A high-throughput capillary isoelectric focusing immunoassay for fingerprinting protein sialylation

Markely, Lam Raga A.; Cheung, Lila; Choi, Young Jun; Estes, Scott; Prajapati, Shashi; Turyan, Iva; Frenkel, Ruth; Sosic, Zoran; Lambropoulos, James; Tescione, Lia; Ryll, Thomas; Berman, Melissa

doi:10.1002/btpr.2206

Cited by 5 publications

(4 citation statements)

References 36 publications

(61 reference statements)

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“…Additionally, capillary nano-immunoassay, based on the isoelectric point (pI) of protein, requires very small amounts of cell samples and provides the information of different isomers, which exhibits higher sensitivity than traditional western blotting assay. 22 , 23 Because pI value is sensitive to different isomers, the mutant or post-transcriptional modified proteins exhibit the different peak signal, and the size of the area under the curve presents the expression levels of protein.…”

Section: Discussionmentioning

confidence: 99%

Novel diagnostic approaches for Fanconi anemia (FA) by single-cell sequencing and capillary nano-immunoassay

Chang

Gao

et al. 2021

Blood Science

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Next-generation sequencing technology has been widely utilized for the diagnosis of Fanconi anemia (FA). However, mixed cell sequencing and chimerism of FA patients may lead to unconfirmed genetic subtypes. Herein, we introduced two novel diagnostic methods, including single-cell sequencing and capillary nano-immunoassay. One FA case with FANCM c.4931G>A p.R1644Q and FANCD1 c.6325G>A p.V2109I was studied. The DNA of 28 cells was amplified and eight types of cells were observed after Sanger sequencing. There were two homozygous mutations ( FANCM / FANCD1 ). Furthermore, the capillary nano-immunoassay was conducted to analyze the expression profile of FA-associated proteins. Abnormal FANCM and FANCD1 expressions simultaneously existed. This case was thus diagnosed as FA-D1/FA-M dual subtype. Compared with mixed cell sequencing, single-cell sequencing data shows more accuracy for the FA subtype evaluation, while the capillary nano-immunoassay is a good method to detect the expression profile of abnormal or modified FA protein.

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Section: Discussionmentioning

confidence: 99%

Novel diagnostic approaches for Fanconi anemia (FA) by single-cell sequencing and capillary nano-immunoassay

Chang

Gao

et al. 2021

Blood Science

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“…This assay, also called Simple Western™ Charge, can similarly be applied to proteins separated by size, called Simple Western Size [13]. Simple Western Charge was employed to rapidly characterize sialylation patterns without requiring protein purification [14]. In this study, we introduce a proofof-concept for CDT analysis that combines the charge-based separation of icIEF with the sensitivity of immunodetection, both of which are performed seamlessly back to back on a single Simple Western platform (Peggy Sue™ from ProteinSimple, CA, USA).…”

Section: Graphical Abstractmentioning

confidence: 99%

“…Finally, a luminol-peroxide solution is applied for chemiluminescence imaging of the whole capillary. More information on the principles of this assay may be found in a similar study that characterized protein sialylation patterns [14]. Following sample preparation, all steps are automatically performed on the Peggy Sue instrument without the need for analyst supervision.…”

Section: Workflow Overviewmentioning

confidence: 99%

A Proof-of-concept Analysis of Carbohydrate-deficient Transferrin by Imaged Capillary Isoelectric Focusing and In-capillary Immunodetection

Haitjema

Heger

et al. 2020

BioTechniques

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Carbohydrate-deficient transferrin (CDT) is a reliable biomarker for chronic alcohol abuse. We developed a method for CDT analysis by capillary isoelectric focusing, followed by immunodetection directly in the capillary, in an automated fashion and on a single platform (Peggy Sue™; ProteinSimple, CA, USA). Transferrin glycoforms in serum samples, including disialo-transferrin, were separated and their apparent isoelectric points and relative percentages were determined. The relative CDT values (percent of total transferrin) matched expected values for both healthy and alcoholic samples. Because the method leveraged the sensitivity of an immunoassay, CDT was measured when serum samples were diluted up to 1200-fold, reducing the volume of serum required. Finally, the process is fully automated, with up to 96 samples analyzed per batch.

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“…A long-standing dilemma in the biopharmaceutical industry has been the comparison and evaluation of orthogonal methods to characterize and quantify covalent modifications of proteins such as deamidation, oxidation, and glycation . These modifications are considered critical quality attributes (CQAs) due to their ability to affect drug efficacy. − To reach a comprehensive view of these and other protein modifications, MS analyses are often performed on both intact proteins and the peptide products of a proteolytic digestion (bottom-up).…”

Section: Introductionmentioning

confidence: 99%

Orthogonal Comparison of Analytical Methods by Theoretical Reconstruction from Bottom-up Assay Data

Nichols

Kil

Mahan

et al. 2021

J. Am. Soc. Mass Spectrom.

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In the never-ending endeavor to produce stable and efficacious protein therapeutics, biopharmaceutical companies often employ numerous analytical techniques to characterize and quantify a drug candidate’s stability. Mass spectrometry, due to the information-rich data it produces, is commonly used in its numerous configurations to ascertain chemical and structural stability. At issue is the comparison of the various configurations utilized, that is, comparing bottom-up methods such as proteolytic digest followed by reversed phase LC-MS with intact LC-MS methods. Similar issues also arise when using capillary isoelectric focusing to see how charge variants change over time, that is, monitoring the progression of charge altering modifications like deamidation. To this end, site-specific degradations as quantified from bottom-up methods like peptide mapping can be used to build reconstructions of both theoretical intact mass spectra as well as theoretical electropherograms. The result can then be superimposed over the experimental data to qualitatively, and perhaps quantitatively, evaluate differences. In theory, if both experimental bottom-up data and intact data are accurate, the theoretical reconstruction produced from the bottom-up data should perfectly overlay with that of the experimental data. Valuable secondary information can also be ascertained from reconstructions, such as whether modifications are stochastic, as well as a detailed view of all possible combinations of modifications and their quantities used in the reconstruction. This comparison is also useful in determining unknown mass differences in deconvoluted intact protein spectra that may be a result of multiple modifications in combination. The comparison of data from alternate sources provides a holistic and more comprehensive view of the molecule under study.

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A high-throughput capillary isoelectric focusing immunoassay for fingerprinting protein sialylation

Cited by 5 publications

References 36 publications

Novel diagnostic approaches for Fanconi anemia (FA) by single-cell sequencing and capillary nano-immunoassay

Novel diagnostic approaches for Fanconi anemia (FA) by single-cell sequencing and capillary nano-immunoassay

A Proof-of-concept Analysis of Carbohydrate-deficient Transferrin by Imaged Capillary Isoelectric Focusing and In-capillary Immunodetection

Orthogonal Comparison of Analytical Methods by Theoretical Reconstruction from Bottom-up Assay Data

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