高效液相色谱-非特异性同位素稀释质谱联用技术定量分析全血中总血红蛋白含量

建立了基于同位素稀释(ID)的高效液相色谱(HPLC)-电感耦合等离子体质谱(ICP-MS)联用技术, 通过测量血红蛋白(HGB)中铁(Fe)元素含量实现了全血中总HGB准确定量的策略. 该策略包括2种方法, 第一种方法为在线HPLC-ID-ICP-MS定量技术: 全血中HGB经液相色谱分离后, 通过柱后在线添加浓缩⁵⁴Fe同位素稀释剂的方式, 与液相色谱洗脱液经三通混合后进入ICP-MS在线测量⁵⁴Fe/⁵⁶Fe的同位素比值, 并根据同位素稀释质谱法公式及蛋白中Fe的含量计算HGB的浓度. 针对全血样品中存在的其它主要含铁蛋白[如转铁蛋白(Tf)], 在实现色谱完全分离的基础上, 通过在线优化改变⁵⁴Fe稀释剂流速实现了HGB和Tf的同时定量. 在另一种方法中, 先对全血样品进行消解并通过ID-ICP-MS测定其中Fe的总量, 然后根据全血样品的HPLC-ICP-MS分析结果, 通过计算HGB质谱峰面积占总峰面积的比值从而得到HGB中Fe含量占总Fe含量的比例, 实现了其中HGB的准确定量. 2种定量方法得到的结果分别为(115.3±2.4) mg/g和(115.5±2.1) mg/g, 结果吻合良好, 方法的检出限为1.0×10^?7 mg/g, 方法精密度RSD均<3%. 2种方法均经过HGB标准物质IRMM/IFCC-467进行验证, 测量结果在标准值不确定度范围之内, 可作为临床检验领域HGB分析的参考测量方法.     

A novel approach for analysis of oligonucleotide-cisplatin interactions by continuous elution gel electrophoresis coupled to isotope dilution inductively coupled plasma mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry

In this work we present a novel approach for in vitro studies of cisplatin interactions with 8-mer oligonucleotides. The approach is based on the recently developed coupling of continuous elution gel electrophoresis (GE) to an inductively coupled plasma-sector field mass spectrometer (ICP-SFMS) with the aim of monitoring the interaction process between this cytostatic drug and the nucleotides. In contrast to existing methods, the electrophoretic separation conditions used here allow both the determination of the reaction kinetics in more detail as well as the observation of dominant intermediates. Two different nucleotides sequences have been investigated for comparison purposes, one containing two adjacent guanines (5'-TCCGGTCC-3') and one with a combination of thymine and guanine (5'-TCCTGTCC-3'), respectively. In order to gain further structural information, MALDI-TOF MS measurements have been performed after fraction collection. This allows for identification of the intermediates and the final products and confirms the stepwise coordination of cisplatin via monoadduct to bisadduct formation. Furthermore, the ICP-MS results were quantitatively evaluated in order to calculate the kinetics of the entire process.     

基体辅助激光解吸/电离质谱测定小相对分子质量物质的方法进展

MALD I-MS分析具有高灵敏度、高分辨率、高质量准确度、制样快速、操作简单及高通量等优点,具有使各种不同类型物质离子化的能力。目前已广泛应用于化学、化工、材料、环境、地质、能源、刑侦、药物、生命科学等领域中。MALD I-MS最早主要用于分析各种生物大分子及聚合物。近年     

电离辐射诱导T淋巴细胞的差异蛋白质组学研究

应用电离辐射诱导人类T淋巴细胞白血病Jurkat细胞株, 再经二维凝胶电泳分离不同照射剂量的细胞总蛋白, 研究电离辐射诱导的人类T淋巴细胞白血病细胞蛋白的差异表达. 应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)进行处理, 得到肽质量指纹(PMF)图谱, 确认6个差异表达蛋白点.     

An attempt to differentiate HPLC-ICP-MS selenium speciation in natural and selenised Agaricus mushrooms using different species extraction procedures

Total determination and speciation analysis of Se in commercial and selenised Agaricus mushrooms have been performed to investigate the Se species naturally occurring in non-enriched mushrooms as well as those present in specimens grown in a Se-enriched medium. Mushroom aqueous and enzymatic extracts have been analysed by three complementary chromatographic separation mechanisms (size-exclusion, anion-exchange and reversed-phase) coupled to an inductively coupled plasma mass spectrometer with an octopole reaction system. Post-column isotope dilution analysis has been used on-line with the separations for quantification of the Se species eluted. The ⁷⁸Se-to-⁷⁷Se isotope ratio was monitored after adequate corrections for both total determinations and Se species quantitative speciation. The results showed marked differences not only in total Se contents but also in Se species found in the two types of Agaricus mushrooms investigated. Selenomethionine was detected in both of them (free in commercial mushrooms and incorporated into proteins in selenised ones) together with a number of unknown selenocompounds.     

