香豆素类新基质在基质辅助激光解吸／电离飞行时间质谱测定多糖和糖蛋白中的应用

将几种香豆素类新基质(香豆素、3-羟基香豆素(3-HC)、3-氨基香豆素(3-AC)、3-羧基香豆素(3-CC)和4-甲基-7-羟基香豆素(4-M-7-HC))分别应用于基质辅助激光解吸/电离飞行时间质谱(MALDI TOF-MS)测定葡聚糖和3种糖蛋白的研究.香豆素和3-羟基香豆素分别与2,5-二羟基苯甲酸(DHB)混合组成2种二元基质,极大地改善了基质和葡聚糖样品的共结晶状况,样品分布更加均匀.葡聚糖样品更易解吸/电离,每个激光点照射样品均能产生较强的质谱信号,且谱图重现性更好,得到了理想的MALDI TOF-MS谱图.当香豆素类基质用于分析糖蛋白时:3-HC和4-M-7-HC是测定糖蛋白A的优异基质,能检测到m/z 为66 672 Da 的离子信号.而3-AC测定糖蛋白B的基质效果比糖类分析常用基质2,5-二羟基苯甲酸更好.因此,这些香豆素类化合物将为MALDI TOF-MS分析多糖和糖蛋白提供更多新基质选择.     

3,4-二氨基苯基苯甲酮在基质辅助激光解析离子化质谱分析磷脂中的应用

考察了3,4-二氨基苯基苯甲酮(DABP)作为基质辅助激光解吸离子化飞行时间质谱(MALDI-TOF-MS)新型基质分析磷脂的能力.与传统基质2,4,6-三羟基苯乙酮(THAP)、2,5-二羟基苯甲酸(DHB)、α-氰基-4-羟基肉桂酸(CHCA)相比,用优化后的DABP基质溶液体系McOH-H2O-HCI(80:20...     

壳聚糖的基质辅助激光解吸电离飞行时间质谱分析

利用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)分析甲壳素脱乙酰化降解产物——壳聚糖,对基质、制样方法等影响MALDI-TOF-MS测定结果的因素进行了研究。实验发现,以2,5-二羟基苯甲酸(DHB)为基质,二次结晶法制样分析壳聚糖,既获得了壳聚糖的分子量信息,又可以推断壳聚糖的脱乙酰度,对壳聚糖的制备及其质量与性能控制有着十分重要的指导意义。     

应用MALDI-FTICRMS技术研究气相中含钨系列离子簇

以Keggin型磷钨酸为原料, 2,5-二羟基苯乙酮(DHAP)和2,4,6-三羟基苯乙酮(THAP)为基质, 使用基质辅助激光解吸/电离-傅里叶变换离子回旋共振质谱(MALDI-FTICRMS)技术研究气相中金属相关簇的稳定性及气相离子-分子反应. 在气相中获得不同系列的-1价含钨离子簇, 准确确定各系列离子簇的组成及Magic Numbers物种, 并发现基质与钨相互作用生成有机-无机杂化离子簇系列. 结果表明, MALDI-FTICRMS技术适用于研究气相中团簇离子的稳定性及气相离子-分子反应.     

UPLC-Q Exactive Plus-MS分析高粱壳化学成分及黄嘌呤氧化酶体外抑制活性筛选

采用高效液相色谱串联四极杆静电场轨道阱质谱对高粱壳中的化学成分进行分析鉴定，并通过高效液相色谱法筛选抑制黄嘌呤氧化酶活性成分。该实验使用Hypersil GOLD C₁₈色谱柱(2.1 nm×30 nm,3μm)，以0.1%甲酸水(A)-乙腈(B)进行梯度洗脱，流速1.0 m L/min，进样量10μL。质谱分析采用ESI离子源，在正离子模式下采集数据，利用Xcalibur 4.1软件结合数据库对所得数据进行分析，共鉴定出27种化学成分。对刺槐素、葛根素、芹菜素、根皮苷、异鼠李素和3,3’,4’,7-四羟基黄酮6种化学成分进行活性筛选，发现芹菜素和3,3’,4’,7-四羟基黄酮对黄嘌呤氧化酶(XO)表现出较强的抑制活性，IC₅₀分别为18.11和34.59μmol/L。     

