Comparative ESI FT-MS and MALDI-TOF structural analyses of representative human <Emphasis Type="Italic">N</Emphasis>-linked glycans

Modern glycan analysis is primarily based on mass spectrometry, where instruments based on electrospray or matrix-assisted laser desorption ionization are currently the most frequently used. In the present study, electrospray ionization (ESI) coupled with a high-resolution Fourier transform mass spectrometer (LTQ Orbitrap) and matrix-assisted laser desorption/ionization (MALDI) coupled with a time-of-flight (TOF/TOF) detector were used to analyze two N-glycan standards with intact free reducing ends (disialo biantennary and asialo triantennary) and representative PA-labeled human serum N-glycan structures isolated by hydrophilic interaction anion-exchange chromatography (HIAX), confirmed by ¹H NMR analysis and consequently compared with the ProteinScape Glycome database. Different combinations of ion sources with fragmentation devices results in various fragmentation patterns and adducts. Also, the effect of sample derivatization on the acquired signals is discussed. Compared to the MALDI technique, free glycans did not lose labile sialic acids easily in the ESI source. On the other hand, fluorescent PA-labeling leads to improved core fragmentation and signal intensities; linkage-specific ethyl esterification leads to reduced adduct and fragment formation and enhanced stability of sialic acids in the MALDI ion source. Thereby, both methods have their advantages and disadvantages in terms of detection, fragmentation and robustness.     

UHPLC-MS/MS法快速测定水产品中17种磺胺和喹诺酮类药物

建立了UHPLC-MS/MS法测定水产品中10种磺胺类(SAs)和7种喹诺酮类(QNs)药物残留的分析方法。样品用200 g/L盐酸羟胺-乙腈溶液提取,以乙酸铵溶液和乙腈为流动相进行梯度洗脱,电喷雾正离子(ESI+)模式电离,多反应监测模式检测,同时对水产品中10种SAs和7种QNs进行定量和定性。在0.25～4.0μg/kg和0.10～2.0μg/kg范围内两类药物的线性良好(r2>0.99);平均回收率为均为80%～120%,RSD为7.4%～14%;10种磺胺药物的检测限(LOD)均为5.0μg/kg,7种喹诺酮药物检测限(LOD)均为2.0μg/kg。该方法适合水产品中这两类药物残留的确证和定量测定。     

Comparison of electrospray and matrix-assisted laser desorption ionization on the same hybrid quadrupole time-of-flight tandem mass spectrometer: Application to bidimensional liquid chromatography of proteins from bovine milk fraction

Recently, two ionization sources, electrospray (ESI) and matrix-assisted laser desorption (MALDI) have been used in parallel to exploit their complementary nature and to increase proteome coverage. In this study, a method using bidimensional (2D) nanoLC coupled online with ESI quadrupole time-of-flight (Q-TOF) with the simultaneous collection of fractions for analyses by LC–MALDI Q-TOF–MS/MS was developed. A total of 39 bovine proteins were identified to a high degree of confidence. To help in differentiating peptide detection following ESI and MALDI with the same mass spectrometer, we compared physico–chemical characteristics of the peptides (molecular mass, charge and size) by principal component analysis (PCA) and analysis of variance on the results of PCA. More hydrophobic peptides with a wider mass coverage were identified when ESI was used, whereas more basic and smaller peptides were identified when MALDI was used. However, the generally accepted differentiation between ESI and MALDI according to the presence of basic amino acids residues Lys and Arg and the ratio Lys/Arg was not shown as significant in this study. Moreover, we pointed out the importance of the type of mass spectrometer used in complement to both ionization sources for achieving a global increase of proteome coverage.     

丹磺酰氯柱前衍生-超高效液相色谱-串联质谱法测定人体尿样中的环己胺

建立丹磺酰氯柱前衍生-超高效液相色谱-串联质谱法测定人体尿样中环己胺的方法。冷冻样品经解冻、离心后,用丹磺酰氯衍生,固相萃取小柱净化。目标化合物采用 Waters ACQUITY CSHTM C18色谱柱(50 mm×2.1 mm,1.7μm)分离,以甲醇和0.002 mol/L乙酸铵溶液为流动相梯度洗脱,采用电喷雾离子源电离、正离子多反应监测模式质谱检测。环己胺在2.5~200μg/L浓度范围内有较好的线性关系,相关系数大于0.999,回收率为98.7%~102.3%,精密度为3.1%~5.2%,检出限和定量限分别为1.0和3.0μg/L。结果表明,本方法操作简单、准确可靠,可适用于人体尿液中环己胺的定量分析。应用本方法测定200份学生尿液样品,环己胺检出率为34.5%。     

