基于化学衍生-质谱技术的生物与临床样本中核酸修饰分析

除了经典碱基外,核酸(DNA和RNA)中还包含许多化学修饰。迄今为止,已经在核酸中鉴定了超过150多种化学修饰。这些化学修饰不会改变核酸的序列,但会改变它们的结构和生化特性,最终调节基因的时空表达。阐明这些修饰的功能可以促进对生命体生理调控机制的深入认识和理解。然而,核酸修饰在体内的丰度通常很低。因此,高灵敏和特异的检测方法对破译这些修饰的功能至关重要。化学衍生与质谱技术相结合对内源性低丰度核酸修饰展现出很好的分析能力。在过去几年中,研究者建立了多种基于化学衍生-质谱分析的分析方法,用于灵敏、高效地分析核酸修饰。该文总结了通过化学衍生-质谱分析方法来破译核酸修饰的最新进展,希望能促进未来对核酸修饰功能的深入研究。     

Ellagic Acid Suppresses ApoB Secretion and Enhances ApoA-1 Secretion from Human Hepatoma Cells,HepG2

The effect of ellagic acid (EA), a naturally occurring polyphenolic compound, on the secretion of apolipoproteins from human hepatocytes, HepG2, was investigated. The levels of apoB and apoA-1 secreted in the cell culture medium were determined by sandwich ELISA. EA did not affect cell viability at the tested concentrations (up to 50 µM). EA suppressed the secretion of apoB and enhanced that of apoA-1 from HepG2 cells. However, cellular apoB levels were increased, suggesting that EA inhibited the trafficking of apoB during the process of secretion. In contrast, the increase in the cellular levels of apoA-1 was consistent with its secreted levels. These results indicate that EA inhibits the secretion of apoB from hepatocytes and increases the secretion of apoA-1. Both of these effects are beneficial for lipoprotein metabolism in the prevention of lifestyle-related diseases. The detailed mechanism underlying these effects of EA on lipoprotein metabolism should be elucidated in the future, but this naturally occurring polyphenolic compound might be antihyperlipidemic. Based on these results, EA is suggested as a candidate food-derived compound for the prevention of hyperlipidemia.     

质谱技术鉴定细胞中组蛋白翻译后修饰的研究进展*

组蛋白是真核细胞中构成染色质内核小体的主要元件,其翻译后修饰蕴藏着组蛋白密码,是表观遗传学的重要内容,影响染色质的结构和功能,进而调控基因表达。组蛋白翻译后修饰形式的鉴定是揭示组蛋白密码的关键,目前质谱技术已经成为分析组蛋白及其翻译后修饰的重要工具。本文综述了组蛋白翻译后修饰鉴定方法的新进展,介绍了基于质谱技术“bottom up”和“top down”的组蛋白分析策略,及CID、ECD和ETD等鉴定组蛋白修饰位点的质谱碎片裂解技术,并结合当前研究进展,评述了质谱技术在组蛋白翻译后修饰谱的鉴定、组蛋白各种变体的测定、以及在生理过程中组蛋白修饰丰度动态变化的定量分析等方面应用的新进展。     

Matrix-assisted laser desorption/ionization mass spectrometry for the evaluation of the C-terminal lysine distribution of a recombinant monoclonal antibody

Recombinant monoclonal antibodies produced using mammalian cell lines contain multiple chemical modifications. One specific modification resides on the C-terminus of the heavy chain. Enzymes inside the cell can cleave the C-terminal lysine from the heavy-chain molecules, and variants with and without C-terminal lysine can be produced. In order to fully characterize the protein, there is a need for analytical methods that are able to account for the different product variants. Conventional analytical methods used for the measurement of the distribution of the two different variants are based on chemical or enzymatic degradation of the protein followed by chromatographic separation of the degradation products. Chromatographic separations with gradient elution have long run times, and analyses of multiple samples are time-consuming. This paper reports development of a novel method for the determination of the relative amounts of the two C-terminal heavy-chain variants based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) measurements of the cyanogen bromide degraded recombinant monoclonal antibody products. The distribution of the variants is determined from the MALDI-TOF mass spectra by measuring the peak areas of the two C-terminal peptides. The assay was used for the assessment of the C-terminal lysine distribution in different development lots. The method was able to differentiate between the products obtained using the same cell line as well as between products obtained from different cell lines.     

