人血清Alpha-1-酸性糖蛋白的糖基化分析

获取真实准确的蛋白质糖基化信息是全面了解糖基化修饰生物学功能的前提.针对简单蛋白质的糖基化分析通常采用反相高效液相色谱-串联质谱技术在肽的水平上对糖基化信息进行采集和解析.本文以人血清Alpha-1-酸性糖蛋白(AGP)酶解液为对象,发展了一套简单有效的蛋白质糖基化分析方法.本方法分为三个步骤,第一步是建立糖肽的理论m/z值表;第二步是获取糖蛋白酶解液的LC-MS谱图,并将每一个色谱峰中所包含糖肽的实际m/z值与理论m/z值进行人工匹配;第三步是对每个色谱峰的糖肽结构归属进行LC-MS/MS验证.采用本方法,我们从AGP酶解液中共鉴定出172条糖肽.与单独采用Survey模式的方法相比,本方法能够显著提高糖肽的覆盖率.     

The putative use of alpha-1-acid glycoprotein as a non-invasive marker of fibrosis

The acute phase response to injury or infection results in alterations in the expression of the plasma proteins produced by the liver. Many of these biomolecules are glycosylated with oligosaccharide chains covalently attached to the polypeptide backbone and the extent and composition of this glycosylation can be altered in a disease-dependent manner. Of particular interest is the observation that the acute phase glycoprotein, alpha-1-acid glycoprotein (AGP) has altered glycosylation in several physiological and pathological conditions. It is posited that changes induced in liver diseases may reflect disease severity and may therefore act as a non-invasive marker of fibrosis. This study has investigated the glycosylation of AGP in the plasma of people with varying degrees of cirrhosis and fibrosis. Hyperfucosylation was observed in all disease samples in comparison to normal plasma and was significantly increased in cirrhosis. Both sialic acid and N-acetylgalactosamine (GalNAc) were negatively associated with fibrosis. Two samples were found to express GalNAc, which as a constituent of the glycosylation of serum proteins is rare. In conclusion, fucose, sialic acid and other aspects of the glycosylation of AGP are influenced by the degree of fibrosis and as such may prove a valuable prognostic indicator of the development of cirrhosis.     

Analysis of low‐molecular mass aldehydes in drinking waters through capillary electrophoresis with laser‐induced fluorescence detection

The potential of CZE with LIF detection in the separation and determination of low‐molecular mass aldehydes involving precolumn derivatization with fluorescein 5‐thiosemicarbazide was investigated. Different variables that affect derivatization (pH, fluorescein 5‐thiosemicarbazide concentration, time and temperature) and separation (pH and concentration of the BGE, kind and concentration of surfactants at levels higher and lower than CMC, and applied voltage) were studied. The separation was conducted within 16 min by using borate buffer (60 mM; pH 10) with 10 μM polyethylene glycol tert‐octylphenyl ether as modifier. Good linearity relationships (correlation coefficients ranged from 0.9978 to 0.9994 for aldehydes) were obtained between the peak areas and concentration of the analytes (0.5–100 μg/L). The LODs for aldehydes were achieved at submicrogram‐per‐liter level (0.15–0.35 μg/L), which indicated that the proposed method surpassed other electrophoretric alternatives in terms of LOD, in many cases even at ca. 1000‐fold. The inter‐day precision (RSD, %) of the aldehydes ranged from 5.2 to 8.3%. Finally, the method was successfully applied to bottled drinking‐water samples, and the aldehydes were readily detected at 0.6–4.4 μg/L levels with average recoveries ranging from 99.1 to 103.5%.     

