Biarsenical−Tetracysteine Motif as a Fluorescent Tag for Detection in Capillary Electrophoresis

Kottegoda, Sumith; Aoto, Phillip C.; Sims, Christopher E.; Allbritton, Nancy L.

doi:10.1021/ac8003242

Cited by 17 publications

(14 citation statements)

References 36 publications

(83 reference statements)

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“…35,36 A detailed description of the custom-built system can be found in the Supporting Information. Fused silica capillaries [30 μm inner diameter, 360 μm outer diameter (Polymicro Technologies, Phoenix, AZ)] had a total length of 38 cm and an effective length of 21.5 cm.…”

Section: Experimental Sectionmentioning

confidence: 99%

Measurement of Protein Kinase B Activity in Single Primary Human Pancreatic Cancer Cells

et al. 2014

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An optimized peptide substrate was used to measure protein kinase B (PKB) activity in single cells. The peptide substrate was introduced into single cells, and capillary electrophoresis was used to separate and quantify nonphosphorylated and phosphorylated peptide. The system was validated in three model pancreatic cancer cell lines before being applied to primary cells from human pancreatic adenocarcinomas propagated in nude mice. As measured by phosphorylation of peptide substrate, each tumor cell line exhibited statistically different median levels of PKB activity (65%, 21%, and 4% phosphorylation in PANC-1 (human pancreatic carcinoma), CFPAC-1 (human metastatic ductal pancreatic adenocarcinoma), and HPAF-II cells (human pancreatic adenocarcinoma), respectively) with CFPAC-1 cells demonstrating two populations of cells or bimodal behavior in PKB activation levels. The primary cells exhibited highly variable PKB activity at the single cell level, with some cells displaying little to no activity and others possessing very high levels of activity. This system also enabled simultaneous characterization of peptidase action in single cells by measuring the amount of cleaved peptide substrate in each cell. The tumor cell lines displayed degradation rates statistically similar to one another (0.02, 0.06, and 0.1 zmol pg–1 s–1, for PANC-1, CFPAC-1, and HPAF-II cells, respectively) while the degradation rate in primary cells was 10-fold slower. The peptide cleavage sites also varied between tissue-cultured and primary cells, with 5- and 8-residue fragments formed in tumor cell lines and only the 8-residue fragment formed in primary cells. These results demonstrate the ability of chemical cytometry to identify important differences in enzymatic behavior between primary cells and tissue-cultured cell lines.

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Section: Experimental Sectionmentioning

confidence: 99%

Measurement of Protein Kinase B Activity in Single Primary Human Pancreatic Cancer Cells

et al. 2014

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“…Detection of TC‐labeled peptides and proteins can be achieved by micellar electrokinetic chromatography (MEKC)—a capillary electrophoresis system with laser‐induced fluorescence (LIF) detection. This setup allowed detection of as little as 3×10 −20 moles of a FlAsH–FLNCCPGCCMEP complex 142. In another study, capillary zone electrophoresis (CZE) with LIF detection was reported to detect 6×10 −12 mol of the E. coli FabI protein 139…”

Section: Application In Biotechnology and Analytical Biochemistrymentioning

confidence: 99%

Exploration of Biarsenical Chemistry—Challenges in Protein Research

Pomorski

Krężel

2011

ChemBioChem

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The fluorescent modification of proteins (with genetically encoded low-molecular-mass fluorophores, affinity probes, or other chemically active species) is extraordinarily useful for monitoring and controlling protein functions in vitro, as well as in cell cultures and tissues. The large sizes of some fluorescent tags, such as fluorescent proteins, often perturb normal activity and localization of the protein of interest, as well as other effects. Of the many fluorescent-labeling strategies applied to in vitro and in vivo studies, one is very promising. This requires a very short (6- to 12-residue), appropriately spaced, tetracysteine sequence (-CCXXCC-); this is either placed at a protein terminus, within flexible loops, or incorporated into secondary structure elements. Proteins that contain the tetracysteine motif become highly fluorescent upon labeling with a nonluminescent biarsenical probe, and form very stable covalent complexes. We focus on the development, growth, and multiple applications of this protein research methodology, both in vitro and in vivo. Its application is not limited to intact-cell protein visualization; it has tremendous potential in other protein research disciplines, such as protein purification and activity control, electron microscopy imaging of cells or tissue, protein-protein interaction studies, protein stability, and aggregation studies.

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“…The biarsenical labeling was extended to fluorescent detection of analytes in CE 71. Recombinant protein or synthesized peptides containing the optimized tetracysteine motif “‐C‐C‐P‐G‐C‐C‐” were labeled with the biarsenical dyes and then analyzed by MECC.…”

Section: Detection Schemes and Approachesmentioning

confidence: 99%

Electrophoretic and electrochromatographic separation of proteins in capillaries: An update covering 2007–2009

Rassi

2009

Electrophoresis

123

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This review article covers 3-year period from 2007 to 2009 and is a continuation of the review article by V. Dolnik, [Electrophoresis 2008, 29, 143-156]. This article with 125 references describes recent developments in CE and CEC of proteins in capillary format and does not cover the developments of CE and CEC in microchip format, since Tran et al. review the microchip subject in this special issue. The present review article has four major topics including (i) the separation media, (ii) multidimensional separations, (iii) detection, and (iv) applications.

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Biarsenical−Tetracysteine Motif as a Fluorescent Tag for Detection in Capillary Electrophoresis

Cited by 17 publications

References 36 publications

Measurement of Protein Kinase B Activity in Single Primary Human Pancreatic Cancer Cells

Measurement of Protein Kinase B Activity in Single Primary Human Pancreatic Cancer Cells

Exploration of Biarsenical Chemistry—Challenges in Protein Research

Electrophoretic and electrochromatographic separation of proteins in capillaries: An update covering 2007–2009

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