MALDI-TOFMS测定高聚合度葡聚糖及不同基体在测定中的作用比较

应用基体辅助激光解吸电离飞行时间质谱(MALDI-TOFMS)直接对未经分级的高聚合度(n>100)葡聚糖样品进行了测定,检测到的葡聚糖最大分子量达18000以上.对2,5-二羟基苯甲酸(DHB)、1-羟基异喹啉(1-HIQ)及3,4-二羟基肉桂酸(咖啡酸,CA)3种一元基体以及DHB与1-HIQ,DHB与CA两种二元基体在测定中的作用进行了比较.结果表明,DHB基体测定的质量范围较大;DHB+1-HIQ和DHB+CA二元混合基体对样品的解吸电离效果及谱图重现性好;DHB+1-HIQ基体获得的谱图分辨率高.     

极谱法测定蒙药那如三味丸中锗的研究

采用差分脉冲极谱法,在pH 1.50、含3,4 –二羟基苯甲醛(DHB)的H2SO4中,测得无机锗的峰电位为Ep=-0.54 V,Ge(Ⅳ)浓度在1.04×10 5～1.05×10 4 mol·L 1范围内与峰电流呈线性关系,利用该法用于测定蒙药那如三味丸中总锗、无机锗和有机锗,得到了满意的结果.     

有机锗(锗-132)的示差脉冲极谱研究

用示差脉冲极谱法,在pH1.30H_2SO_4和3,4-二羟基苯甲醛(DHB)底液中,获得了有机锗(Ge-132)和无机锗Ge(Ⅳ)二个配位吸附极谱波,波峰分别为E_P2-0.62V和E_pl=-0.47V(υs.AgCI/Ag),Ge-132浓度在2.6×10_(-5)～3.1×10_（-3）mol/L范围内,Ge(Ⅳ)浓度在 3.1×10_（-5）～2.1×10_（-4）mol/L范围内分别与峰电流星线性关系.利用该示差脉冲极谱法可同时测定Ge-132和Ge(Ⅳ),用于有机锗营养口服液中Ge-132和Ge(Ⅳ)的测定结果满意.还探讨了Ge-132极谱波的性质,测定了有关动力学参数.     

固相萃取高效液相色谱-质谱法测定血浆中乙醇醛、甘油醛的含量

采用柱前衍生、固相萃取、高效液相色谱-质谱联用方法,建立了血浆中乙醇醛(GCO)、甘油醛(GCE)含量的测定方法。衍生试剂为3-m ethyl-2-benzoth iazolinone-hydrazone hydrochloride(MBTH);内标物为3-羟基丁醛;HPLC流动相为甲醇-醋酸铵(5 mmol/L),梯度洗脱;质谱电喷雾选择离子检测。比较了糖尿病大鼠和正常大鼠血浆中GCO、GCE的含量,正常大鼠血浆中GCO和GCE的含量分别为0.0574±0.0036μmol/L、0.0186±0.0019μmol/L;糖尿病大鼠血浆中GCO和GCE的含量分别为0.0763±0.0071μmol/L、0.0240±0.0023μmol/L,差异显著。     

基于香豆素的比色探针的合成及对Cu^(2+)的识别EI北大核心CSCD

合成了探针8-(2-肼基苯并噻唑)-4-甲基-7-羟基香豆素(L),通过紫外光谱法研究其识别Cu^(2+)性能,并通过Job’s曲线、红外光谱和质谱研究其对Cu^(2+)的识别机理。结果表明:在二甲基亚砜(DMSO)/水(V∶V=9∶1)溶液体系中,探针溶液中加入Cu^(2+)后,溶液颜色由无色变为浅黄色;探针溶液的紫外可见吸光度与Cu^(2+)浓度在0.125~5μmol/L范围内呈良好的线性关系,线性方程为A=0.037c+0.052,线性相关系数R^(2)=0.9881,检出限为0.02μmol/L,该探针可以定量检测水中的Cu^(2+)。     