Comparison of alpha-1-acid glycoprotein isoforms from healthy and cancer patients by capillary IEF

alpha-1-Acid glycoprotein (AGP) is a glycoprotein that presents different forms in the same individual, depending on the amino acid sequence and/or on the carbohydrate distribution of each form. Changes in these two types of heterogeneities are related to pathophysiological states. The aim of this work is to study the possibility of comparing AGP samples in terms of their CIEF profiles, what would facilitate in a future to perform studies about the role of AGP as a disease marker. In the present study, the CIEF profiles of AGP samples purified from sera of healthy donors and of ovary cancer and lymphoma patients are qualitatively and quantitatively compared. To make possible the comparison of those electrophoretical profiles, reliable assignment of AGP peaks is necessary. A computer program developed in our laboratory is used to select the migration parameters that make possible an accurate assignment of AGP peaks. Percentages of correct assignment of AGP peaks using the migration time of each peak relative to the migration time of an internal standard close to 95% are achieved. After peak assignment, a different distribution of the area percentage of AGP forms is observed when comparing samples from diseased and healthy individuals, the most acidic AGP forms being present in a higher proportion in the samples from cancer patients. Although the number of samples studied is too low to get any clinical significance from these results, this work provides a way to study the role of AGP as a biomarker.     

CIEF with hydrodynamic and chemical mobilization for the separation of forms of alpha-1-acid glycoprotein

Alpha-1-acid glycoprotein (AGP) is a protein that exists in different forms, which is due to variations in the amino acid sequence and/or in the glycosidic part of the protein. These differences confer to these forms, among other characteristics, diverse pIs. Changes in these forms of AGP have been correlated to modifications of the pathophysiological conditions of the individuals. One of the analytical techniques employed for their study has been IEF performed in slab gels. CIEF method with hydrodynamic and chemical mobilization, involving an isotachophoretic process, is developed in this work to separate up to 12 bands of forms of standard AGP, which is proposed as a more reproducible, quantitative, less sample-consuming, and more automated one than conventional IEF. The challenge of this work has been the development of a CIEF method for the separation of bands of a very acidic protein (pI range: 1.8-3.8) in a capillary. Intraday RSD values < or = 1.7% have been achieved for the relative migration time of the AGP bands to that of an internal standard. For intraday area precision, RSD (%) in the range of 2.70-22.71% for AGP zones accounting for more than 10% of total area of AGP sample has been obtained. As a proof of the potential of the methodology proposed, an AGP sample purified from a pool of sera of patients suffering from ovary cancer is analyzed by CIEF.     

CE-LIF analysis of mitochondria using uncoated and dynamically coated capillaries

This report is the first demonstration of the use of uncoated and dynamically coated capillaries for the separation of individual mitochondria via CE. Currently, the analysis of individual mitochondria relies upon fused-silica capillaries coated with a hydrophilic polymer (e.g. poly(acryloylaminopropanol)), which is used to minimize adsorption to the capillary surface. Both uncoated fused-silica capillaries and 0.2% w/w poly(vinyl alcohol) dynamic coating solutions are used to successfully analyze isolated individual mitochondrial particles using CE-LIF. While it was possible to separate mouse liver mitochondria on an uncoated capillary, rat liver mitochondria proved to have strong adsorption characteristics that only allowed them to be adequately separated with a PVA dynamic coating or a poly(acryloylaminopropanol) (AAP) capillary. The possible causes for this adsorption are analyzed and discussed. This study shows that uncoated and dynamically coated capillaries can be used in place of AAP-coated capillaries to analyze mitochondria and suggests the use of these capillaries for the analysis of other organelles, offering a greatly simplified method for the analysis of individual organelles.     

Development of a fast and simple immunochromatographic method to purify alpha 1-acid glycoprotein from serum for analysis of its isoforms by capillary electrophoresis

Alpha 1-acid glycoprotein (AGP) is a very heterogeneous glycoprotein presenting several isoforms due to variations in its polypeptidic and glycosidic moieties. Differences in AGP isoforms between healthy and diseased individuals have been related to different pathological situations such as cancer or cardiovascular diseases, among others. Capillary electrophoresis study of the role of AGP isoforms as biomarkers requires prior purification of AGP from biological samples. Current AGP purification methods are time- and labour-consuming, and generally they have not been proven to be compatible with capillary electrophoresis analysis. In this work, different methods for AGP purification from human serum are developed and compared. The applicability of acidic precipitation and immunoaffinity chromatographic methods for AGP purification are studied. Two different immunoaffinity approaches are employed; in the first one, interferents present in the AGP sample are captured and removed, and in the second one, AGP is retained in a house-made anti-AGP column, being in this way isolated from the rest of interferents of the sample. Best results in AGP purification from human serum to be analyzed by capillary zone electrophoresis (CZE) were obtained when acidic purification was combined with immunoaffinity chromatography (IAC) employing the house-made anti-AGP column. The method was shown not to alter the proportion of AGP peaks due to isoforms existing in AGP samples. The applicability of this fast and easy purification method developed for analyzing by CZE isoforms of AGP from natural serum samples by CZE is demonstrated.     