同位素稀释热电离质谱法测定人血清中痕量铜和锌

采用热电离同位素稀释质谱法(ID-TIMS)准确测定了欧盟标准物质与测量研究院(EC-JRC-IRMM)组织的国际测量评估计划IMEP-17人血清样品中的痕量铜和锌。由于锌和铜都是易受污染的元素,本工作建立了仅用少量硝酸消解的低流程本底和适于热电离质谱测量的生物基体血清中痕量铜和锌的样品前处理方法;采用适当比例的硅胶和磷酸作为电离增强剂,在热电离质谱(TIMS)测量时获得了较高强度且稳定的铜和锌离子束;血清中痕量铜和锌的测量结果可直接溯源到国际单位mole。2种人血清样品中铜和锌测量结果的不确定度(k=2)分别为0.94%、0.83%和0.49%,测量值被EC-JRC-IRMM采用作为该样品的标准值。     

蛋白质分子量测定过程中的酸效应

在基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）和电喷雾质谱（ESI-MS）测定蛋白质分子分子量的过程中,适当提高样品的酸度,可提高分析测试的灵敏度。在选定最佳样品分子浓度的基础上,通过适当加入三氟乙酸（TFA）来调整测试样品的酸度,准确测定了标准蛋白质-溶菌酶（lysozyme）的分子量,并且对蛋白质分子在“软电离”质谱中,受酸效应的影响进行了初步探讨。     

Structural studies of the allelic wheat glutenin subunits 1Bx7 and 1Bx20 by matrix-assisted laser desorption/ionization mass spectrometry and high-performance liquid chromatography/electrospray ionization mass spectrometry

Structural studies of the high molecular mass (HMM) glutenin subunits 1Bx7 (from cvs Hereward and Galatea) and 1Bx20 (from cv. Bidi17) of bread wheat were conducted using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry (RP-HPLC/ESI-MS). For all three proteins, MALDI-TOFMS analysis showed that the isolated fractions contained a second component with a mass about 650 Da lower than the major component. The testing and correction of the gene-derived amino acid sequences of the three proteins were performed by direct MALDI-TOFMS analysis of their tryptic peptide mixture. Analysis of the digest was performed by recording several MALDI mass spectra of the mixture at low, medium and high mass ranges, optimizing the matrix and the acquisition parameters for each mass range. Complementary data were obtained by RP-HPLC/ESI-MS analysis of the tryptic digest. This resulted in coverage of about 98% of the sequences. In contrast to the gene-derived data, the results obtained demonstrate the insertion of the sequence QPGQGQ between Trp716 and Gln717 of subunit 1Bx7 (cv. Galatea) and a possible single amino acid substitution within the T20 peptide of subunit 1Bx20. Moreover, the mass spectrometric data demonstrated that the lower mass components present in all the fractions correspond to the major components but lack about six amino acid residues, which are probably lost from the protein C-terminus. Finally, the results obtained provide evidence for the lack of glycosylation or other post-translational modifications of these subunits.     

Limited proteolysis combined with isotope labeling and quantitative LC-MALDI MS for monitoring protein conformational changes: a study on calcium-binding sites of cardiac Troponin C

Studies of protein-protein and protein-ligand interactions are important for understanding biological functions of proteins. A new technique based on the partial proteolysis of proteins combined with quantitative mass spectrometry is developed as a means of tracking structural changes after the formation of a protein-ligand complex. In this technique, a protein of interest with and without the binding of a ligand is digested with an enzyme to generate a set of peptides, followed by separation of the peptides by liquid chromatography. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) is used to identify chromatographically separated peptides, and locate their sequence alignments in the parent protein. Using an isotopically labeled protein as a sample against an unlabeled protein standard, quantitative information can be gathered. This overcomes the inherent lack of quantitative capability of MALDI MS. The utility of the technique to investigate protein-ligand interactions is demonstrated in a model system involving calcium binding to cardiac Troponin C (cTnC). Using this technique, the general location of the three calcium-binding sites of cTnC can be determined by using several different enzymes to generate overlapping peptide maps of cTnC.     

