Preparation of single cells for imaging/profiling mass spectrometry

Characterizing chemical changes within individual cells is important for determining fundamental mechanisms of biological processes that will lead to new biological insights and improved disease understanding. Analyzing biological systems with imaging and profiling mass spectrometry (MS) has gained popularity in recent years as a method for creating chemical maps of biological samples. To obtain mass spectra that provide relevant molecular information about individual cells, samples must be prepared so that salts and other cell culture components are removed from the cell surface and that the cell contents are rendered accessible to the desorption beam. We have designed a cellular preparation protocol for imaging/profiling MS that removes the majority of the interfering species derived from the cellular growth medium, preserves the basic morphology of the cells, and allows chemical profiling of the diffusible elements of the cytosol. Using this method, we are able to reproducibly analyze cells from three diverse cell types: MCF7 human breast cancer cells, Madin-Darby canine kidney (MDCK) cells, and NIH/3T3 mouse fibroblasts. This preparation technique makes possible routine imaging/profiling MS analysis of individual cultured cells, allowing for understanding of molecular processes within individual cells.     

Comprehensive mass spectrometry based metabolic profiling of blood plasma reveals potent discriminatory classifiers of pancreatic cancer

Poor outcome of pancreatic cancer necessitates development of an early diagnostic method to reduce mortality. No reliable early diagnostic test for pancreatic cancer detection has been developed and validated to date. In the current study, metabolic profiling of plasma samples from selected cancer patients and noncancerous controls was performed to seek novel metabolic biomarkers of pancreatic cancer. A comprehensive mass spectrometry based analytical platform established at the Metabolomics Core of the UC Davis Genome Center allowed detection of multiple compounds previously unreported in plasma from pancreatic cancer patients. It was found that selective amino acids, bile acids, and polar lipids were detected with increased or decreased levels in pancreatic cancer samples compared to controls. These findings on blood plasma levels of the relevant metabolites might be very useful clinical parameters for early diagnosis of pancreatic cancer. Copyright © 2010 John Wiley & Sons, Ltd.     

Metabolite mass spectrometry profiling of cacao genotypes reveals contrasting resistances to Ceratocystis cacaofunesta phytopathogen

Ceratocystis wilt is a lethal disease of cacao, and the search for resistant genotypes may provide the best way to deal with the disease. Resistance or susceptibility behavior of some cacao genotypes when infected by Ceratocystis cacaofunesta is not yet understood. Herein, we report an LC-MS metabolomic screening analysis based on high-resolution MS to obtain comprehensive metabolic profile associated with multivariate data analysis of PLS-DA, which was effective to classify CCN-51 and TSH-1188 as resistant genotypes to C. cacaofunesta fungus, while CEPEC2002 was classified as a susceptible one. Using reversed-phase LC method, electrospray interface, and high-resolution tandem MS by the quadrupole-TOF analyzer, the typical profiles of metabolites, such as phenylpropanoids, flavonoids, lipids, alkaloids, and amino acids, were obtained. Untargeted metabolite profiles were used to construct discriminant analysis by partial least squares (PLS-DA)-derived loading plots, which placed the cacao genotypes into two major clusters related to susceptible or resistant groups. Linolenic, linoleic, oleic, stearic, arachidonic, and asiatic acids were annotated metabolites of infected, susceptible, and resistant genotypes, while methyl jasmonate, jasmonic acid, hydroxylated jasmonic acid, caffeine, and theobromine were annotated as constituents of the resistant genotypes. Trends of these typical metabolites levels revealed that CCN51 is susceptible, CEPEC2002 is moderately susceptible, and TSH1188 is resistant to C. cacaofunesta. Therefore, profiles of major metabolites as screened by LC-MS offer an efficient tool to reveal the level of resistance of cacao genotypes to C. cacaofunesta present in any farm around the world.     

