Post-column fluorescence derivatization of proteins and peptides in capillary electrophoresis with a sheath flow reactor and 488 nm argon ion laser excitation

Naphthalene-2,3-dicarboxyaldehyde (NDA) is commonly used for detection of primary amines in conjunction with their separation with HPLC and CE. The fluorescence of the derivatives can be measured by a conventional fluorometer or via LIF. NDA is a reactive dye, which can replace o-phthaldehyde (OPA) and provides for derivatives which are considerably more stable than OPA derivatives. In addition, NDA can be used to derivatize primary amines at concentrations as low as 100 pM. In this work, HPLC/fluorescence and MEKC/LIF experiments were performed to separate/detect six neuroactive compounds, the amino acids, Gly, Glu, Asp, gamma-aminobutyric acid (GABA) and the catecholamines, dopamine and noradrenaline. The two methods were compared in terms of performance of separation. The amino acids can be separated in HPLC in less than 30 min and an identical separation is obtained in CE using MEKC and lithium salts with greater resolution (the number of theoretical plates was approximately 5000 for HPLC and 200 000 for MEKC). The lowest detected concentration was in the range of 0.1 nM for CE/LIF. The presence of a high salt concentration does not affect the separation of the samples. Examples of the analysis of microdialysate samples as well as amino acids in Ringer's solution are presented.

“…Similarly, CE (CE/LIF) has been widely used for the study of amino acids [15,16], catecholamines [17] as well as peptides and proteins ( [18], for a review see [19]). …”

Section: Introductionmentioning

confidence: 99%

HPLC-fluorescence detection and MEKC-LIF detection for the study of amino acids and catecholamines labelled with naphthalene-2,3-dicarboxyaldehyde

Siri¹,

Lacroix

Garrigues

et al. 2006

“…While postcolumn derivatization of proteins has been demonstrated for the proteins separated in the free solution, i.e. CZE [21][22][23][24], there has been no report on postcolumn derivatization of proteins separated by CSE. In postcolumn derivatization of amino groups, o-phthaldialdehyde [21] and naphthalene-2,3-dicarbaldehyde (NDA) [25] are frequently employed as the fluorogenic labeling reagents.…”

Section: Introductionmentioning

confidence: 96%

“…Differences in the LOD for the standard proteins may be attributed to labeling at multiple sites, e.g. a protein, which has lysine residues to be reacted with NDA, produces high fluorescence intensity [24]. The protein derivatives produced in NDA-2-mercaptethanol system cannot be isolated, since they are instable [31].…”

mentioning

confidence: 99%

Postcolumn derivatization of proteins in capillary sieving electrophoresis/laser‐induced fluorescence detection

Kaneta¹,

Yamamoto²,

Imasaka³

2009

The separation methods for proteins with high resolution and sensitivity are absolutely important in the field of biological sciences. Capillary sieving electrophoresis (CSE) is an excellent separation technique for DNA and proteins with high resolution, while LIF permits the most sensitive detection in CSE. Therefore, proteins have to be labeled with fluorescent or fluorogenic reagent to produce fluorescent derivatives. Both precolumn and oncolumn derivatization have been employed for the labeling of proteins in CSE. However, there is no report on the postcolumn derivatization due to the limitation in the use of a standard migration buffer, despite it being a promising method for sensitive detection of proteins. Here, we show a novel postcolumn derivatization method for protein separation by CSE, using a tertiary amine as a buffer component in the running buffer. Tris, which is commonly used as a base in CSE separation buffers, was substituted by tertiary amines, 2-(diethylamino)ethanol and triethanolamine. A buffer solution containing 2-(diethylamino)ethanol or triethanolamine can be used for the CSE separation followed by the postcolumn derivatization of proteins, since both reagents are unreactive toward a fluorogenic labeling reagent, naphthalene-2,3-dicarbaldehyde. Thus, LIF detection using the postcolumn derivatization permits significant reduction in the LOD (by a factor of 2.4-28) of proteins, compared with conventional absorbance detection.

“…Dynamic coatings using buffer additives are becoming increasingly popular as they are used to control EOF by adjusting the concentration of the additives and improve the selectivity by interacting in different extent with the proteins [17][18][19][20][21][22][23]. Additives that have been used include didodecyldimethylammonium bromide (DDAB), CTAB, and tetradecyltrimethylammonium bromide [17][18][19]. In addition to these cationic surfactants, cationic polyelectrolyes, such as polyethyleneimine, Polybrene, and poly-(diallyldimethylammonium chloride) (PDDA) have been performed [20][21][22][23][24].…”

Section: Introductionmentioning

confidence: 99%

“…Dynamic coating is simple to perform through hydrogen bonding, coulombic attraction, and hydrophobic interaction, but requires repeated replenishment for reproducible operations [15,16]. Dynamic coatings using buffer additives are becoming increasingly popular as they are used to control EOF by adjusting the concentration of the additives and improve the selectivity by interacting in different extent with the proteins [17][18][19][20][21][22][23]. Additives that have been used include didodecyldimethylammonium bromide (DDAB), CTAB, and tetradecyltrimethylammonium bromide [17][18][19].…”

Section: Introductionmentioning

confidence: 99%

Online concentration and separation of basic proteins using a cationic polyelectrolyte in the presence of reversed electroosmotic flow

Tseng

2006

We report an online concentration and separation method for basic proteins using poly(diallyldimethylammonium chloride) (PDDA) solutions in the presence of reversed EOF. Using a capillary dynamically coated with 2% PDDA containing 0.1 M NaCl and filled with 1.2% PDDA under neutral conditions (10 mM phosphate, pH 7.0), we have demonstrated the separation of six basic proteins with peak efficiencies ranging from 175 000 to 616 000 plates/m and RSDs of migration time less than 0.4%. Additionally, high-speed separation of six basic proteins (<7 min) was achieved using a short capillary filled with 0.6% PDDA solutions. Under injection of the large-volume sample (210 nL), the LODs at S/N of 3 for basic proteins are down to nanomolar range. For example, the LOD for lysozyme is 1.2 nM, which is a 260-fold sensitivity enhancement compared with conventional injection method. The proposed method has been applied to the stacking of lysozyme in human saliva samples. Without any pretreatment, we also demonstrated the capability of this method to detect low amounts of peptide samples through the stacking of tryptic peptide of myoglobin. The experimental results indicate that our proposed method has great potential for use in clinical diagnosis and proteomics applications.