Simultaneous Determination of Haloacetic Acids in Environmental Waters using Electrospray Ionization Liquid Chromatography Mass Spectrometry

Negative ionization electrospray liquid chromatography mass spectrometry was developed for the simultaneous determination of all nine haloacetic acids containing bromine and chlorine. Haloacetic acids were separated on a crosslinked polystyrene resin column using 3% acetic acid dissolved in acetonitrile:water (20:80) as mobile phase. The precision of this method varied from ±2.2 to ±7.1% for nine haloacetic acids. In addition, quantitative results obtained with spiked water samples at three different concentrations are described. The limit of detection of the proposed method using 200 mL of water samples was between 0.003 and 0.070 μg/L. This method was successfully applied to the trace determination of haloacetic acids in waste water, river water, and seawater.     

The relative influence of phosphorylation and methylation on responsiveness of peptides to MALDI and ESI mass spectrometry

Qualitative and quantitative analysis of post‐translational protein modifications by mass spectrometry is often hampered by changes in the ionization/detection efficiencies caused by amino acid modifications. This paper reports a comprehensive study of the influence of phosphorylation and methylation on the responsiveness of peptides to matrix‐assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry. Using well‐characterized synthetic peptide mixtures consisting of modified peptides and their unmodified analogs, relative ionization/detection efficiencies of phosphorylated, monomethylated, and dimethylated peptides were determined. Our results clearly confirm that the ion yields are generally lower and the signal intensities are reduced with phosphopeptides than with their nonphosphorylated analogs and that this has to be taken into account in MALDI and ESI mass spectrometry. However, the average reduction of ion yield caused by phosphorylation is more pronounced with MALDI than with ESI. The unpredictable impact of phosphorylation does not depend on the hydrophobicity and net charge of the peptide, indicating that reliable quantification of phosphorylation by mass spectrometry requires the use of internal standards. In contrast to phosphorylation, mono‐ and dimethylated peptides frequently exhibit increased signal intensities in MALDI mass spectrometry (MALDI‐MS). Despite minor matrix‐dependent variability, MALDI methods are well suited for the sensitive detection of dimethylated arginine and lysine peptides. Mono‐ and dimethylation of the arginine guanidino group did not significantly influence the ionization efficiency of peptides in ESI‐MS. Copyright © 2009 John Wiley & Sons, Ltd.     

On‐chip solid‐phase extraction pre‐concentration/focusing substrates coupled to atmospheric pressure matrix‐assisted laser desorption/ionization ion trap mass spectrometry for high sensitivity biomolecule analysis

Atmospheric pressure matrix‐assisted laser desorption/ionization (AP‐MALDI) has proven a convenient and rapid method for ion production in the mass spectrometric (MS) analysis of biomolecules. AP‐MALDI and electrospray ionization (ESI) sources are easily interchangeable in most mass spectrometers. However, AP‐MALDI suffers from less‐than‐optimal sensitivity due to ion losses during transport from the atmosphere into the vacuum of the mass spectrometer. Here, we study the signal‐to‐noise ratio (S/N) gains observed when an on‐chip dynamic pre‐concentration/focusing approach is coupled to AP‐MALDI for the MS analysis of neuropeptides and protein digests. It was found that, in comparison with conventional AP‐MALDI targets, focusing targets showed (1) a sensitivity enhancement of approximately two orders of magnitude with S/N gains of 200–900 for hydrophobic substrates, and 150–400 for weak cation‐exchange (WCX) substrates; (2) improved detection limits as low as 5 fmol/µL for standard peptides; (3) significantly reduced matrix background; and (4) higher inter‐day reproducibility. The improved sensitivity allowed successful tandem mass spectrometric (MS/MS) sequencing of dilute solutions of a derivatized tryptic digest of a protein standard, and enabled the first reported AP‐MALDI MS detection of neuropeptides from Aedes aegypti mosquito heads. Copyright © 2009 John Wiley & Sons, Ltd.     