Mass spectrometry profiling analysis enables the identification of new modifications in ribosomal RNA

Ribosomal RNAs (rRNAs) provide the structural framework of ribosomes and play critical roles in protein translation. In ribosome biogenesis, rRNAs acquire various modifications that can influence the structure and catalytic activity of ribosomes. However, rRNA modifications in plants have yet to be fully defined. Herein, we proposed a method to purify rRNAs by a successive isolation with different strategies, including polyA-based mRNA depletion and agarose gel electrophoresis-based purification, with which highly pure rRNAs could be obtained. In addition, we developed a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method to systematically profile and characterize modifications from the isolated highly pure plant 18S rRNA and 25S rRNA. LC-ESI-MS/MS analysis showed that 10 and 12 kinds of modifications were present in plant 18S rRNA and 25S rRNA, respectively. Notably, among these identified modifications, 2 kinds of modifications of N²,N²-dimethylguanosine (m^2,2G) and N⁶,N⁶-dimethyladenosine (m^6,6A) in 18S rRNA, and 4 kinds of modifications of m^2,2G, m^6,6A, N7-methylguanosine (m⁷G) and 3-methyluridin (m³U) in 25S rRNA, were first reported to be present in plants. Moreover, exposure of Arabidopsis thaliana to cadmium (Cd) led to significant changes of modifications in both 18S rRNA and 25S rRNA of plants, indicating that rRNA modifications play important roles in response to environmental stress. The discovery of new modifications in plant rRNAs improves the spectra of plant rRNA modifications and may promote the investigation of the functional roles of plant ribosomes in regulating gene expression.     

Stable isotopic labelling-assisted untargeted metabolic profiling reveals novel conjugates of the mycotoxin deoxynivalenol in wheat

An untargeted screening strategy for the detection of biotransformation products of xenobiotics using stable isotopic labelling (SIL) and liquid chromatography–high resolution mass spectrometry (LC-HRMS) is reported. The organism of interest is treated with a mixture of labelled and non-labelled precursor and samples are analysed by LC-HRMS. Raw data are processed with the recently developed MetExtract software for the automated extraction of corresponding peak pairs. The SIL-assisted approach is exemplified by the metabolisation of the Fusarium mycotoxin deoxynivalenol (DON) in planta. Flowering ears were inoculated with 100 μg of a 1?+?1 (v/v) mixture of non-labelled and fully labelled DON. Subsequent sample preparation, LC-HRMS measurements and data processing revealed a total of 57 corresponding peak pairs, which originated from ten metabolites. Besides the known DON and DON-3-glucoside, which were confirmed by measurement of authentic standards, eight further DON-biotransformation products were found by the untargeted screening approach. Based on a mass deviation of less than ±5 ppm and MS/MS measurements, one of these products was annotated as DON-glutathione (GSH) conjugate, which is described here for the first time for wheat. Our data further suggest that two DON-GSH-related metabolites, the processing products DON-S-cysteine and DON-S-cysteinyl-glycine and five unknown DON conjugates were formed in planta. Future MS/MS measurements shall reveal the molecular structures of the detected conjugates in more detail.     

Quantitative metabolic profiling of 21 endogenous corticosteroids in urine by liquid chromatography-triple quadrupole-mass spectrometry

Since corticosteroid metabolism may be affected by disease states, the accurate and precise measurement of endogenous corticosteroids in urine is necessary to understand their biochemical roles. An efficient quantitative profiling of 21 endogenous corticosteroids in urine has been validated by liquid chromatography-tandem mass spectrometry (LC-MS/MS). After enzymatic hydrolysis with β-glucuronidase, samples were purified using a solid-phase extraction cartridge and then separated through a sub-2 μm particle C18 column (2.1 mm × 50 mm, 1.9 μm) and quantified within 12.1 min using a triple quadrupole MS with electrospray ionization in positive ion mode. All corticosteroids resulted in the base-line separation, which is even achieved for stereo-isomers, such as α-/β-cortol, α-/β-cortolone, and allo-tetrahydrocortisol/tetrahydrocortisol. Overall recoveries ranged from 85% to 106% with limit of quantification ranged from 0.5 to 2.0 ng mL⁻¹ for the corticosteroids examined. The precision (% CV) and accuracy (% bias) of the assay were 1.7-7.8% and 95.1-105.4%, respectively, in 0.5-200 ng mL⁻¹ calibration ranges (r² > 0.9903), for quality-control samples containing 21 endogenous corticosteroids at three different urinary concentrations. Clinical application included quantitative analysis from patients with both prostate cancer and benign prostatic hyperplasia with altered cortisol concentrations. The described LC-MS/MS method eliminates interference from other urine components, has excellent chromatographic resolution achieved by a small particle LC column with a sufficient sensitivity to allow the profiling of both gluco- and mineralo-corticosteroids at a time.     