Analysis of the interaction between alpha-1-acid glycoprotein and tamoxifen and its metabolites

Tamoxifen is administered for the treatment of breast cancer; however resistance to therapy is commonplace. Postulated mechanisms of resistance to tamoxifen include altered pharmacology of the drug, changes in the structure and function of the oestrogen receptor and expression of genes that function to support the growth of cells resistant to tamoxifen. However, binding of drugs to proteins found in the plasma is known to affect the efficacy of drugs and alter their distribution. It is already known that tamoxifen is bound 99% to albumin. We investigated the interaction between the plasma protein, alpha-1-acid glycoprotein (AGP), and tamoxifen, since if binding did occur then the free plasma concentration of the drug would be reduced, resulting in the minimum effective concentration of tamoxifen not being attained. Using a recently described intrinsic fluorescence technique for the study of drug-protein interactions, the extent of binding between tamoxifen citrate and AGP was determined. Furthermore, analysis of binding of the known active metabolites of tamoxifen (4-hydroxytamoxifen, N-desmethyltamoxifen, N-desdimethyltamoxifen, cis-alpha-hydroxytamoxifen and trans-alpha-hydroxytamoxifen) to AGP was conducted. Tamoxifen citrate and metabolites were shown to bind AGP, however the level of interaction was low and negligible at the concentration of the drug found in the plasma.     

On-line immunoaffinity capillary electrophoresis based on magnetic beads for the determination of alpha-1 acid glycoprotein isoforms profile to facilitate its use as biomarker

An immunoaffinity purification method coupled on-line to capillary electrophoresis (IACE) which allows the determination of several isoforms of intact alpha-1 acid glycoprotein (AGP) in serum samples using UV detection is developed. The immunoaffinity step is based on anti-AGP antibodies (Abs) covalently bound to magnetic beads (MBs) which are captured at the inlet end of the capillary using permanent magnets placed inside the cartridge of the CE instrument. The on-line method includes injection of the MBs with the Ab bound (MBs–Ab) and their trapping by the magnets at the entrance of the separation column, injection of serum sample and capture of AGP by the Abs, release of captured AGP, focus of desorbed protein, separation of AGP isoforms, and removal of MBs–Ab. The optimization of the different factors involved in each step allowed purification, separation and detection of AGP isoforms in a single electrophoretic analysis in about 1 h. Automation, sample and reagents consumption as well as analysis time was improved compared to off-line alternatives which use purification of AGP in an immunochromatographic column and CE separation of AGP isoforms in two independent operations. The analytical methodology developed allows the separation of 10 AGP isoforms in serum samples from a healthy donor. For a serum sample, precision (expressed as relative standard deviation) in terms of corrected area percentage was better than 0.5% for each peak accounting for more than 10% of total AGP and it was better than 4.0% in terms of relative migration time of each AGP isoform considering the whole process.     

Glycosylation site analysis of human alpha-1-acid glycoprotein (AGP) by capillary liquid chromatography-electrospray mass spectrometry

A new anionic surfactant (RapiGest SF) was successfully used for site-specific analysis of glycosylation in human alpha-1-acid glycoprotein (AGP). By means of this analytical approach combined with capillary HPLC-mass spectrometry (and tandem mass spectrometry), the N-linked glycosylation pattern of AGP was explored. On the basis of mass matching and MS/MS experiments ca 80 different AGP-derived glycopeptides were identified. Glycosylation shows a markedly different pattern for the various glycosylation sites. At sites I and II, triantennary complex-type oligosaccharides predominate and at sites III, IV and V, tetra-antennary complex-type oligosaccharides predominate. Sites IV and V show the presence of additional N-acetyl lactosamine (Gal-GlcNAc) units (even higher degree of branching and/or longer antennae are also present).     