Trace iodine quantitation in biological samples by mass spectrometric methods: The optimum internal standard

Accurate quantitation of iodine in biological samples is essential for studies of nutrition and medicine, as well as for epidemiological studies for monitoring intake of this essential nutrient. Despite the importance of accurate measurement, a standardized method for iodine analysis of biological samples is yet to be established. We have evaluated the effectiveness of ⁷²Ge, ¹¹⁵In, and ¹²⁹I as internal standards for measurement of iodine in milk and urine samples by induction coupled plasma mass spectrometry (ICP-MS) and of ³⁵Cl¹⁸O₄⁻, ¹²⁹I⁻, and 2-chlorobenzenesulfonate (2-CBS) as internal standards for ion chromatography-tandem mass spectrometry (IC-MS/MS). We found recovery of iodine to be markedly low when IC-MS/MS was used without an internal standard. Percent recovery was similarly low using ³⁵Cl¹⁸O₄ as an internal standard for milk and unpredictable when used for urine. 2-Chlorobenzebenzenesulfonate provided accurate recovery of iodine from milk, but overestimated iodine in urine samples by as much as a factor of 2. Percent recovery of iodine from milk and urine using ICP-MS without an internal standard was ∼120%. Use of ¹¹⁵In predicted approximately 60% of known values for both milk and urine samples. ⁷²Ge provided reasonable and consistent percent recovery for iodine in milk samples (∼108%) but resulted in ∼80% recovery of iodine from urine. Use of ¹²⁹I as an internal standard resulted in excellent recovery of iodine from both milk and urine samples using either IC-MS/MS and ICP-MS.     

Iron isotope fractionation between liquid and vapor phases of iron pentacarbonyl

Iron isotope fractionation between liquid and vapor iron pentacarbonyl was measured in a closed system at ∼0 and ∼21 °C to determine if Fe isotope analysis of iron pentacarbonyl vapor is viable using electron-impact, gas-source mass spectrometry. At the 2σ level, there is no significant Fe isotope fractionation between vapor and liquid under conditions thought to reflect equilibrium. Experiments at ∼0 °C indicate iron pentacarbonyl vapor is ∼0.05 per mil (‰) greater in ⁵⁶Fe/⁵⁴Fe than liquid iron pentacarbonyl, which is just resolvable at the 1σ level. Partial decomposition of iron pentacarbonyl vapor or liquid to an iron oxide or iron metal shows that significant isotopic fractionation occurs, where the decomposed product has a lower ⁵⁶Fe/⁵⁴Fe ratio as compared to the starting iron pentacarbonyl. It follows that methods to decompose iron pentacarbonyl must be quantitative to obtain accurate isotope values.     

Fabrication of nanostructured silicon by metal-assisted etching and its effects on matrix-free laser desorption/ionization mass spectrometry

A matrix-free, high sensitivity, nanostructured silicon surface assisted laser desorption/ionization mass spectrometry (LDI-MS) method fabricated by metal-assisted etching was investigated. Effects of key process parameters, such as etching time, substrate resistance and etchant composition, on the nanostructured silicon formation and its LDI-MS efficiency were studied. The results show that the nanostructured silicon pore depth and size increase with etching time, while MS ion intensity increases with etching time to 300 s then decreases until 600 s for both low resistance (0.001–0.02 Ω cm) and high resistance (1–100 Ω cm) silicon substrates. The nanostructured silicon surface morphologies were found to directly affect the LDI-MS signal ion intensity. By characterizing the nanostructured silicon surface roughness using atomic force microscopy (AFM) and sample absorption efficiency using fluorescence microscopy, it was further demonstrated that the nanostructured silicon surface roughness was highly correlated to the LDI-MS performance.     

High-efficiency protein extraction from polyacrylamide gels for molecular mass measurement by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry

A simple and fast method of protein extraction from Coomassie Brilliant Blue (CBB)-stained polyacrylamide gels suited for molecular mass measurement of proteins by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) is reported. Proteins in CBB-stained gel pieces were extracted by a 10-min soaking in 0.1 M NaOH at 25 degrees C. The recovery of this one-step extraction method was 34-73% for proteins <67 kDa. CBB adduction to proteins during mass spectrometric analysis was avoided by a destaining step before the alkaline extraction. The molecular mass values of the extracted proteins coincided with those of purified proteins within +/-0.01-0.10% deviation for all the proteins <36 kDa. Because of the high extraction recovery, mass measurement was possible for the proteins extracted from CBB-stained gels with loaded protein quantities as little as 34 ng for cytochrome c, alpha-lactalbumin, myoglobin, beta-lactoglobulin, trypsinogen, and carbonic anhydrase (12.4-29.0 kDa), 340 ng for glyceraldehyde-3-phosphate dehydrogenase (35.6 kDa) and albumin (66.3 kDa). This method provides a highly efficient approach to utilize CBB-stained one- or two-dimensional gels for whole protein analysis using MALDI-TOF-MS.     

