Hydrophobic interaction chromatography coupled with atomic fluorescence spectrometric detectionEffect of the denaturation on the determination of thiolic proteins (original) (raw)
Related papers
Hydrophobic interaction chromatography coupled with atomic fluorescence spectrometric detection
Talanta, 2004
Hydrophobic interaction chromatography coupled online with chemical vapour atomic fluorescence spectrometry (HIC-CVGAFS) has been optimized for the analysis of thiolic proteins in denaturing conditions. Proteins are pre-column simultaneously denatured and derivatized in phosphate buffer solution containing 8.0 mol dm −3 urea and p-hydroxymercurybenzoate (PHMB) and the derivatized denatured proteins are separated on a silica HIC Eichrom Propyl column in the presence of 8.0 M urea in the mobile phase. Post-column online reaction of derivatized denatured proteins with bromine, generated in situ by KBr/KBrO 3 in HCl medium, allowed the fast conversion of the uncomplexed PHMB and of the PHMB bound to proteins to inorganic mercury also in presence of urea. Hg 2+ , present in solution as Hg 2+ -urea complex, is selectively detected by AFS in a Ar/H 2 miniaturized flame after sodium borohydride reduction to Hg. Under optimized conditions, online bromine treatment gives a 100 ± 2% recovery of both free and protein-complexed PHMB. Denatured glyceraldehyde-3-phosphate dehydrogenase, aldolase, lactate dehydrogenase, trioso phosphate isomerase and -lactoglobulin have been examined. As the sensitivity and limit of detection of proteins in the HIC-CVGAFS apparatus depends on number of -SH groups reacting with PHMB, the denaturation process, which increases the number of PHMB-reactive thiolic groups in proteins, improves the analytical performances of the described system in protein analysis. The detection limit for the denatured proteins examined was found in the range of 10 −10 -10 −12 mol dm −3 , depending on the considered protein, with linear calibration curves spanning over four decades of concentration.
Analytical and Bioanalytical Chemistry, 2004
Hydrophobic-interaction chromatography coupled on-line with chemical-vapor-generation atomicfluorescence spectrometry (HIC-CVGAFS), optimized recently for the analysis of thiol-containing proteins under denaturing conditions, has been used to study the chemical reduction of denatured proteins. Four proteins chosen as models (human serum albumin (HSA), bovine serum albumin (BSA), a-lactalbumin (a-Lac) from bovine milk, and lysozyme from chicken egg (Lys)) were denatured with urea and reduced with dithiothreitol (DTT), with selenol as catalyst. The method is based on derivatization of the -SH groups of proteins with p-hydroxymercurybenzoate (PHMB), followed by HIC separation and post-column on-line reaction of the derivatized reduced, denatured proteins with bromine generated in situ. Hg II , derived from rapid conversion of uncomplexed and protein-complexed PHMB, is selectively detected by AFS in an Ar/H 2 miniaturized flame after sodium borohydride (NaBH 4 ) reduction to Hg°. The yield of the reduction was studied as a function of reductant concentration, reduction time (t red ), and urea concentration. Results showed that the optimum values for DTT and selenol concentrations and for t red were between 1 and 100 mmol L À1 and between 1 and 20 min, respectively, depending on the protein studied. The percentage disulfide bond reduction increases as the urea concentration used for protein denaturation increases, giving a single-step sigmoid increment for single-domain, low-MW proteins (a-Lac and Lys), and a two-step sigmoid increment for multi-domain, high MW proteins (HSA and BSA). The shapes of plots of percentage reduced disulfide against urea concentration are characteristic of each protein and are correlated with the location of S-S in the protein. Under the adopted conditions complete protein denaturation is the conditio sine qua non for obtaining 100% S-S reduction. The detection limit for denatured, reduced proteins examined under the optimized conditions was found to be in the range 1-5·10 À12 mol L À1 (10-30 pg), depending on the protein considered.
Detection of reversible protein thiol modifications in tissues
Analytical Biochemistry, 2006
Oxidation/reduction reactions of protein thiol groups (PSH) have been implicated in many physiological and pathological processes. Although many new techniques for separation and identiWcation of modiWed cysteinyl residues in proteins have been developed, critical assessment of reagents and sample processing often are overlooked. We carefully compared the eVectiveness of N-ethylmaleimide (NEM), iodoacetamide (IAM), and iodoacetic acid (IAA) in alkylating protein thiols and found that NEM required less reagent (125 vs. 1000 mol:mol excess), required less time (4 min vs. 4 h), and was more eVective at lower pHs (4.3 vs. 8.0) in comparison with IAM and IAA. The relative eYcacy of dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP) for reducing protein disulWdes suspended in NaPO 4 buVer or MeOH was assessed, and no diVerences in total normalized Xuorescence were detected at the concentrations tested (10-100 mM); however, individual band resolution appeared better in samples reduced with DTT in MeOH. In addition, we found that oxidation ex vivo was minimized in tissue samples that were homogenized in aqueous buVers containing excess molar quantities of NEM compared with samples homogenized in MeOH containing NEM. Using NEM for thiol alkylation, DTT for disulWde reduction, and mBBr for labeling the reduced disulWde and Xuorimetric detection, we were able to generate an in-gel standard curve and quantitate total disul-Wde contents within biological samples as well as to identify changes in speciWc protein bands by scanning densitometry. We demonstrated that reagents and techniques we have identiWed for disulWde detection in complex samples are also applicable to two-dimensional electrophoresis separations.