Solid-support fluorescent derivatization of picomoles of protein at low concentration with FITC

A new method based on solid-support reaction is described to realize fluorescent derivatization of proteins at concentrations as low as 10⁻⁸ M. A simple, low-cost homemade capillary C18 cartridge was fabricated as the solid-support reactor. Using bovine serum albumin (BSA) as a test protein, we demonstrated that the protein can be captured by this reactor and then labeled by fluorescein isothiocyanate (FITC, isomer I) on solid-support. Unwanted fluorescent intruder (excrescent FITC and products of secondary reactions) were removed from target easily. The analysis by nano-HPLC with laser-induced fluorescence (LIF) detection was described. The effect of reaction conditions on the derivatization has been evaluated and discussed. The use of the solid-support reactor allows easy handling of as little as 8.5 pmol of BSA. A fraction from weak anion-exchange chromatography (WAX) of human liver extract was used as an illustrative example of application to real samples.     

The influence of alpha1-acid glycoprotein on collagenase-3 activity in early rheumatoid arthritis

The concentration and glycosylation of alpha(1)-acid glycoprotein (AGP) alter significantly during inflammation. A definitive physiological role for AGP remains elusive and is the subject of extensive investigation. This study investigated the influence of AGP on the activity of collagenase-3, an important mediator of cartilage destruction in rheumatoid arthritis. AGP was isolated from normal and rheumatoid plasma. Fucosylation was determined by high pH anion-exchange chromatography; sialylation was assessed following enzymatic digest. Rheumatoid AGP displayed elevated fucosylation and sialylation compared with normal. The influence of each sample on collagenase-3 activity was measured fluorometrically. AGP influenced collagenase-3 catalysis and collagen binding, with catalytic activity correlating with fucosylation. Rheumatoid AGP exhibited less efficient inhibition than normal plasma AGP. It is hypothesized that AGP within rheumatoid synovial fluid may be inadequate to prevent excessive cartilage destruction and hence may exacerbate the disease process.     

Analysis of N‐acetylaminosugars by CE: A comparative derivatization study

N‐linked or O‐linked glycans derived from glycoprotein processing carry, an N‐acetylglucosamine or an N‐acetylgalactosamine respectively, at their reducing termini. The presence of the N‐acetylamino group on C‐2 of reducing sugar residues has been reported to hamper the derivatization reaction with a chromophore at the anomeric centre. In this paper N‐acetyllactosamine, N‐acetylglucosamine, N‐acetylgalactosamine and several other neutral monosaccharides are coupled to three different dyes (4‐aminobenzonitrile, 2‐aminopyridine, 2‐aminobenzoic acid (2‐AA)) by reductive amination and analysed by CE with UV detection. The 2‐AA derivatives showed the lowest concentration detection limits, varying approximately in the 2–3 μM range for the saccharides tested including the N‐acetamido ones. The possibility to separate and detect with the same sensitivity ten 2‐AA‐labelled monosaccharides mainly found in mammalian or plant glycoproteins in a single CE run is highlighted. The analysis has been carried out in less than 25 min using the borate‐complexation method in CZE mode. The influence of the strength of the acid used as catalyst in the chemical modification of the sugars with 2‐AA is also shortly addressed.     

A fluorescent derivatization method of proteins for the detection of low‐level impurities by microchip capillary gel electrophoresis

A novel pre‐chip fluorescent derivatization method is presented for protein sizing and quantification by microchip CGE. The derivatization reaction employed a water‐soluble and stable fluorescent dye and was performed under conditions that favored the formation of homogeneous reaction products. The method delivered in terms of protein sizing similar results as microchip CGE with on‐chip staining but showed an extended linear dynamic range for protein quantification encompassing four orders of magnitude. The sensitivity of the method was similar to standard silver‐stained planar gels. The characterization of derivatization reaction products by MS and preparative isoelectric focusing indicated that a constant degree of dye molecule tagging was obtained over a broad range of protein/dye ratios. The method allowed detecting and quantifying an impurity spiked into an antibody preparation down to a level of 0.05%. Advantages of this method compared with CGE approaches with pre‐column derivatization include a shorter analysis time and an increased robustness and ease of use.     