Alkaline cleavage of covalent bonds in chicken insulin and bovine alpha-lactalbumin analyzed by matrix-assisted laser desorption/ionization-mass spectrometry

In the course of searching methods to extract proteins from Coomassie blue-stained polyacrylamide gels, we found proteins are extracted in relatively high recovery when the gel pieces are soaked in alkaline solutions. However, alkaline conditions are known to cause decomposition of proteins, especially peptide bond cleavage and disulfide degradation. We studied the effects of alkaline on two purified proteins, chicken insulin and bovine alpha-lactalbumin, both containing four disulfide bonds in their structure. The process of covalent bond cleavage was traced by analyzing the mass spectra of the proteins using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). When the proteins are kept at pH 13 in the presence of 0.1% dithithreitol (DTT), peptide bonds at the C-terminal side of asparaginyl residues are preferably cleaved producing succinimides, whereas cysteinyl residues are not decomposed. In the absence of DTT, the disulfide bonds of the proteins are decomposed by alkaline and the cleavage of the peptide bonds are less obvious, possibly because the conformation of the proteins are partially retained until the full decomposition of disulfide bonds. These results identified for the first time the cleavage sites of proteins under alkaline treatment and further suggested the general tendency of the reactions, both in the presence and absence of DTT.     

Alternative approaches for the detection of various phospholipid classes by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The detection of phospholipids (PLs) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was demonstrated nearly a decade ago. However, its use as a conventional tool for PL analysis has been hindered by ambiguities in peak assignments caused by spectral overlaps and difficulties in the detection of some PL classes when analytes with positively charged head groups, such as sphingomyelins (SMs) and phosphatidylcholines (PCs) are present. In this work, either a strong cation-exchange resin or CsCl crystals were added directly to the PL samples to reduce spectral complexity and enhance sensitivity. The quantitative exchange resulted in virtually only protonated or Cs+ adducts. To alleviate difficulties in the detection and identification of PL classes with ionization efficiencies lower than those of SMs and PCs, improvements in the sensitivity of negative-ion mass spectra were sought. For this purpose, several neutral and basic matrices were tried. Among them, p-nitroaniline (PNA) proved to be an advantageous alternative to the use of 2,5-dihydroxybenzoic acid (DHB), the most commonly used matrix in PL analysis. Because of its lower acidity, PNA increased the relative amount of deprotonated species and improved the sensitivity of negative-ion mass spectra. It was possible to confirm peak assignments for PL classes that normally give weak signals when DHB is used. Noteworthy is the detection (in both positive and negative modes) and conclusive identification of species in natural mixtures of phosphatidylethanolamines (PEs) and PE plasmalogens (PEps). PNA allowed the identification of PEs and PEps even in mixtures containing SMs and PCs. Although some cations related to PCs and PEs overlapped in positive-ion spectra, these interferences were eliminated in the negative mode as only the deprotonated forms of PEs and PEps were detectable and those of SMs and PCs were absent owing to their neutrality.     

Positive and negative‐mode laser desorption/ionization‐mass spectrometry (LDI‐MS) for the detection of indigoids in archaeological purple

Laser‐based ionization techniques have demonstrated to be a valuable analytical tool to study organic pigments by mass spectrometric analyses. Though laser‐based ionization techniques have identified several natural and synthetic organic dyes and pigments, they have never been used in the characterization of purple. In this work, positive and negative‐mode laser desorption/ionization mass spectrometry (LDI‐MS) was used for the first time to detect indigoids in shellfish purple. The method was used to study organic residues collected from archaeological ceramic fragments that were known to contain purple, as determined by a classical high‐performance liquid chromatography‐based procedure. LDI‐MS provides a mass spectral fingerprint of shellfish purple, and it was found to be a rapid and successful tool for the identification of purple. In addition, a comparison between positive and negative mode ionization highlighted the complementarity of the two ionization modes. On the one hand, the negative‐ion mode LDI‐MS showed a better selectivity and sensitivity to brominated molecules, such as 6,6'‐dibromoindigo, 6‐monobromoindigo, 6,6'‐dibromoindirubin, 6‐ and 6’‐monobromoindirubin, thanks to their electronegativity, and produced simpler mass spectra. On the other hand, negative‐ion mode LDI‐MS was found to have a lower sensitivity to non‐brominated compounds, such as indigo and indirubin, whose presence can be established in any case by collecting the complementary positive‐ion LDI mass spectrum. Copyright © 2013 John Wiley & Sons, Ltd.     