Optimal preparation methods for automated matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry profiling of low molecular weight proteins and peptides

Mass spectrometry (MS) profiling of the proteome and peptidome for disease‐associated patterns is a new concept in clinical diagnostics. The technique, however, is highly sensitive to external sources of variation leading to potentially unacceptable numbers of false positive and false negative results. Before MS profiling can be confidently implemented in a medical setting, standard experimental methods must be developed that minimize technical variance. Past studies of variance have focused largely on pre‐analytical variation (i.e., sample collection, handling, etc.). Here, we examined how factors at the analytical stage including the matrix and solid‐phase extraction influence MS profiling. Firstly, a standard peptide/protein sample was measured automatically by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) MS across five consecutive days using two different preparation methods, dried droplet and sample/matrix, of four types of matrix: α‐cyano‐4‐hydroxycinnamic acid (HCCA), sinapinic acid (SA), 2,5‐dihydroxybenzoic acid (DHB) and 2,5‐dihydroxyacetophenone (DHAP). The results indicated that the matrix preparation greatly influenced a number of key parameters of the spectra including repeatability (within‐day variability), reproducibility (inter‐day variability), resolution, signal strength, background intensity and detectability. Secondly, an investigation into the variance associated with C8 magnetic bead extraction of the standard sample prior to automated MS profiling demonstrated that the process did not adversely affect these same parameters. In fact, the spectra were generally more robust following extraction. Thirdly, the best performing matrix preparations were evaluated using C8 magnetic bead extracted human plasma. We conclude that the DHAP prepared according to the dried‐droplet method is the most appropriate matrix to use when performing automated MS profiling. Copyright © 2009 John Wiley & Sons, Ltd.     

A Novel MALDI Matrix for Analyzing Peptides and Proteins: Paraffin Wax Immobilized Matrix

制作出一种新型的石蜡固化基质,并将其用于多肽以及蛋白质的MALDI-FTMS分析。在其制作过程中通过加热将固体石蜡融化并均匀涂敷在不锈钢靶板上,在不同温度下滴加基质溶液于石蜡涂层及不锈钢靶板上,分别制得不同形式的基质点。三维视频显微镜表征各基质点的表观形貌,并对比其上样前后的形貌变化。通过在不同基质点上分析多肽混合液以及牛血清白蛋白酶解液,以评价该固化基质的性能。MALDI-FTMS分析结果表明石蜡涂层内固化基质方法检测灵敏度明显优于其它几种状况下的样品分析方法。     

A binary matrix for improved detection of phosphopeptides in matrix‐assisted laser desorption/ionization mass spectrometry

Application of matrix‐assisted laser‐desorption/ionization mass spectrometry (MALDI MS) to analysis and characterization of phosphopeptides in peptide mixtures may have a limitation, because of the lower ionizing efficiency of phosphopeptides than nonphosphorylated peptides in MALDI MS. In this work, a binary matrix that consists of two conventional matrices of 3‐hydroxypicolinic acid (3‐HPA) and α‐cyano‐4‐hydroxycinnamic acid (CCA) was tested for phosphopeptide analysis. 3‐HPA and CCA were found to be hot matrices, and 3‐HPA not as good as CCA and 2,5‐dihydroxybenzoic acid (DHB) for peptide analysis. However, the presence of 3‐HPA in the CCA solution with a volume ratio of 1:1 could significantly enhance ion signals for phosphopeptides in both positive‐ion and negative‐ion detection modes compared with the use of pure CCA or DHB, the most common phosphopeptide matrices. Higher signal intensities of phosphopeptides could be obtained with lower laser power using the binary matrix. Neutral loss of the phosphate group (?80 Da) and phosphoric acid (?98 Da) from the phosphorylated‐residue‐containing peptide ions with the binary matrix was decreased compared with CCA alone. In addition, since the crystal shape prepared with the binary matrix was more homogeneous than that prepared with DHB, searching for ‘sweet’ spots can be avoided. The sensitivity to detect singly or doubly phosphorylated peptides in peptide mixtures was higher than that obtained with pure CCA and as good as that obtained using DHB. We also used the binary matrix to detect the in‐solution tryptic digest of the crude casein extracted from commercially available low fat milk sample, and found six phosphopeptides to match the digestion products of casein, based on mass‐to‐charge values and LIFT TOF‐TOF spectra. Copyright © 2009 John Wiley & Sons, Ltd.     

