Emerging affinity-based techniques in proteomics

Xie, Shengnan; Moya, Colby; Bilgin, Betul; Jayaraman, Arul; Walton, S. Patrick

doi:10.1586/epr.09.74

Cited by 31 publications

(32 citation statements)

References 118 publications

(104 reference statements)

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“…Both of these problems are overcome by modern LC-MS/MS techniques [95, 195–197]. However, the MS-based techniques are not inherently quantitative because proteolytic peptides exhibit a wide range of physicochemical properties (size, charge, hydrophobicity) that lead to large differences in mass spectrometric response.…”

Section: Proteomicsmentioning

confidence: 99%

Proteomics of Plant Pathogenic Fungi

González-Fernández

Prats

Jorrín‐Novo

2010

Journal of Biomedicine and Biotechnology

139

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Plant pathogenic fungi cause important yield losses in crops. In order to develop efficient and environmental friendly crop protection strategies, molecular studies of the fungal biological cycle, virulence factors, and interaction with its host are necessary. For that reason, several approaches have been performed using both classical genetic, cell biology, and biochemistry and the modern, holistic, and high-throughput, omic techniques. This work briefly overviews the tools available for studying Plant Pathogenic Fungi and is amply focused on MS-based Proteomics analysis, based on original papers published up to December 2009. At a methodological level, different steps in a proteomic workflow experiment are discussed. Separate sections are devoted to fungal descriptive (intracellular, subcellular, extracellular) and differential expression proteomics and interactomics. From the work published we can conclude that Proteomics, in combination with other techniques, constitutes a powerful tool for providing important information about pathogenicity and virulence factors, thus opening up new possibilities for crop disease diagnosis and crop protection.

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Section: Proteomicsmentioning

confidence: 99%

Proteomics of Plant Pathogenic Fungi

González-Fernández

Prats

Jorrín‐Novo

2010

Journal of Biomedicine and Biotechnology

139

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“…Kinetic Analysis of Affinity Reagents. Knowledge about the kinetics of binding between antibodies and antigens is important for all immunoassays (3,19). The wide range in limit of detection among the analytes studied in this report led us to further explore the relationship between kinetics and assay performance.…”

mentioning

confidence: 91%

“…While incompatibility with low-affinity antibodies is a major drawback for PLA, it is not restricted to this assay alone. The availability of high-affinity antibodies or other capture reagents is a general limitation for all sandwich immunoassays, because the sensitivity is ultimately determined by the quality of the reagents used (19).…”

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confidence: 99%

Streamlined circular proximity ligation assay provides high stringency and compatibility with low-affinity antibodies

Jalili

Horecka

Swartz

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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Proximity ligation assay (PLA) is a powerful tool for quantitative detection of protein biomarkers in biological fluids and tissues. Here, we present the circular proximity ligation assay (c-PLA), a highly specific protein detection method that outperforms traditional PLA in stringency, ease of use, and compatibility with low-affinity reagents. In c-PLA, two proximity probes bind to an analyte, providing a scaffolding that positions two free oligonucleotides such that they can be ligated into a circular DNA molecule. This assay format stabilizes antigen proximity probe complexes and enhances stringency by reducing the probability of random background ligation events. Circle formation also increases selectivity, since the uncircularized DNA can be removed enzymatically. We compare this method with traditional PLA on several biomarkers and show that the higher stringency for c-PLA improves reproducibility and enhances sensitivity in both buffer and human plasma. The limit of detection ranges from femtomolar to nanomolar concentrations for both methods. Kinetic analyses using surface plasmon resonance (SPR) and biolayer interferometry (BLI) reveal that the variation in limit of detection is due to the variation in antibody affinity and that c-PLA outperforms traditional PLA for low-affinity antibodies. The lower background signal can be used to increase proximity probe concentration while maintaining a high signal-to-noise ratio, thereby enabling the use of low-affinity reagents in a homogeneous assay format. We anticipate that the advantages of c-PLA will be useful in a variety of clinical protein detection applications where high-affinity reagents are lacking.

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“…Antibody-based multiplex sensing assays have been developed in various planar and bead array formats and are becoming accepted methods for protein expression and interaction studies (MacBeath and Schreiber 2000; Schweitzer et al 2002; Xie et al 2009). However, the function of these arrays may be impaired by the high local concentrations of surface-bound antibodies and non-specific interactions between the substrate and the antibodies or analytes (Zichi et al 2008).…”

Section: Introductionmentioning

confidence: 99%

Development of a dual-aptamer-based multiplex protein biosensor

Xie

Walton

2010

Biosensors and Bioelectronics

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Parallel biosensors for proteins are becoming more essential for the thorough and systematic investigation of complex biological processes. These tools also enable improved clinical diagnoses relative to single protein analyses due to their greater information content. If implemented correctly, affinity-based techniques can provide unique advantages in terms of sensitivity and flexibility. Aptamers are increasingly being used as the affinity reagents of choice for protein biosensing applications. Here, we describe the development and characterization of an aptamer-based method for parallel protein analyses that relies on recognition of the target protein by two unique aptamers targeting different epitopes on the protein. Our results show that the technique achieved simultaneous and quantitative detection of thrombin and platelet derived growth factor-BB (PDGF-BB) with high specificity both in buffered solutions and in serum samples.

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Emerging affinity-based techniques in proteomics

Cited by 31 publications

References 118 publications

Proteomics of Plant Pathogenic Fungi

Proteomics of Plant Pathogenic Fungi

Streamlined circular proximity ligation assay provides high stringency and compatibility with low-affinity antibodies

Development of a dual-aptamer-based multiplex protein biosensor

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