Quantitative determination of melphalan DNA adducts using HPLC - inductively coupled mass spectrometry

For the quantification of Melphalan DNA adducts, an analytical approach based on the detection of phosphorus using liquid chromatography combined with inductively-coupled-plasma mass spectrometry (ICP-MS) was developed. In reaction mixtures of native 2'-deoxynucleotides-5'-monophosphates and Melphalan, which were separated using reversed phase chromatography, phosphate adducts were found as the most abundant modifications. Besides the phosphate adducts, several base alkylated adducts were observed. In calf thymus DNA incubated with Melphalan and enzymatically digested using Nuclease P1, the phosphate adducts as well as monoalkylated dinucleotides were found. The most abundant single Melphalan adduct observed in DNA was a ring-opened adenosine monophosphate. Some dinucleotide adducts and the adenosine adduct were identified using electrospray ionization mass spectrometry (ESI-MS).     

Deciphering nucleic acid modifications by chemical derivatization-mass spectrometry analysis

Chemical derivatization in combination with mass spectrometry (MS) analysis is a promising strategy for the sensitive and effective analysis of nucleic acid modifications. In this review, we summarize the recent advances for deciphering modifications in DNA and RNA by chemical derivatization-MS analysis.     

Multiresidue Screening of Veterinary Drugs in Meat,Milk, Egg,and Fish Using Liquid Chromatography Coupled with Ion Trap Time-of-Flight Mass Spectrometry

New approaches to veterinary drug screening based on liquid chromatography-mass spectrometry (LC-MS/MS) and time-of-flight mass spectrometry (ToF/MS) are rapid and have high selectivity and sensitivity. In this study, we developed a multiresidue method for screening over 100 veterinary drug residues using ion trap (IT)-ToF/MS. The screened compounds comprised major drug classes used in veterinary practice, representing the following: amphenicols, anthelmintics, benzimidazoles, β-lactams, coccidiostats, ionophores, macrolides, non-steroidal anti-inflammatory drugs, quinolones, sulfonamides, tetracyclines, and tranquilizers. The method was developed based on chromatographic retention time, specific accurate mass, isotope distribution, and fragment data. Each compound was validated at three levels, and the mass accuracy, accuracy, and repeatability were calculated. All parameters showed acceptable values and conformed to the Commission Decision 2002/657/EC criteria. This screening method can simultaneously analyze over 100 veterinary drugs in meat, milk, eggs, and fish in a single analytical run.     

A strategy for distinguishing modified peptides based on post-digestion 18O labeling and mass spectrometry

The simultaneous identification of multiple different protein modifications, with or without known mass changes, is a challenging application of mass spectrometry. In this contribution, a strategy for distinguishing modified peptides within a large background of unmodified peptides was demonstrated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis of cytochrome c (Cyt-c) modified with 4-hydroxy-2-nonenal (HNE), based on post-digestion 18O labeling. Labeling of control Cyt-c peptides obtained from in-solution or in-gel digestion with 18O, prior to mixing in the ratio of 1:1 with peptides derived from a modified sample, identified more HNE modifications than a method based on a known mass increment search (Isom AL, Barnes S, Wilson L, Kirk M, Coward L, Darley-Usmar V. J. Am. Soc. Mass Spectrom. 2004; 15: 1136), demonstrating the potential of this strategy to enhance the detection of modified peptides by mass spectrometry. A virtue of the strategy is that it obviates the need for isotopic labeling of the modifier, making the method applicable to the detection of modifications occurring in vivo. Additionally, this technique identified protease auto-cleavage peptides by their altered mass isotopomer distribution due to incomplete 18O exchange, and modified peptides containing 'protein carbonyls' by partial 18O exchange, allowing these peptides to be differentiated during data analysis.     