超高效液相色谱-三重四极杆质谱法测定银杏叶提取物及其制剂中银杏酸

建立了超高效液相色谱-串联三重四极杆质谱测定银杏叶提取物及其制剂中银杏酸的方法。采用Agilent Poroshell 120 EC-C18色谱柱(50 mm×3.0 mm, 2.7 μ m),以甲醇-1%冰醋酸溶液(90 : 10, v/v)为流动相,在电喷雾离子化负离子模式下,以多反应监测方式(MRM)检测。银杏酸C13 : 0、C15 : 1、C17 : 1在2~200 μ g/L范围内呈良好的线性关系,相关系数均大于0.999;在5、20和100 μ g/L加标水平下的平均加样回收率为86.3%~114.3%,相对标准偏差(RSD)为0.5%~13.6%;检出限为0.003~0.08 μ g/g,定量限为0.01~0.19 μ g/g。本方法已应用于实际样品的测定。     

Ultrasound ionization of biomolecules

To date, mass spectrometric analysis of biomolecules has been primarily performed with either matrix‐assisted laser desorption/ionization (MALDI) or electrospray ionization (ESI). In this work, ultrasound produced by a simple piezoelectric device is shown as an alternative method for soft ionization of biomolecules. Precursor ions of proteins, saccharides and fatty acids showed little fragmentation. Cavitation is considered as a primary mechanism for the ionization of biomolecules. Copyright © 2010 John Wiley & Sons, Ltd.     

固相萃取-超高效液相色谱-电喷雾串联质谱法同时测定地表水中16种全氟有机化合物

建立了固相萃取-超高效液相色谱-电喷雾串联三重四极杆质谱联用技术分析水中16种全氟有机化合物的高通量检测方法。样品经WAX固相萃取柱富集和净化后,采用Waters ACQUITY UPLC^TM BEH C₁₈色谱柱,含2 mmol/L乙酸铵的甲醇和含2 mmol/L乙酸铵的水作为流动相进行梯度洗脱,质谱采用电喷雾负离子电离,多反应监测模式检测。16种全氟有机化合物在0.5~100 μg/L或1.0~100 μg/L浓度范围内线性关系良好,相关系数为0.9987~0.9999,方法的检出限为0.06~0.46 ng/L;高、中、低3个添加水平的回收率为67.6%~103%,相对标准偏差为2.94%~12.0%。结果表明,该方法灵敏、准确且检测范围广,分析速度快,是一种适于实际水样中全氟有机化合物检测分析的方法。     

Electrospray ionization and atmospheric pressure matrix‐assisted laser desorption/ionization mass spectrometry of antioxidants applied in lubricants

The aim of this study was to investigate the utility of ion trap mass spectrometry (ITMS) in combination with the two desorption/ionization methods, electrospray (ESI) and atmospheric pressure matrix‐assisted laser desorption/ionization (AP‐MALDI), for the detection of antioxidants which are applied in lubricants. These experiments should form the base for future investigations of antioxidants in tribologically formed thin layers on the surface of frictional systems. Seventeen different antioxidants were selected out of the group of hindered phenolic and aromatic aminic compounds. Practically all antioxidants could be characterized by positive ion ESI‐ and AP‐MALDI‐ITMS, forming various types/species of molecular ions (e.g. [M]^{+ .} , [M+H]⁺, [M+Na]⁺ or [M–2H+H]⁺). A few compounds could be analyzed by negative ion ESI‐MS, too, but none by negative ion AP‐MALDI‐MS. The influence of target materials in AP‐MALDI‐MS (gold‐ and titanium nitride (TiN)‐covered stainless steel, micro‐diamond‐covered hard metal, hand‐polished and sand‐blasted stainless steel targets) with respect to the molecular ion intensity and type of molecular ion of two selected antioxidants was evaluated. The surface properties are of particular interest because in friction tests different materials with different surface characteristics are used. However, the MS results indicate that optimal target surfaces have to be found for individual antioxidants in AP‐MALDI‐MS but in general smooth surfaces were superior to rough surfaces. Finally the gold‐covered stainless steel MALDI target provided the best mass spectra and was selected for all the antioxidants investigated. Copyright © 2009 John Wiley & Sons, Ltd.     