Global profiling of ultraviolet-induced metabolic disruption in Melissa officinalis by using gas chromatography-mass spectrometry

Melissa officinalis contains various secondary metabolites that have health benefits. Generally, irradiating plants with ultraviolet (UV)-B induces the accumulation of secondary metabolites in plants. To understand the effect of UV-B irradiation on the metabolism of M. officinalis, metabolomics based on gas chromatography-mass spectrometry (GC-MS) was used in this study. The GC-MS analysis revealed 37 identified metabolites from various chemical classes, including alcohols, amino acids, inorganic acids, organic acids, and sugars. The metabolite profiles of the groups of M. officinalis irradiated with UV-B were separated and differentiated according to their irradiation times (i.e., 0, 1, and 2 h), using principal component analysis (PCA) and hierarchical clustering analysis (HCA), respectively. The PCA score plots of PC1 and PC2 showed that the three groups with different irradiation times followed a certain trajectory with increasing UV-B irradiation. HCA revealed that metabolic patterns differed among the three groups, and the 1 h-irradiated group was more similar to the control group (0 h) than the 2 h-irradiated group. In particular, UV-B irradiation of plants led to a decrease in sugars such as fructose, galactose, sucrose, and trehalose and an increase in metabolites in the tricarboxylic acid cycle, the proline-linked pentose phosphate pathway, and the phenylpropanoid pathway. This study demonstrated that metabolite profiling with GC-MS is useful for gaining a holistic understanding of UV-induced changes in plant metabolism.     

液相色谱-质谱联用系统在肝硬化不同阶段代谢轮廓研究中的应用

采用高效液相色谱-轨道离子阱质谱联用(HPLC-LTQ Orbitrap XL MS)代谢组学研究平台分析不同阶段肝硬化病人和健康人群的血清标本,获取代谢轮廓。采用模式识别方法结合非参数检验对数据进行分析。研究发现,由肝硬化A级组、B级组、C级组和健康对照组的代谢轮廓构建的正交偏最小二乘判别分析(OPLS-DA)模型(R2（Y）=90.1%, Q2=66.7%),对检测组数据的预测准确率达到93.8%,具有很好的判别能力。从代谢轮廓中可以鉴别出用于区分不同疾病阶段的特异性代谢标志物,如溶血磷脂酰胆碱、甘氨鹅去氧胆酸、半胱氨酸、甘氨酸、氨基己二酸、哌可酸等。研究结果表明: 利用代谢组学方法获得的血清代谢轮廓可以用来构建区分模型和寻找代谢标志物,为乙肝肝硬化的诊断和监测提供支持和依据。     

Direct peptide profiling of single neurons by matrix-assisted laser desorption–ionization mass spectrometry

Peptide profiles of single neurons in Lymnaea stagnalis were directly characterized by matrix-assisted laser desorption–ionization mass spectrometry. The mass analysis was performed after minor pretreatment and without any separation steps. Good-quality spectra were obtained of several cell types and also other tissues. The results were compared with the results of conventional peptide chemical methods. In addition to many known peptides, several new peptides were identified. The method provides new opportunities for studying peptide compositions at the single-cell level, which is shown to have many advantages.     

Nonacid cleavable detergents applied to MALDI mass spectrometry profiling of whole cells

Although cleavable detergents were first synthesized a number of years ago, they have only recently been successfully applied to problems involving biological molecules. Recent reports have demonstrated that these compounds are useful for applications involving both 2D PAGE and mass spectrometry. However, most cleavable surfactants have utilized acid-labile functional groups to affect cleavage. In applications where extreme pH is required, acid cleavable detergents have limited usefulness. We report the synthesis of fluoride cleavable silane compounds and photolabile cinnamate esters as cleavable detergents having alternative cleavage chemistries than previously reported cleavable detergents. These compounds were applied to whole cell analysis using MALDI mass spectrometry, and it was demonstrated that their use results in an increase in the number of proteins analyzed by increasing protein solubility.     