Affinity and chemical enrichment strategies for mapping low‐abundance protein modifications and protein‐interaction networks

Protein post‐translational modifications and protein interactions are the central research areas in mass‐spectrometry‐based proteomics. Protein post‐translational modifications affect protein structures, stabilities, activities, and all cellular processes are achieved by interactions among proteins and protein complexes. With the continuing advancements of mass spectrometry instrumentations of better sensitivity, speed, and performance, selective enrichment of modifications/interactions of interest from complex cellular matrices during the sample preparation has become the overwhelming bottleneck in the proteomics workflow. Therefore, many strategies have been developed to address this issue by targeting specific modifications/interactions based on their physical properties or chemical reactivities, but only a few have been successfully applied for systematic proteome‐wide study. In this review, we summarized the highlights of recent developments in the affinity enrichment methods focusing mainly on low stoichiometric protein lipidations. Besides, to identify potential glyoxal modified arginines, a small part was added for profiling reactive arginine sites using an enrichment reagent. A detailed section was provided for the enrichment of protein interactions by affinity purification and chemical cross‐linking, to shed light on the potentials of different enrichment strategies, along with the unique challenges in investigating individual protein post‐translational modification or protein interaction network.     

Precipitation and selective extraction of human serum endogenous peptides with analysis by quadrupole time-of-flight mass spectrometry reveals posttranslational modifications and low-abundance peptides

The endogenous peptides of human serum may have regulatory functions, have been associated with physiological states, and their modifications may reveal some mechanisms of disease. In order to correlate levels of specific peptides with disease alongside internal standards, the polypeptides must first be reliably extracted and identified. Endogenous blood peptides can be effectively enriched by precipitation of the serum with organic solvents followed by selective extraction of peptides using aqueous solutions modified with organic solvents. Polypeptides on filter paper were assayed with Coomasie brilliant blue binding. The polypeptides were resolved by detergent tricine polyacrylamide electrophoresis and visualized by diamine silver staining. Peptides in the extracts were collected by C18 and analyzed by matrix-assisted laser desorption/ionization and liquid chromatography–electrospray ionization–tandem mass spectrometry (MS/MS) quadrupole time-of-flight MS/MS. Peptides were resolved as multiple isotopic peaks in MS mode with mass deviation of 0.1 Da or less and similar accuracy for fragments. The sensitivity of MS and MS/MS analysis was estimated to be in the picomolar range or less. The peptide composition of the extracts was dependent on solvent formulation. Multiple peptides from apolipoproteins, complement proteins, coagulation factors, and many others were identified by X!Tandem with high mass accuracy of peptide ions and fragments from collision-induced dissociation. Many previously unreported posttranslational modifications of peptides including phosphorylations, oxidations, glycosylations, and others were detected with high mass accuracy and may be of clinical importance. About 4,630 redundant peptides were identified with 99% confidence separately, and together some 1,251 distinct proteins were identified with 99% confidence or greater using the Paragon algorithm.     

Adding a new separation dimension to MS and LC–MS: What is the utility of ion mobility spectrometry?

Ion mobility spectrometry is an analytical technique known for more than 100 years, which entails separating ions in the gas phase based on their size, shape, and charge. While ion mobility spectrometry alone can be useful for some applications (mostly security analysis for detecting certain classes of narcotics and explosives), it becomes even more powerful in combination with mass spectrometry and high‐performance liquid chromatography. Indeed, the limited resolving power of ion mobility spectrometry alone can be tackled when combining this analytical strategy with mass spectrometry or liquid chromatography with mass spectrometry. Over the last few years, the hyphenation of ion mobility spectrometry to mass spectrometry or liquid chromatography with mass spectrometry has attracted more and more interest, with significant progresses in both technical advances and pioneering applications. This review describes the theoretical background, available technologies, and future capabilities of these techniques. It also highlights a wide range of applications, from small molecules (natural products, metabolites, glycans, lipids) to large biomolecules (proteins, protein complexes, biopharmaceuticals, oligonucleotides).     

