdle Andrew J AJ Li Ming-Shi MS Kaforou Myrsini M Langford Paul R PR Levin Michael M

PloS one 20201223 12

A novel approach to recover and identify immune complexes (ICs) was developed using size exclusion chromatography (SEC) and affinity chromatography on immunoglobulin binding columns (HiTrap Protein G). The purification process was monitored by 1D SDS-PAGE, protein staining, Western blotting and, finally, liquid chromatography tandem mass spectrometry (LC MS/MS) was used to identify the recovered antigens. This approach was applied to serum with artificially created immune complexes (ICs) compris ...[more]

PMID: 33362259

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Project description:Sour passion fruit (Passiflora edulis Sims) has economic and social relevance and is an alternative crop mainly for family farming agriculture. The use of micropropagation by somatic embryogenesis offers scale-up clonal propagation with high phytosanitary quality. The aim of this work was to evaluate the influence of polyethylene glycol (PEG) on the maturation of somatic embryos associated with differential accumulation of proteins and changes in the endogenous polyamines (PAs) content during somatic embryogenesis of P. edulis ‘UENF Rio Dourado’. Maturation of somatic embryos was performed using embryogenic callus in MS culture medium with PEG 6% or without PEG (control). PEG 6% promoted the maturation of a significantly higher number of somatic embryos at globular and cotyledonary stages when compared to control treatment. The higher somatic embryo formation induced by PEG 6% was associated with an increase in endogenous contents of free spermine, a PA with an important role in the maturation process of somatic embryogenesis cultures. PEG also promoted a significantly higher content of putrescine and total free PAs at the end of maturation, which was relevant for the increase in the number of somatic embryos. Comparative proteomic analyses of PEG 6%/control revealed that PEG 6% treatment induced the up-accumulation of proteins related to the glycolytic process, generation of precursor metabolites energy, and response to light stimulus, we can highlight the enolase ENO1, triosephosphate isomerase (TPI), ribulose bisphophate carboxylase/oxygenase activase chloroplastic (RCA), glyceraldehyde-3-phosphate dehydrogenase GAPCP-1, chloroplastic (GAPCP-1), succinate dehydrogenase [ubiquinone] flavoprotein subunit1, mitochondrial (SDH1-1), pyruvate dehydrogenase E1 component subunit alpha, mitochondrial (IAR4), ATP synthase CF1 beta subunit (PB), malate dehydrogenase [NADP], chloroplastic (AT5G58330) and chlorophyll a-b binding protein 21, chloroplastic (LHB1B1), and the down-accumulated proteins related mainly to the cellular metabolic process with proteins such as NADP-dependent malic enzyme (NADP-ME4), phosphoenolpyruvate carboxylase 2 (PPC1), UDP-glucose 6-dehydrogenase 1 and 4 (UGD2) and sucrose synthase 2 isoform X2 (SSA) and cell division cycle protein 48 homolog (ATCDC48B). The use of PEG induced thematuration and development of somatic embryos of P. edulis Sims ‘UENF Rio Dourado’ by the differential accumulation of proteins and modulation of endogenous contents of PAs

Project description:Background: The first step in SARS-CoV-2 infection is binding of the virus to angiotensin converting enzyme 2 (ACE2) on the airway epithelium. Asthma affects over 300 million people world-wide, many of whom may encounter SARS-CoV-2. Epidemiologic data suggests that asthmatics who get infected may be at increased risk of more severe disease. Our objective was to assess whether maintenance inhaled corticosteroids (ICS), a major treatment for asthma, is associated with airway ACE2 expression in asthmatics. Methods: Large airway epithelium (LAE) of asthmatics treated with maintenance ICS (ICS+), asthmatics not treated with ICS (ICS-), and healthy controls (controls) was analyzed for expression of ACE2 and other coronavirus infection-related genes using microarrays. Results: AsResults: As a group, there was no difference in LAE ACE2 expression in all asthmatics vs controls. In contrast, subgroup analysis demonstrated that LAE ACE2 expression was higher in asthmatics ICS+ compared to ICS‾ and ACE2 expression was higher in male ICS+ compared to female ICS+ and ICS‾ of either sex. ACE2 expression did not correlate with serum IgE, absolute eosinophil level, or change in FEV1 in response to bronchodilators in either ICS- or ICS+. Conclusion: Airway ACE2 expression is increased in asthmatics on long-term treatment with ICS, an observation that should be taken into consideration when assessing the use of inhaled corticosteroids during the pandemic. a group, there was no difference in LAE ACE2 expression in all asthmatics vs controls. In contrast, subgroup analysis demonstrated that LAE ACE2 expression was higher in asthmatics ICS+ compared to ICS‾ and ACE2 expression was higher in male ICS+ compared to female ICS+ and ICS‾ of either sex. ACE2 expression did not correlate with serum IgE, absolute eosinophil level, or change in FEV1 in response to bronchodilators in either ICS- or ICS+. Conclusion: Airway ACE2 expression is increased in asthmatics on long-term treatment with ICS, an observation that should be taken into consideration when assessing the use of inhaled corticosteroids during the pandemic.

Dataset Information

A proteomics-based method for identifying antigens within immune complexes

Publications

A proteomics-based method for identifying antigens within immune complexes.

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