Discovery of the serum biomarker proteins in severe preeclampsia by proteomic analysis

Preeclapsia (PE) is a severe disorder that occurs during pregnancy, leading to maternal and fetal morbidity and mortality. PE affects about 3-8% of all pregnancies. In this study, we conducted liquid chromatography- mass spectrometry/mass spectrometry (LC-MS/MS) to analyze serum samples depleted of the six most abundant proteins from normal and PE-affected pregnancies to profile serum proteins. A total of 237 proteins were confidently identified with <1% false discovery rate from the two groups of duplicate analysis. The expression levels of those identified proteins were compared semiquantitatively by spectral counting. To further validate the candidate proteins with a quantitative mass spectrometric method, selective reaction monitoring (SRM) and enzyme linked immune assay (ELISA) of serum samples collected from pregnant women with severe PE (n = 8) or normal pregnant women (n = 5) was conducted. α2- HS-glycoprotein (AHSG), retinol binding protein 4 (RBP4) and α-1-microglobulin/bikunin (AMBP) and Insulin like growth factor binding protein, acid labile subunit (IGFBP-ALS) were confirmed to be differentially expressed in PE using SRM (P<0.05). Among these proteins, AHSG was verified by ELISA and showed a statistically significant increase in PE samples when compared to controls.     

Applications of Tandem Mass Spectrometry (MS/MS) in Protein Analysis for Biomedical Research

Mass Spectrometry (MS) allows the analysis of proteins and peptides through a variety of methods, such as Electrospray Ionization-Mass Spectrometry (ESI-MS) or Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS). These methods allow identification of the mass of a protein or a peptide as intact molecules or the identification of a protein through peptide-mass fingerprinting generated upon enzymatic digestion. Tandem mass spectrometry (MS/MS) allows the fragmentation of proteins and peptides to determine the amino acid sequence of proteins (top-down and middle-down proteomics) and peptides (bottom-up proteomics). Furthermore, tandem mass spectrometry also allows the identification of post-translational modifications (PTMs) of proteins and peptides. Here, we discuss the application of MS/MS in biomedical research, indicating specific examples for the identification of proteins or peptides and their PTMs as relevant biomarkers for diagnostic and therapy.     

Two-dimensional electrophoresis protein profiling as an analytical tool for human acute leukemia classification

Two-dimensional electrophoresis (2-DE) was used to profile the proteins of leukemic cells from 61 cases of akute leukemia (AL) characterized by the French-American-British (FAB) classification. The differentially expressed protein spots were identified by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and electrospray ionization-tandem MS (ESI-MS/MS). The distinct protein profiles (DPPs) of AL FAB subtypes were explored successfully, including acute myeloid leukemia (AML), its subtypes (M2, M3, and M5), and acute lymphoid leukemia (ALL), which were homogeneous within different samples of the same subgroup but clearly differed from all other subgroups. We also found a group of proteins differentially expressed between AL cells and normal white blood cells. Among the DPPs of AL subtypes, some proteins have been reported, but most of them were first reported here to mark AML differentiation and to discriminate AML from ALL. These data show that 2-DE protein profiling could be used as an analytical tool for facilitating molecular definition of human AL classification and understanding the mechanism of leukemogensis, and the extension of the present analysis to the currently less well-defined AL will identify additional subgroups and may promote the identification of new targets for specific treatment approaches.     

MoS2/Ag nanohybrid: A novel matrix with synergistic effect for small molecule drugs analysis by negative-ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

This paper reports a facile synthesis of molybdenum disulfide nanosheets/silver nanoparticles (MoS₂/Ag) hybrid and its use as an effective matrix in negative ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The nanohybrid exerts a strong synergistic effect, leading to high performance detection of small molecule analytes including amino acids, peptides, fatty acids and drugs. The enhancement of laser desorption/ionization (LDI) efficiency is largely attributed to the high surface roughness and large surface area for analyte adsorption, better dispersibility, increased thermal conductivity and enhanced UV energy absorption as compared to pure MoS₂. Moreover, both Ag nanoparticles and the edge of the MoS₂ layers function as deprotonation sites for proton capture, facilitating the charging process in negative ion mode and promoting formation of negative ions. As a result, the MoS₂/Ag nanohybrid proves to be a highly attractive matrix in MALDI-TOF MS, with desired features such as high desorption/ionization efficiency, low fragmentation interference, high salt tolerance, and no sweet-spots for mass signal. These characteristic properties allowed for simultaneous analysis of eight different drugs and quantification of acetylsalicylic acid in the spiked human serum. This work demonstrates for the first time the fabrication and application of a novel MoS₂/Ag hybrid, and provides a new platform for use in the rapid and high throughput analysis of small molecules by mass spectrometry.