Identification of unexpected modifications of fluorescein-labeled oligodeoxynucleotides by nuclease P1 digestion and mass spectrometric techniques

Fluorescein-labeled oligodeoxynucleotides (ODNs) from automated synthesis commonly produce multiple peaks in high performance liquid chromatography (HPLC) chromatograms. We found that these peaks are due to chemical modifications of the ODNs instead of the common perception of isomers. To identify the modifications, a model ODN, fluorescein-T(25), was synthesized and five compounds were isolated. Nuclease P1 (NP1) digestion was employed to cleave these compounds into nucleotides and fluorescein-nucleotides in order that the modifications be determined by mass spectrometry (MS). Analyses of NP1 digestion products containing fluorescein by MS revealed the expected product F1-T (M) and four unexpected compounds with MWs at M-1, M-17, M-16 and M + 16, respectively. Collision-induced dissociation (CID) spectra of these digestion products indicate that all modifications occur on the thiourea linkage [-NH-C( = S)-NH-] to the fluorescein moiety and the adjacent phosphate group, and the modifications were determined. The modifications were also confirmed by accurate mass measurement with Fourier transform mass spectrometry (FT-MS), by the synthesis of a reference compound, and by a mechanistic study using model compounds. These results demonstrate the power of the mass spectrometric techniques by determining the structures of two pairs of ODNs with MW difference of 1 Da. The results also suggest that fluorescein phosphoramidite with a thiourea linkage is not appropriate for the automated synthesis of fluorescein-labeled ODNs of high purity.     

Analysis of multiple pesticide residues in tobacco using pressurized liquid extraction, automated solid-phase extraction clean-up and gas chromatography-tandem mass spectrometry

A new method was developed for the analysis of pesticide residues in tobacco. The objective was to significantly increase the number of samples that can be processed by the laboratory and to enable the extension of the current coverage to additional pesticides. A new analytical approach was therefore defined based on two main axes, the automation of the sample preparation and the selectivity of the analyte detection using tandem mass spectrometry. This latter aspect reduces the stringency of the requirements placed on the clean-up of the extracts and on the chromatographic resolution when less selective detectors are used. The extraction of the analytes from the matrix is performed using the pressurized liquid extraction technique. Tobacco samples are extracted at elevated temperature and pressure (100 C and 100 atm; 1 atm = 101,325 Pa) using acetone as an extraction solvent. The resulting extract is then concentrated using a Vortex evaporator. Three different solid-phase extraction (SPE) procedures, adjusted to the chemical properties of the different active ingredients to be measured, are applied to the concentrated extract, thus leading to three extract fractions. The first fraction contains such main classes of active ingredients as organohalogenated and 2,6-dinitroaniline compounds while the second one collects the organophosphorus and acylalanines residues; these two fractions are analyzed by capillary gas chromatography coupled to tandem mass spectrometry using negative chemical ionization and electron impact ionization in the positive mode, respectively. The third extract fraction gathers the N-methylcarbamates residues which are analyzed by HPLC with post-column derivatization and fluorescence detection. The different sample preparation stages from extraction to SPE clean-up have been automated through the use of recent analytical technologies. In combination with the analysis by tandem mass spectrometry, this provided a potential for a high sample throughput.     

