Identification of proteins in single-cell capillary electrophoresis fingerprints based on comigration with standard proteins.
AuthorsHu S, Le Z, Newitt R, Aebersold R, Kraly JR, Jones M, Dovichi NJ,
JournalAnal Chem
PubMed ID14570203
'In the previous paper in this Journal, we reported the use of capillary sieving electrophoresis to characterize proteins expressed by single cancer cells at specific phases in the cell cycle. Analysis of the data revealed one component with cell cycle-dependent changes in expression at the 99% confidence limit. However, the ... More
Kinetics and apparent activation energy of the reaction of the fluorogenic reagent 5-furoylquinoline-3-carboxaldehyde with ovalbumin.
JournalJ Chromatogr B Analyt Technol Biomed Life Sci
PubMed ID12880858
'Incomplete labeling of proteins by a derivatizing reagent usually results in the formation of a large number of products, which can produce unacceptable band broadening during electrophoretic analysis. In this paper, we report on the reaction of the fluorogenic reagent 5-furoylquinoline-3-carboxaldehyde (FQ) with the lysine residues of ovalbumin. Mass spectrometry ... More
Solid-phase fluorescent labeling reaction of picomole amounts of insulin in very dilute solutions and their analysis by capillary electrophoresis.
AuthorsPinto DM, Arriaga EA, Sia S, Li Z, Dovichi NJ
JournalElectrophoresis
PubMed ID7588523
'The fluorescent labeling of peptides at concentrations as low as 10(-8) M can be achieved by using a solid-phase reactor. Using oxidized insulin chain B as a test peptide, we demonstrate the use of an Immobilon CD membrane to capture and preconcentrate peptides. Insulin chain B can then be labeled ... More
MitoTracker Green labeling of mitochondrial proteins and their subsequent analysis by capillary electrophoresis with laser-induced fluorescence detection.
AuthorsPresley AD, Fuller KM, Arriaga EA
JournalJ Chromatogr B Analyt Technol Biomed Life Sci
PubMed ID12880861
'MitoTracker Green (MTG) is a mitochondrial-selective fluorescent label commonly used in confocal microscopy and flow cytometry. It is expected that this dye selectively accumulates in the mitochondrial matrix where it covalently binds to mitochondrial proteins by reacting with free thiol groups of cysteine residues. Here we demonstrate that MTG can ... More
Analysis of aminophospholipid molecular species by methyl-beta-cyclodextrin modified micellar electrokinetic capillary chromatography with laser-induced fluorescence detection.
AuthorsZhang L, Hu S, Cook L, Dovichi NJ
JournalElectrophoresis
PubMed ID12207317
'Micellar electrokinetic capillary chromatography (MEKC) with laser-induced fluorescence detection is used for the analysis of three classes of aminophospholipids: phosphatidylethanolamine (PE), phosphatidylserine (PS), and lysophosphatidylethanolamine (LPE) molecular species. 3-(2-Furoyl) quinoline-2-carboxaldehyde (FQ), a fluorogenic dye, was employed for labeling of these phospholipids. The FQ-labeled lipid species were then separated by sodium ... More
Recent advances in methods for the analysis of catecholamines and their metabolites.
AuthorsTsunoda M
JournalAnal Bioanal Chem
PubMed ID16924378
'Catecholamines, for example epinephrine, norepinephrine, and dopamine, are widely distributed and are important neurotransmitters and hormones in mammalian species. Several methods have been developed for analysis of catecholamines and related compounds. Determination of catecholamines in biological fluids has enabled us to clarify the physiological role played by these amines. Catecholamine ... More
Quantification of carbonylated proteins in rat skeletal muscle mitochondria using capillary sieving electrophoresis with laser-induced fluorescence detection.
AuthorsFeng J, Arriaga EA,
JournalElectrophoresis
PubMed ID18064596
'Carbonyl-modified proteins are markers of oxidative damage. Here, we report a new method for detecting and quantifying carbonylated proteins by capillary sieving electrophoresis (CSE) with LIF detection (CSE-LIF). Alexa 488 hydrazide is used for the specific labeling of carbonyls while 3-(2-furoyl) quinoline-2-carboxaldehyde (FQ) is used for protein labeling. BSA subjected ... More
Capillary sodium dodecyl sulfate-DALT electrophoresis of proteins in a single human cancer cell.
AuthorsHu S, Zhang L, Cook LM, Dovichi NJ
JournalElectrophoresis
PubMed ID11699905
'Capillary Sodium dodlecyl sulfate (SDS)-DALT an (abbreviation for Dalton) electrophoresis was applied to analysis of proteins in single HT29 human colon adenocarcinoma cells. A vacuum pulse was employed to introduce a single cell into the coated capillary. Once the cell was lysed, proteins were denatured with SDS, fluorescantly labeled with ... More
Picomolar analysis of proteins using electrophoretically mediated microanalysis and capillary electrophoresis with laser-induced fluorescence detection.
'We report a method for the analysis of picomolar concentration proteins using electrophoretically mediated microanalysis (EMMA) to label proteins on-column with a fluorogenic reagent. Labeling is followed by capillary zone electrophoresis separation and postcolumn detection based on laser-induced fluorescence. The method provides a concentration detection limit (3 sigma) of 3 ... More
Sodium dodecyl sulfate-capillary electrophoresis of proteins in a sieving matrix utilizing two-spectral channel laser-induced fluorescence detection.