Observation of gas-phase molecular dications formed from neutral organics in solution via qemical electron-transfer reactions by using electrospray Ionization Mass Spectrometry

The first observation of organic dications formed by multiple electron loss in electrospray mass spectra is reported. The dications of β-carotene, canthaxanthine, cobalt(II) octaethylporphyrin, and nicke(II) octaethylporphyrin were created in solution via chemical electrontransfer reactions and detected in the gas phase by electrospray ionization mass spectrometry (ES-MS) using a flow-injection experiment. The analytes were injected into a flowing solvent-oxidant stream (10 μL/min) composed of dried methylene chloride containing ≈ 0.1% by volume trifluoroacetic acid and 0.1% by volume antimony pentafluoride (SbF₅). The dications created in this oxidizing solvent system were preserved for detection by rapidly transferring them from the reactive solvent-oxidant system to the gas phase, where, in the absence of the solvent system, they were “long-lived” and amenable to mass analysis. This work demonstrates means to produce ions novel to ES-MS and means to detect and study by ES-MS species that are short-lived in solution. In addition, this work shows that electrospray ionization can potentially be used to generate gas-phase dications for mass spectrometric study that are difficult to produce directly from gas-phase neutrals by other ionization techniques (e.g., M²⁺ from β-carotene).     

PSMA@Ag复合微球的制备及其在MALDI-MS分析小分子化合物中的应用研究

基质辅助激光解吸电离质谱(MALDI-MS)作为一种有力的分析手段,在生物分子分析中有着广泛的应用,但很难应用于分子量小于500的待测物的分析。该文利用聚多巴胺修饰还原法制备了核壳结构的聚苯乙烯-马来酸酐共聚物@银纳米壳层(PSMA@Ag)复合微球。采用傅立叶红外光谱法验证了聚多巴胺(PDA)的成功修饰。结合扫描电子显微镜(SEM)和紫外-可见光谱(UV-Vis)分析结果,发现Ag纳米壳层成功地包覆在PSMA微球的表面。将制备的PSMA@Ag复合微球作为辅助基质直接应用于MALDI-MS,成功地从0.5μL待测物样品中检测到2 pmol脯氨酸(M_w=115)和1 pmol丝氨酸(M_w=105)。研究结果证明PSMA@Ag微球对MALDI的离子化过程有促进作用,为解决MALDI-MS在分析小分子待测物时背景噪声过大,信号无法分辨的问题提供了一个有效途径。     

Analysis of serum protein precipitated with antiserum by matrix-assisted laser desorption ionization/time-of-flight and electrospray ionization mass spectrometry as a clinical laboratory test

Serum transferrin precipitated with specific antisera was analyzed by matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF-MS) and electrospray ionization-mass spectrometry (ESI-MS). When analyzed by MALDI, transferrin showed signal peaks that clearly could be separated from ions of IgG present in the immunoprecipitate. By ESI-MS, when the immunoprecipitates were loaded through a microcapillary polymeric reversed-phase column connected to the electrospray ionization probe, the mass spectra of transferrin were observed with a high signal-to-noise ratio and good resolution. By MALDI/TOF-MS, the observed molecular weight of normal transferrin was ～ 1.2 ku smaller when analyzed in the reflectron mode than in the linear mode. The observed molecular weight of transferrin treated with sialidase was approximately the same in both modes. A comparison between the results obtained in both modes may help to estimate the number of sialic acids on the protein molecule. A transferrin isoform with a molecular weight of ～2.2 ku less than the normal species was identified in the serum of patients with a carbohydrate-deficient glycoprotein syndrome as well as in heavy alcohol consumers.     

超高效液相色谱-串联质谱法测定水稻基质中阿维菌素残留量

建立了超高效液相色谱-串联质谱法(UPLC-MS/MS)测定水稻基质中阿维菌素残留量,考察了基质效应,并对实际样品进行了检测.稻田土、稻壳、糙米和稻杆经乙腈振荡提取,稻田水经乙酸乙酯液液分配提取后,用C18固相萃取小柱或弗罗里硅土柱净化,采用UPLC-MS/MS正离子扫描测定残留的阿维菌素.稻田土、稻田水和糙米的3种添加浓度(1.0,10.0和100 μg/kg或μg/L)的平均回收率为84％～107％,相对标准偏差为4.7％～13.6％.稻壳和稻杆的2档添加浓度(10.0和100 μg/kg)的平均回收率为90％和103％,相对标准偏差为8.4％～12.9％.本方法在稻田水、糙米和稻田土中的检出限为0.3μg/kg在稻壳和稻杆中检出限为3.0 μg/kg,低于欧盟和日本在稻米中制定的阿维菌素最大残留限量值.阿维菌素在2.0～100 μg/L范围内线性关系良好( r＞ 0.999).     