N-Terminal peptide labeling strategy for incorporation of isotopic tags: a method for the determination of site-specific absolute phosphorylation stoichiometry

Determining the phosphorylation stoichiometry at specific sites in a phosphoprotein is a very challenging task. We describe here a novel mass spectrometry based method that is capable of measuring the absolute phosphorylation stoichiometry at specific sites without the need for specific internal standards, phospho-site antibodies or radioactivity. The method is based on a gentle chemical labeling strategy which specifically and differentially labels the N-terminus of all peptides in a sample with either a D(5)- or D(0)-propionyl group and measures the ratio of the abundance of the D(5)/D(0) peptide pairs simultaneously using mass spectrometry. Using matrix-assisted laser desorption/ionization (MALDI), the method can measure absolute stoichiometry to within at least 10% and can be applied to both in vitro and in vivo phosphorylated peptides and proteins. Furthermore, this method can potentially be applied to the quantitative study of other types of protein post-translational modifications, and the profiling of protein expression on the proteome level.     

Glycomics and glycoproteomics: Approaches to address isomeric separation of glycans and glycopeptides

Changes in the glycome of human proteins and cells are associated with the progression of multiple diseases such as Alzheimer's, diabetes mellitus, many types of cancer, and those caused by viruses. Consequently, several studies have shown essential modifications to the isomeric glycan moieties for diseases in different stages. However, the elucidation of extensive isomeric glycan profiles remains challenging because of the lack of analytical techniques with sufficient resolution power to separate all glycan and glycopeptide iso‐forms. Therefore, the development of sensitive and accurate approaches for the characterization of all the isomeric forms of glycans and glycopeptides is essential to tracking the progression of pathology in glycoprotein‐related diseases. This review describes the isomeric separation achievements reported in glycomics and glycoproteomics in the last decade. It focuses on the mass spectrometry–based analytical strategies, stationary phases, and derivatization techniques that have been developed to enhance the separation mechanisms in liquid chromatography systems and the detection capabilities of mass spectrometry systems.     

Quaternary ammonium pesticides: A review of chromatography and non-chromatography methods for determination of pesticide residues in water samples

The monitoring of pesticide residues in water sources is essential because of their increased worldwide demand in agriculture and their subsequent detection in waters. Pesticide residues in water matrices are traditionally determined by multiresidue methodologies based on chromatography coupled to mass spectrometry. However, for quaternary ammonium pesticide residues, as highly polar compounds, the chromatographic approach frequently fails, requiring modifications in the separation method, or even an alternative technique for analyte quantification. Therefore, to solve this analytical limitation for these residues, several authors proposed unusual methodologies, such as those based on spectroscopic or electroanalytical approaches. This review intends to offer an overview of the analysis of quaternary ammonium pesticide residues in different water sources, focusing on advances in sample preparation before chromatographic separations and alternative analytical techniques, such as spectroscopy and electroanalytical methods.     

Electrospray ionization tandem mass spectrometry of biphenyl-modified oligo(deoxy)ribonucleotides

Antisense oligonucleotides and aptamers are important candidates for future therapeutic applications. Different structural modifications are introduced into oligonucleotides to obtain high affinity and binding specificity. Sequence elucidation of oligonucleotides incorporating a wide variety of modifications presents an analytical challenge, as the standard protocols cannot be applied. Mass spectrometry has the potential to solve complex structural problems. However, a better understanding of the fundamental aspects of gas-phase dissociation of modified DNA and RNA is needed. In this work the influence of specific chemical modifications on backbone dissociation is pointed out. Biphenyl-modified oligo(deoxy)ribonucleotides, which incorporate C-glycosidic bound abasic nucleobase substitutes, were subjected to collision-induced dissociation in an electrospray tandem mass spectrometer, with the goal to investigate the role of nucleobase loss on backbone dissociation. DNA bearing biphenyl nucleobase substitutes show abundant [a-B]- and w-ions generated by cleavage of the 3'-C-O bonds, except for the phosphodiester groups adjacent to the biphenyl modifications. At these positions no dissociation was observed, demonstrating the dependence of DNA backbone dissociation on nucleobase loss. Also, no evidence for a base loss independent mechanism responsible for formation of w-ions was found. RNA incorporating biphenyl nucleobase substitutes fragment into c- and y-ions resulting from cleavage of the 5'-P-O bond. Adjacent to the biphenyl modifications no altered dissociation behavior was found. This leads to the conclusion that dissociation of RNA is independent of the 1'-modification and, therefore, independent of nucleobase loss.     