Total nucleotide analysis of Hydra DNA and RNA by MEKC with LIF detection and 32P‐postlabeling

The model organism Hydra has been used for molecular studies for more than 20 years, however, its DNA base composition has not been determined yet. We have analyzed DNA and total RNA of the freshwater polyp Hydra magnipapillata with two independent procedures of high accuracy and sensitivity – fluorescence labeling of nucleotides followed by CE‐LIF detection and ³²P‐postlabeling. DNA of Hydra was digested either to deoxyribonucleoside‐5′‐monophosphates or deoxyribonucleoside‐3′‐monophosphates selectively conjugated with the fluorescent dye 4,4‐difluoro‐5,7‐dimethyl‐4‐bora‐3a,4a‐diaza‐s‐indacene‐3‐propionyl ethylene diamine hydrochloride (BODIPY FL EDA) separated and detected using CE‐LIF. Both versions of the assay revealed a high A+T composition of 78 and 71%, whereas total DNA methylation (5‐methyldeoxycytidine) was 2.6 and 3.1%. Total Hydra RNA showed highest base levels for guanine (33%) and a level of 1.4% for pseudouracil. All values were in good agreement with those determined by the ³²P‐postlabeling method.     

Capillary electrophoresis‐laser‐induced fluorescence detection of rat brain catecholamines with microwave‐assisted derivatization

In this study, a rapid and sensitive method is described for the catecholamines detection in rat brain. CE with LIF detection for the determination of FITC derivatized catecholamines (dopamine, epinephrine, and norepinephrine) was demonstrated. Conventional water bath and microwave‐assisted derivatization methods were employed and a significant reduction in the derivatization time from 2 h for the conventional water bath at room temperature (ca. 25°C) to 2 min for the microwave‐assisted derivatization was achieved. Online sample concentration of field‐amplified sample stacking (FASS) method was employed to achieve higher sensitivities (the detection limits obtained in the normal injection mode ranged from 2.6 to 4.5 ng L^?1 and in the FASS mode ranged from 22 to 34 pg L^?1). Furthermore, this microwave‐assisted derivatization CE–LIF method successfully determined catecholamines in rat brain with as low as 100 ng L^?1 (FASS mode) to 10 μg L^?1 (normal injection mode). This CE–LIF method provided better detection ability when compared to the best reports on catecholamines analyses.     

Design, characterization, and utilization of a fast fluorescence derivatization reaction utilizing o-phthaldialdehyde coupled with fluorescent thiols

We have developed a chemical derivatization scheme for primary amines that couples the fast kinetic properties of o-phthaldialdehyde (OPA) with the photophysical properties of visible, high quantum yield, fluorescent dyes. In this reaction, OPA is used as a cross-linking reagent in the labeling reaction of primary amines in the presence of a fluorescent thiol, 5-((2-(and-3)-S-(acetylmercapto)succinoyl)amino)fluorescein (SAMSA fluorescein), thereby incorporating fluorescein (epsilon = 78 000 M(-1), quantum yield of 0.98) into the isoindole product. Detection is based on excitation and emission of the incorporated fluorescein using the 488 nm laser line of an Ar(+) laser rather than the UV-excited isoindole, thereby eliminating the UV light sources for detection. Using this method, we have quantitatively labeled biologically important primary amines in less than 10 s. Detection limits for analysis of glutamate, glycine, GABA, and taurine were less than 2 nM. We present the characterization of OPA/SAMSA-F reaction and the potential utility of the derivatization reaction for dynamic chemical monitoring of biologically relevant analytes using CE.     