MALDI-TOF mass spectrometry of a combinatorial peptide library: effect of matrix composition on signal suppression

The effect of matrix composition on signal suppression caused by a dominant compound under MALDI ionization was studied using the combinatorial TQTXT pentapeptide library as a model system. The peptide library is composed of 19 components with all proteinogenic amino acids except cysteine in position X. From these compounds, only the Arg peptide (TQTRT) was detected with sufficient intensity in the MALDI-TOF mass spectrum under typical MALDI conditions (CCA matrix). The analysis of a set of compounds utilized as different matrix components, additives and a cationizing agent revealed that the composition of the matrix is a critical point in signal suppression. Highly improved ion yields were achieved by using a CCA/DHB mixture as a matrix. The addition of K(+) as a cationizing agent to the CCA matrix resulted in MALDI-TOF mass spectra with relative ion intensities very similar to those obtained by electrospray ionization.     

A 2,5-dihydroxybenzoic acid/N,N-dimethylaniline matrix for the analysis of oligosaccharides by matrix-assisted laser desorption/ionization mass spectrometry

The use of a novel 2,5-dihydroxybenzoic acid/N,N-dimethylaniline (DHB/DMA) matrix-assisted laser desorption/ionization (MALDI) matrix for detection and quantitative analysis of native N-linked oligosaccharides was investigated in this study. Substantial improvements in sensitivity were observed relative to the signals obtained with a traditional DHB matrix. Moreover, the morphology of the matrix crystal layer was very uniform, unlike that of DHB. This resulted in highly homogeneous sample distribution throughout the spot, allowing reproducible and consistent mass spectra to be obtained without spot-to-spot variations in signal. Here, we also demonstrate an approach for performing sensitive and accurate quantitative analysis of native N-linked glycans with this novel matrix using an internal standard method.     

A binary matrix of 2,5-dihydroxybenzoic acid and glycerol produces homogenous sample preparations for matrix-assisted laser desorption/ionization mass spectrometry

We introduce a two-component matrix for ultraviolet matrix-assisted laser desorption/ionization mass spectrometry (UV-MALDI-MS) that consists of 2,5-dihydroxybenzoic acid (DHB) and glycerol. Upon slow evaporation of residual water/methanol solvents in a pre-vacuum chamber sample preparations are obtained that exhibit a homogeneous morphology with analyte-matrix crystals evenly distributed over the whole sample spot. At a molar DHB/glycerol ratio of approximately 1:5, the crystals range in length from approximately 100 to 300 microm and are about 15-30 microm wide. Mass spectra of peptides, proteins, and an oligosaccharide are presented and compared with those recorded from standard dried-droplet DHB matrix. The ion signals show a reproducibility of the order of 10-15% when scanning the surface of an individual sample or even different samples that contain the same amount of peptide, A close to linear relationship between peptide concentration and the corresponding peptide ion signal is found over three orders of magnitude of sample prepared. However, when a fixed position is irradiated with a large number of laser pulses, a monotonous decay of peptide ion signal with time is observed. Potentially, the binary matrix will be especially useful for the analysis of samples that are stabilized in buffered aqueous glycerol solution and preliminary results addressing this aspect are shown.     