Characterisation of archaeological waterlogged wood by pyrolytic and mass spectrometric techniques

Two techniques based on analytical pyrolysis and mass spectrometry, direct exposure-MS (DE-MS) and pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS), were used to characterise waterlogged archaeological wood and to study degradation patterns of wood in aqueous environments. The two techniques were applied to samples from the excavation of the Site of the Ancient Ships of Pisa San Rossore in Pisa (Italy), and data were compared to those relative to native sound wood of the same species (pine, elm, beech). Both the methods result valuable in the analysis of ancient wood artefacts, avoiding the long wet-chemical procedures that are commonly used in wood analysis, and allowing us to use a minimal sample size. DE-MS achieves a global mass spectral fingerprint of lignin and polysaccharides pyrolysis compounds in few minutes, and the results have been interpreted with the support of principal component analysis (PCA) of mass spectra. Py-GC/MS permits detailed molecular analysis of pyrolysis compounds and highlights some chemical modifications of lignin in archaeological samples, as demethylation of both guaiacyl and syringyl lignin units. Both the techniques demonstrate consistent loss of polysaccharides in archaeological wood.     

Structural characterization of intact antibodies by high‐resolution LTQ Orbitrap mass spectrometry

Recombinant monoclonal antibodies (MAbs) can be heterogeneous due to modifications that can occur during expression, purification or during storage. These large multichain proteins (～150 kDa) are structurally challenging for detailed characterization to identify the sites of modifications. We report the use of LTQ Orbitrap mass spectrometry to accurately measure the average masses of individual glycoforms by direct infusion of an intact antibody. To identify the site‐specific modification of methionines in the antibody caused by forced oxidation, we used a ‘middle‐down’ approach. The antibody was subjected to limited digestion using the endoproteinase Lys‐C and reduced to generate Fab heavy chain, single chain Fc and light chain fragments (～25 kDa each). These species were subjected to on‐line liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) analysis using an LTQ Orbitrap, where these large precursors were dissociated by higher‐energy collisions in the C‐trap. High resolution and accuracy achieved for resulting fragments allowed us to show in a site‐specific manner that only the methionines in the Fc heavy chain were oxidized under the studied conditions. Copyright © 2009 John Wiley & Sons, Ltd.     

Solid-state glycation of beta-lactoglobulin by lactose and galactose: localization of the modified amino acids using mass spectrometric techniques

The Maillard reaction is commonly encountered during food processing or storage, and also in human nutrition, hence there is a need for analytical methodologies to identify and characterize the modified proteins. This paper reports specific methods using mass spectrometric techniques to localize protein modifications induced by lactose and galactose on beta-lactoglobulin (beta-Lg) under solid-state glycation conditions. The extent of glycation was first determined by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). The specific identification of lactose-modified amino acid residues was realized using both NanoESI-MS, NanoESI-MS/MS (neutral loss scanning modes) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) (with and without guanidination of lysine residues) on unfractionated digests. The results indicated that, after 8.25 h of incubation, the lysine residues were the main targets of lactose-induced modification. In addition to the 15 lysine residues, Leu1 (NH2 terminal) and the Arg124 were also found to be modified, thus leading to a total of 17 different modified amino acid residues (versus 15 found by LC/ESI-MS measurement). In a second set of experiments, different strategies consisting of constant neutral loss and precursor ion scanning were compared to characterize galactose-induced modifications. Owing to the high level of beta-Lg glycation, the combined use of these different strategies appeared to be necessary for determining the galactose-modified sites after 8.25 h of incubation. Thus, among the 22 galactose adducts deduced from the LC/ESI-MS measurement, apart from the N-terminal and classical lysine residues, we also observed a few arginine residues (Arg40, Arg124 and Arg148) that were modified, and also dialkylations on specific lysine residues (Lys47, Lys75).