AuthorsCraig DB, Polakowski RM, Arriaga E, Wong JC, Ahmadzadeh H, Stathakis C, Dovichi NJ
JournalElectrophoresis
PubMed ID9761200
We report a method for protein labeling, separation by capillary electrophoresis in a polymer sieving matrix, and detection by laser-induced fluorescence. Different dyes are used to label standard and sample proteins. A two-spectral channel detector resolves fluorescence from the sample and standards. Comparison of the migration time of the sample ... More
Application of 3-(2-furoyl)quinoline-2-carbaldehyde as a fluorogenic reagent for the analysis of primary amines by liquid chromatography with laser-induced fluorescence detection.
AuthorsBeale SC, Hsieh YZ, Wiesler D, Novotny M
JournalJ Chromatogr
PubMed ID2324216
3-(2-Furoyl)quinoline-2-carbaldehyde (FQCA) has been synthesized and characterized as a fluorogenic derivatizing reagent for liquid chromatography. The reagent forms highly fluorescent isoindoles upon reaction with primary amines. The spectral properties and optimum reaction conditions for the formation of isoindoles have been investigated. The utility of FQCA for the high-sensitivity analysis of ... More
Labeling effects on the isoelectric point of green fluorescent protein.
We studied the effects of fluorescent labeling on the isoelectric points (pI values) of proteins using capillary isoelectric focusing with laser-induced fluorescence detection (cIEF-LIF). Specifically, we labeled green fluorescent protein (GFP) from the jellyfish Aequorea victoria with the fluorogenic dye 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ). cIEF-LIF was used to monitor the native fluorescence ... More
Non-SELEX: selection of aptamers without intermediate amplification of candidate oligonucleotides.
AuthorsBerezovski MV, Musheev MU, Drabovich AP, Jitkova JV, Krylov SN
JournalNat Protoc
PubMed ID17406423
Aptamers are typically selected from libraries of random DNA (or RNA) sequences through systematic evolution of ligands by exponential enrichment (SELEX), which involves several rounds of alternating steps of partitioning of candidate oligonucleotides and their PCR amplification. Here we describe a protocol for non-SELEX selection of aptamers--a process that involves ... More
Capillary sodium dodecyl sulfate-DALT electrophoresis with laser-induced fluorescence detection for size-based analysis of proteins in human colon cancer cells.
Capillary sodium dodecyl sulfate (SDS)-DALT electrophoresis (SDS-DALT-CE) refers to CE separation of proteins based on their size; DALT is the abbreviation for Dalton, the unit used to describe molecular weight. In this work, seven proteins from 18 to 116 kDa were denatured by SDS, labeled by 3-(2-furoyl) quinoline-2-carboxaldehyde, separated by ... More
Methyl-beta-cyclodextrin modified micellar electrokinetic capillary chromatography with laser-induced fluorescence for separation and detection of phospholipids.
AuthorsLe Zhang Krylov SN, Hu S, Dovichi NJ
JournalJ Chromatogr A
PubMed ID11100855
Micellar electrokinetic capillary chromatography (MECC) with laser-induced fluorescence detection was applied to the separation of amino group-containing phospholipids including phosphatidylethanolamine (PE), phosphatidylserine (PS), lysophosphatidylethanolamine (LysoPE), and lysophosphatidylserine (LysoPS). A fluorogenic dye, 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ), was successfully used to fluorescently label these phospholipids. 4-Fluoro-7-nitrobenzofurazan only produced fluorescent product from LysoPE and PE; ... More
Heterogeneity of protein labeling with a fluorogenic reagent, 3-(2-furoyl)quinoline-2-carboxaldehyde.
AuthorsStoyanov AV, Ahmadzadeh H, Krylov SN
JournalJ Chromatogr B Analyt Technol Biomed Life Sci
PubMed ID12401353
Fluorogenic reagents are used for protein labeling when high-sensitivity fluorescence detection is required. Similar to traditional labeling with activated fluorescent dyes, such as fluorescein isothiocyanate, a fluorogenic reaction is expected to change the physical-chemical properties of proteins. Knowledge of these changes may be essential for efficient separation and identification of ... More
Repeatability of chemical cytometry: 2-DE analysis of single RAW 264.7 macrophage cells.
AuthorsSobhani K, Fink SL, Cookson BT, Dovichi NJ
JournalElectrophoresis
PubMed ID17557367
This report presents the use of 2-DE with ultrasensitive fluorescence detection as a chemical cytometry tool to characterize the protein and biogenic amine content of single cells from the RAW 264.7 murine macrophage cell line. Cells were sorted by cell cycle prior to 2-DE analysis. Cells in the G2/M phase ... More
MEKC-LIF of gamma-amino butyric acid in microdialysate: systematic optimization of the separation conditions by factorial analysis.
AuthorsEhlen JC, Albers HE, Breyer ED
JournalJ Neurosci Methods
PubMed ID15979724
Micellar electrokinetic chromatography allows the efficient separation of biogenic amines and amino acids in biological samples. Analytes of interest, sample composition, and sample matrix may vary between studies, which necessitates optimization of separations to meet the requirements and conditions of an experiment. Factorial analysis is an efficient tool to accomplish ... More