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Selection of appropriate protein assay method for a paper microfluidics platform.

ABSTRACT: Background:Paper-analytical devices (PADs) have gained popularity as a simple and low-cost alternative for determining a wide range of analytes including proteins. Even though several colorimetric PADs methods for protein estimation are reported in literature, they lack justification for the chosen method and parameters therein. Aim:Major aim of this work was to thoroughly evaluate the most commonly used colorimetric protein assays and recommend the most appropriate method for PADs platform. Method:We performed following six colorimetric protein assays on PADs: biuret, lowry, bicinchoninic acid, bradford, bromocresol green, and tetrabromophenol blue. We obtained assay signal by analyzing images of the PADs and then assessed analytical figures of merit. Result:Precision, accuracy, LOD, and LOQ of PADs protein assay methods ranged from 1.2 to 6.4%, 73.3-102.4%, 0.3-3.8 ?mg/mL, and 1.2-12.8 ?mg/mL, respectively. Out of six methods, we determined bromocresol green and tetrabromophenol blue as the best methods for serum and urine samples, respectively based on their optimized parameters and analytical figures of merit. The total average serum and urine protein in human samples were found to be 94.6 ?± ?16.2 ?mg/mL and 2.1 ?± ?1.5 ?mg/mL, respectively using PADs methods. The PADs result on human samples moderately correlated with the results from spectrophotometric determination (r2 ?> ?0.6). Conclusion:Paper-based protein assays were easy to perform and were completed with thousand-fold less volume of samples/reagents without a spectrophotometer compared to conventional assay methods. After testing human samples, we found one protein assay method may not be appropriate for all types of samples.

SUBMITTER: Pokhrel P

PROVIDER: S-EPMC7248653 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Selection of appropriate protein assay method for a paper microfluidics platform.

Pokhrel Pravin P Jha Shashank S Giri Basant B

Practical laboratory medicine 20200516

<h4>Background</h4>Paper-analytical devices (PADs) have gained popularity as a simple and low-cost alternative for determining a wide range of analytes including proteins. Even though several colorimetric PADs methods for protein estimation are reported in literature, they lack justification for the chosen method and parameters therein.<h4>Aim</h4>Major aim of this work was to thoroughly evaluate the most commonly used colorimetric protein assays and recommend the most appropriate method for PAD ...[more]

PMID: 32478161

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Project description:Often plant tissues are recalcitrant and, due to that, methods relying on protein precipitation, such as TCA/acetone precipitation and phenol extraction, are usually the methods of choice for protein extraction in plant proteomic studies. However, the addition of precipitation steps to protein extraction methods may negatively impact protein recovery, due to problems associated with protein re-solubilization. Moreover, we show that when working with non-recalcitrant plant tissues, such as young maize leaves, protein extraction methods with precipitation steps compromise the maintenance of some labile post-translational modifications (PTMs), such as phosphorylation. Therefore, a critical issue when studying PTMs in plant proteins is to ensure that the protein extraction method is the most appropriate, both at qualitative and quantitative levels. In this work, we compared five methods for protein extraction of the C4-photosynthesis related proteins, in the tip of fully expanded third-leaves. These included: TCA/Acetone Precipitation; Phenol Extraction; TCA/Acetone Precipitation followed by Phenol Extraction; direct extraction in Lysis Buffer (a urea-based buffer); and direct extraction in Lysis Buffer followed by Cleanup with a commercial kit. Protein extraction in Lysis Buffer performed better in comparison to the other methods. It gave one of the highest protein yields, good coverage of the extracted proteome and phosphoproteome, high reproducibility, and little protein degradation. This was also the easiest and fastest method, warranting minimal sample handling. We also show that this method is adequate for the successful extraction of key enzymes of the C4-photosynthetic metabolism, such as PEPC, PPDK, PEPCK, and NADP-ME. This was confirmed by MALDI-TOF/TOF MS analysis of excised spots of 2DE analyses of the extracted protein pools. Staining for phosphorylated proteins in 2DE revealed the presence of several phosphorylated isoforms of PEPC, PPDK, and PEPCK.

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Selection of appropriate protein assay method for a paper microfluidics platform.

Publications

Selection of appropriate protein assay method for a paper microfluidics platform.

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