Development of a new magnetic beads-based immunoprecipitation strategy for proteomics analysis

Vila, A. Molares; Arrilucea, P. Rupérez Pérez de; Peláez, E. Caso; Gago-Martı́nez, Ana

doi:10.1016/j.jprot.2010.02.015

Cited by 7 publications

(2 citation statements)

References 26 publications

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“…To investigate the morphology and seeding activity of mHTT aggregates formed in zQ175 mouse brains, we first established an antibody-based immunoprecipitation (IP) method using magnetic beads ( Vila et al, 2010 ). We then systematically tested 8 different anti-HTT antibodies for their ability to IP mHTT aggregates under native conditions from crude brain extracts.…”

Section: Resultsmentioning

confidence: 99%

Small, Seeding-Competent Huntingtin Fibrils Are Prominent Aggregate Species in Brains of zQ175 Huntington’s Disease Knock-in Mice

et al. 2021

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The deposition of mutant huntingtin (mHTT) protein aggregates in neurons of patients is a pathological hallmark of Huntington’s disease (HD). Previous investigations in cell-free and cell-based disease models showed mHTT exon-1 (mHTTex1) fragments with pathogenic polyglutamine (polyQ) tracts (>40 glutamines) to self-assemble into highly stable, β-sheet-rich protein aggregates with a fibrillar morphology. HD knock-in mouse models have not been extensively studied with regard to mHTT aggregation. They endogenously produce full-length mHTT with a pathogenic polyQ tract as well as mHTTex1 fragments. Here, we demonstrate that seeding-competent, fibrillar mHTT aggregates can be readily detected in brains of zQ175 knock-in HD mice. To do this, we applied a highly sensitive FRET-based protein amplification assay that is capable of detecting seeding-competent mHTT aggregate species down to the femtomolar range. Furthermore, we show that fibrillar structures with an average length of ∼200 nm can be enriched with aggregate-specific mouse and human antibodies from zQ175 mouse brain extracts through immunoprecipitations, confirming that such structures are formed in vivo. Together these studies indicate that small, fibrillar, seeding-competent mHTT structures are prominent aggregate species in brains of zQ175 mice.

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Section: Resultsmentioning

confidence: 99%

Small, Seeding-Competent Huntingtin Fibrils Are Prominent Aggregate Species in Brains of zQ175 Huntington’s Disease Knock-in Mice

et al. 2021

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“…Among the different solid supports used for IP, magnetic beads are widely used. They have demonstrated to be effective and to have lower nonspecific binding and a better orientation of coupled antibodies than others, such as agarose resins …”

Section: Introductionmentioning

confidence: 99%

Shotgun Characterization of the Circulating IgM Antigenome of an Infectious Pathogen by Immunocapture-LC–MS/MS from Dried Serum Spots

Abad,

Coronado,

Vincelle-Nieto

et al. 2024

J. Proteome Res.

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One of the main challenges in compiling the complete collection of protein antigens from pathogens for the selection of vaccine candidates or intervention targets is to acquire a broad enough representation of them to be recognized by the highly diversified immunoglobulin repertoire in human populations. Dried serum spot sampling (DSS) retains a large repertoire of circulating immunoglobulins from each individual that can be representative of a population, according to the sample size. In this work, shotgun proteomics of an infectious pathogen based on DSS sampling coupled with IgM immunoprecipitation, liquid chromatography–mass spectrometry (LC–MS/MS), and bioinformatic analyses was combined to characterize the circulating IgM antigenome. Serum samples from a malaria endemic region at different clinical statuses were studied to optimize IgM binding efficiency and antibody leaching by varying serum/immunomagnetic bead ratios and elution conditions. The method was validated using Plasmodium falciparum extracts identifying 110 of its IgM-reactive antigens while minimizing the presence of human proteins and antibodies. Furthermore, the IgM antigen recognition profile differentiated between malaria-infected and noninfected individuals at the time of sampling. We conclude that a shotgun proteomics approach offers advantages in providing a high-throughput, reliable, and clean way to identify IgM-recognized antigens from trace amounts of serum. The mass spectrometry raw data and metadata have been deposited with ProteomeXchange via MassIVE with the PXD identifier PXD043800.

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Automation of Immunoglobulin Glycosylation Analysis

Hendel

Gardner

Spencer

2021

Experientia Supplementum

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Development of a new magnetic beads-based immunoprecipitation strategy for proteomics analysis

Cited by 7 publications

References 26 publications

Small, Seeding-Competent Huntingtin Fibrils Are Prominent Aggregate Species in Brains of zQ175 Huntington’s Disease Knock-in Mice

Small, Seeding-Competent Huntingtin Fibrils Are Prominent Aggregate Species in Brains of zQ175 Huntington’s Disease Knock-in Mice

Shotgun Characterization of the Circulating IgM Antigenome of an Infectious Pathogen by Immunocapture-LC–MS/MS from Dried Serum Spots

Automation of Immunoglobulin Glycosylation Analysis

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