Proceedings of the National Academy of Sciences, 1975
Activated thiol-Sepharose [agarose-(glutathione-2-pyridyl disulfide) conjugate] has been used to immobilize proteins with a single or a few thiol groups via disulfide bridges. The immobilized proteins were subsequently proteolytically degraded. After washing, the thiol-containing peptides were eluted with a reducing agent. A single preparative paper electrophoresis, occasionally after a modification such as oxidation, was sufficient to obtain pure peptides in good yields. The method was applied to the major parvalbumin from hake muscle (a protein with 108 amino acid residues and one cysteine residue), to mercaptalbumin from bovine serum (565 residues and one cysteine), and to human serum ferroxidase [EC 1.16.3.1; iron (II):oxygen oxidoreductase] (ceruloplasmin) (1065 residues and three cysteines). The use of the technique, e.g., as a simple means of obtaining homologous peptides in related proteins, is discussed.
Highly selective assay of proteins in dilute solutions
Talanta, 2002
A very simple, highly selective assay of proteins has been developed. Solutions of 6 M hydrochloric acid containing 0-20 mg ml − 1 protein are subjected to acid hydrolysis, in the presence of air, during their evaporation to dryness on a steam bath. Then, the resulted amino acids are determined with a 2,2-dihydroxy-1,3-indanedione reagent. To eliminate any interference and to increase in selectivity of the assay, a blank with the unhydrolyzed protein is carried out. The protein being analyzed or alanine is used to plot calibration curves. The absorbance of the colored solution is read at 516 nm. The color system obeys Beer's law in the range 0.1-20 mg ml − 1 protein. The molar absorptivity for alanine was found to be 1.69 × 10 3 (99.7) l mol − 1 cm − 1 . Also, the relative molar absorptivity for peptone was found to be 1.23 ×103 ( 96.5) l mol − 1 cm − 1 . The lowest limits for the estimation of serum albumin, gelatin, and peptone are around 0.25, 0.19 and 0.31 mg ml − 1 , respectively. The method is highly selective and the determination is little affected by the presence of other substances. All other important analytical parameters were studied and practical applicability of the method has been verified by the analysis of some biological materials.
Methods for the determination and quantification of the reactive thiol proteome
Free Radical Biology and Medicine, 2009
Protein thiol modifications occur under both physiological and pathological conditions and have been shown to contribute to changes in protein structure, function, and redox signaling. The majority of protein thiol modifications occur on cysteine residues that have a low pKa; these nucleophilic proteins comprise the “reactive thiol proteome.” The most reactive members of this proteome are typically low-abundance proteins. Therefore, sensitive and quantitative methods are needed to detect and measure thiol modifications in biological samples. To accomplish this, we have standardized the usage of biotinylated and fluorophore-labeled alkylating agents, such as biotinylated iodoacetamide (IAM) and N-ethylmaleimide (NEM) and BODIPY-labeled IAM and NEM, for use in one- and two-dimensional proteomic strategies. Purified fractions of cytochrome c and glyceraldehyde-3-phosphate dehydrogenase were conjugated to a known amount of biotin or BODIPY fluorophore to create an external standard that can be run on standard SDS–PAGE gels, which allows for the quantification of protein thiols from biological samples by Western blotting or fluorescence imaging. A detailed protocol is provided for using thiol-reactive probes and making external standards for visualizing and measuring protein thiol modifications in biological samples.
Analytical Biochemistry, 2010
Serum low-molecular-weight proteins (LMWPs, molecular weight <30 kDa) are closely related to the body physiological and pathological situations, whereas many difficulties are encountered when enriching and fractionating them. Using C 18 absorbent (100 Å) enrichment and fractionation under urea/dithiothreitol (DTT) denatured environment followed by 60% acetonitrile (ACN) elution, serum LMWPs could be enriched more than 100-fold and were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2-DE), and isotope-coded affinity tag (ICAT) labeling quantification. Proteins existing in human serum at low nanograms/milliliter (ng/ml) levels, such as myeloid-related proteins (MRPs), could be identified directly from 2-DE coupled with matrixassisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS) and LTQ-Orbitrap MS. Sixteen proteins were confidentially identified and quantified using ICAT labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS). By virtue of its easy operation and high reproducibility to process large quantity complex serum samples, this method has potential uses in enriching LMWPs either in serum or in cell and tissue samples.