Incoherent production reactions of positive and negative ions in matrix-assisted laser desorption/ionization

Utilizing synchronized dual-polarity matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, we found good evidence of the incoherent production of positive and negative matrix ions. Using thin, homogeneous 2,5-dehydroxybenzoic acid (DHB) matrix films, positive and negative matrix ions were found to appear at different threshold laser fluences. The presence of molecular matrix ions of single charge polarity suggests that the existence of DHB ion-pairs may not be a prerequisite in MALDI. Photoelectrons induced by the laser excitation may assist the production of negative DHB ions, as shown in experiments conducted with stainless steel and glass substrates. At high laser fluences, the relative yield of positive and negative matrix ions remained constant when homogeneous matrix films were used, but it fluctuated significantly with inhomogeneous crystal morphology. This result is also inconsistent with the hypothesis that matrix ion-pairs are essential primary ions. Evidence from both low and high laser fluences suggests that the productions of positive and negative matrix ions in MALDI may occur via independent pathways.     

Ultrasensitive determination of 1,4-dihydroxybenzene based on fluorescence resonance energy quenching of luminescent quantum dots modified on surface of silica nanoparticles

An ultrasensitive solid-phase fluorescence resonance energy quenching (FREQ) method for determination of 1,4-dihydroxybenzene (DHB) using mercaptosuccinic acid (MSA)-capped CdTe quantum dots (QDs) immobilized on silica nanoparticles (NPs) as donors was developed. In the method, silica NPs were first modified with 3-aminopropyltriethoxysilane (APTS). Then, MSA-capped CdTe QDs were immobilized on the surface of the APTS-modified silica NPs. Finally, DHB in the solution was attached to the empty sites on the surface of silica NPs with QDs through electrostatic interaction. The fluorescence emission of the QDs was quenched by the proximal DHB molecules on the silica NPs. The quenching efficiency of the solid-phase FREQ method was 200-times higher than that of the solution-phase FREQ method. Using the ultrasensitive solid-phase FREQ method, DHB as low as 2.4 × 10⁻¹² mol/L could be detected. The method was applied to quantify trace DHB in water samples.     

Combining tissue extraction and off-line capillary electrophoresis matrix-assisted laser desorption/ionization Fourier transform mass spectrometry for neuropeptide analysis in individual neuronal organs using 2,5-dihydroxybenzoic acid as a multi-functional agent

In this study we report an improved protocol that combines simplified sample preparation and micro-scale separation for mass spectrometric analysis of neuropeptides from individual neuroendocrine organs of crab Cancer borealis. A simple, one-step extraction method with commonly used matrix-assisted laser desorption/ionization (MALDI) matrix, 2,5-dihydroxybenzoic acid (DHB), in saturated aqueous solution, is employed for improved extraction of neuropeptides. Furthermore, a novel use of DHB as background electrolyte for capillary electrophoresis (CE) separation in the off-line coupling of CE to MALDI-Fourier transform mass spectrometric (FT-MS) detection is also explored. The new CE electrolyte exhibits full compatibility with MALDI-MS analysis of neuropeptides in that both the peptide extraction process and MALDI detection utilize DHB. In addition, enhanced resolving power and improved sensitivity are also observed for CE-MALDI-MS of peptide mixture analysis. Collectively, the use of DHB has simplified the extraction and reduced the sample loss by elimination of homogenizing, drying, and desalting processes. In the mean time, the concurrent use of DHB as CE separation buffer and subsequent MALDI matrix offers improved spectral quality by eliminating the interferences from typical CE electrolyte in MALDI detection.     

基于金纳米棒-壳聚糖复合膜的葡萄糖生物传感器

本文采用金纳米棒-壳聚糖复合膜固定葡萄糖氧化酶构建电流型葡萄糖生物传感器.通过电化学交流阻抗法和循环伏安法对酶膜状态进行了表征,得到了相应的等效电路和动力学参数.实验结果表明,金纳米棒-壳聚糖复合膜可以辅助电子传递,提高电极的电流响应,并使生物传感器的使用温度范围有很大的扩展.此传感器表现出对葡萄糖溶液浓度的优良响应,线性范围在2.78×10^-5mol/L—2.22×10^-3mol/L,响应灵敏度约为7.819μA·cm^-2(mmol/L)^-1,表观米氏常数为10mmol/L.本工作还研究了温度和溶液pH值对电极电流响应的影响.