Gold and silver nanoparticles‐based laser desorption/ionization mass spectrometry method for detection and quantification of carboxylic acids

A comparison of ionization efficiency for gold and silver nanoparticles used as an active media of matrix‐less laser desorption/ionization (LDI) mass spectrometry (MS) methods was made for carboxylic acids including fatty acids. The matrix‐assisted laser desorption/ionization (MALDI)‐type targets containing monoisotopic cationic ¹⁰⁹Ag nanoparticles (¹⁰⁹AgNPs) and Au nanoparticles (AuNPs) were used for rapid MS measurements of 10 carboxylic acids of different chemical properties. Carboxylic acids were directly quantified in experiments with 10 000‐fold concentration change conditions ranging from 1 mg/ml to 100 ng/ml which equates to 1 μg to 100 pg of carboxylic acids per measurement spot.     

液相色谱-电喷雾电离三重四级杆质谱测定畜禽肝脏中维吉尼亚霉素M1的残留量

建立了动物肝脏组织中维吉尼亚霉素M1残留的液相色谱-电喷雾电离三重四级杆质谱(LC-ESI-MS/MS)的测定方法。样品经0.2 mol/L NH4H2PO4和乙酸乙酯提取,分散型固相萃取PSA填料净化,采用正离子扫描多反应监测(MRM)模式下液质联用仪进行定性和定量分析。该方法省去耗时的固相萃取过程,回收率在67.2%～90.3%之间,RSD<10.0%;检测限为0.5μg/kg,定量限为1.0μg/kg。     

Applications of mass spectrometry to food proteins and peptides

The application of mass spectrometry (MS) to large biomolecules has been revolutionized in the past decade with the development of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) techniques. ESI and MALDI permit solvent evaporation and sublimation of large biomolecules into the gaseous phase, respectively. The coupling of ESI or MALDI to an appropriate mass spectrometer has allowed the determination of accurate molecular mass and the detection of chemical modification at high sensitivity (picomole to femtomole). The interface of mass spectrometry hardware with computers and new extended mass spectrometric methods has resulted in the use of MS for protein sequencing, post-translational modifications, protein conformations (native, denatured, folding intermediates), protein folding/unfolding, and protein-protein or protein-ligand interactions. In this review, applications of MS, particularly ESI-MS and MALDI time-of-flight MS, to food proteins and peptides are described.     

Analysis of sugar epimers using mass spectrometry: N-acetyllactosamine-6,6'-disulfate and the 2'-epimer

Galbeta1-4GlcNAc-6,6'-disulfate and 2'-epimer corresponding to Galbeta1- 4ManNAc-6,6'-disulfate were distinguished by mass spectrometry by utilizing fast atom bombardment (FAB), electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) methods. As for the steric information, negative-ion ESI mass spectrometry/mass spectrometry (MS/MS) provides the most extensive data, but FAB MS/MS also reveals detailed structural information of interest in our case, where MALDI MS is not yet fully equipped with post-source decay.     

超高效液相色谱三重四极杆质谱联用法快速检测尿液和血浆中鹅膏毒肽和鬼笔毒肽

建立了同时快速检测尿液和血浆中3种鹅膏毒肽和2种鬼笔毒肽的超高效液相色谱三重四极杆质谱联用分析方法。尿液样品直接进样,血浆样品经乙腈沉淀除蛋白后,在UPLCHSST3色谱柱上分离,正离子电喷雾多反应监测(MRM)模式检测,基体匹配标准外标法定量。尿液和血浆样品的线性范围分别为2～100和1～100μg/L;加标回收率分别在92.0%～108.0%和85.0%～100.0%的范围内;相对标准偏差为1.0%～22.0%和2.0%～22.0%(n=6);样品的检出限为0.2～1.0μg/L和0.1～0.5μg/L(S/N=3)。本方法灵敏,简单,快速,特异性强。