Automated derivatization and analysis of malondialdehyde using column switching sample preparation HPLC with fluorescence detection

Analyte derivatization is advantageous for the analysis of malondialdehyde (MDA) as a biomarker of oxidative stress in biological samples. Conventionally, however, derivatization is time consuming, error-prone and has limited options for automation. We have addressed these challenges for the solid phase analytical derivatization of MDA from small volume tissue homogenate samples. A manual derivatization method was first developed using Amberlite XAD-2 (12 mg) as the solid phase. Subsequently an automated column switching process was developed that provided simultaneous derivatization and extraction of the MDA-DH hydrazone product on a cartridge packed with XAD-2, followed by quantitative elution of the product to an analytical LC column (Waters NovoPak C18, 3.9 x 150 mm). The LOD was 0.02 microg/mL and recovery was quantitative. The method was linear (r(2) >0.999) with precision < 5% from the LOQ (0.06 microg/mL) to at least 35 microg/mL. The method was successfully applied to the analysis of small volume (30 microL) mouse tissue homogenate samples. Endogenous levels of MDA in the tissues ranged from 20 to 40 nmol/g tissue (ca. 0.1-0.2 microg/mL homogenate). Compared to conventional MDA analyses, the current method has advantages in automation, selectivity, precision and sensitivity for analysis from very small sample volumes.     

Fluorescence determination of N-acetylaspartic acid in the rat cerebrum homogenate using high-performance liquid chromatography with pre-column fluorescence derivatization

N-acetyl-L-aspartic acid (NAA) is an endogenous compound, and its brain concentration is suggested to be altered in neurological disorders. In the present study, a fluorescence determination method for NAA was developed by employing reversed-phase high-performance liquid chromatography (HPLC) with pre-column fluorescence derivatization using 4-N,N-dimethylaminosulfonyl-7-N-(2-aminoethyl)amino-2,1,3-benzoxadiazole (DBD-ED). Using methylsuccinic acid as the internal standard, a linear calibration curve for NAA was constructed in the range 125-1000 microM (n=3). The detection limit on the column was approximately 5.0 fmol (signal-to-noise ratio 3). The proposed HPLC method was applied to determine NAA in the rat cerebrum homogenate. Cerebrum NAA was successfully determined using 10 microL of the homogenate, and the validation data for the proposed HPLC method demonstrated satisfactory results. Intra- and inter-day precision and accuracy were within 1.1-7.0 and -8.1-6.3%, respectively. The concentration of NAA in the male rat cerebrum (13 weeks old) was 84+/-4.6 micromol/mg protein (n=3).     

Quantification of polyamines in human erythrocytes using a new near-infrared cyanine 1-(epsilon-succinimidyl-hexanoate)-1'-methyl-3,3,3',3'-tetramethyl-indocarbocyanine-5,5'-disulfonate potassium with CE-LIF detection

A novel near-infrared (NIR) cyanine 1-(epsilon-succinimidyl-hexanoate)-1'-methyl-3,3,3',3'-tetramethyl-indocarbocyanine-5,5'-disulfonate potassium (MeCy5-OSu) has been developed in our laboratory. Simultaneous determination of MeCy5-OSu-derivatized polyamines spermine (Spm), spermidine (Spd), cadaverine (Cad), and putrescine (Put) based on the separation by CE combined with diode LIF detection has been accomplished. The highest derivatization efficiency was achieved in 0.2 mol/L borate buffer (pH 8.8) for 20 min at 25 degrees C. Polyamine derivatives were separated within 14 min in the phosphate running buffer (pH 3) containing 50 mmol/L phosphoric acid, 40 mmol/L SDS, and 35% methanol v/v. Linearity of response was obtained in the range of 10-200 nmol/L. The detection limits (S/N = 3) for Spm, Spd, Cad, and Put were 0.8, 1, 3, and 2 nmol/L, respectively. The proposed method has been successfully applied to the analysis of polyamines in erythrocytes of two healthy persons and one cancer patient. Average recoveries for erythrocyte samples were 93.6-106% and coefficients of variation ranged from 1.8 to 5.4%. The analysis of polyamines in erythrocytes can be used for studying the relationship between their changes and the carcinogenesis process involved in erythrocytes.