Mass spectrometric analysis reveals O-methylation of pyruvate kinase from pancreatic cancer cells

Pyruvate kinase (PK) is an important glycolytic enzyme that catalyzes the dephosphorylation of phosphoenolpyruvate to pyruvate. Human PK isozyme M2 (PKM2), a splice variant of M1, is overexpressed in many cancer cells, and PKM2 has been investigated as a potential tumor marker for diagnostic assays and as a target for cancer therapy. To facilitate identification and characterization of PK, we studied the enzyme from pancreatic cancer cells and normal pancreatic duct cells by electrophoresis and mass spectrometry, and identified multiple O-methylated residues from PK. These findings advance our knowledge of the biochemical properties of PK and will be important in understanding its biological function in cells.

Raman spectroscopic identification of single bacterial cells under antibiotic influence

The identification of pathogenic bacteria is a frequently required task. Current identification procedures are usually either time-consuming due to necessary cultivation steps or expensive and demanding in their application. Furthermore, previous treatment of a patient with antibiotics often renders routine analysis by culturing difficult. Since Raman microspectroscopy allows for the identification of single bacterial cells, it can be used to identify such difficult to culture bacteria. Yet until now, there have been no investigations whether antibiotic treatment of the bacteria influences the Raman spectroscopic identification. This study aims to rapidly identify bacteria that have been subjected to antibiotic treatment on single cell level with Raman microspectroscopy. Two strains of Escherichia coli and two species of Pseudomonas have been treated with four antibiotics, all targeting different sites of the bacteria. With Raman spectra from untreated bacteria, a linear discriminant analysis (LDA) model is built, which successfully identifies the species of independent untreated bacteria. Upon treatment of the bacteria with subinhibitory concentrations of ampicillin, ciprofloxacin, gentamicin, and sulfamethoxazole, the LDA model achieves species identification accuracies of 85.4, 95.3, 89.9, and 97.3 %, respectively. Increasing the antibiotic concentrations has no effect on the identification performance. An ampicillin-resistant strain of E. coli and a sample of P. aeruginosa are successfully identified as well. General representation of antibiotic stress in the training data improves species identification performance, while representation of a specific antibiotic improves strain distinction capability. In conclusion, the identification of antibiotically treated bacteria is possible with Raman microspectroscopy for diverse antibiotics on single cell level.

基于液相色谱与质谱联用的代谢组学及磷脂轮廓分析在糖代谢异常研究中的应用

糖代谢异常由于其发病率的升高和影响人类的生活质量而日益受到科学研究者的关注。实验中利用液相色谱与质谱(LC-MS)联用技术对糖代谢异常分别进行了代谢组学和磷脂轮廓分析,研究了糖代谢异常中的两个阶段——空腹血糖受损(IFG)和初诊糖尿病(NDD)的代谢差异情况。首先从LC-MS采集到血浆中代谢组学分析及磷脂轮廓分析的原始谱图,通过软件的峰匹配等步骤得到峰表,之后利用多种统计分析方法进行数据分析,通过正交校正的偏最小二乘法(OSC-PLS)对样品进行分型,根据模型的变量重要因子(VIP)、显著性差异检验结果等筛选出差异性代谢物。结果显示: NDD组比IFG组与对照组(N组)比较存在更明显的代谢差异,发生变化的化合物主要为游离脂肪酸、溶血磷脂酰胆碱、磷脂酰乙醇胺、鞘磷脂和磷脂酰胆碱等。     

Lipid profiling of mammalian cells with in situ matrix-assisted laser desorption ionization-mass spectrometry

The analysis of cellular lipids and phospholipids has been of continuously increasing research interest due to the importance of these molecules in psychological process. In this work, a mass spectrometry-based method for direct, in situ analysis of lipids in cells was reported. Mammalian cells were directly cultured on ITO-coated glass and then analyzed by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). The matrix application process was achieved by electrospray coating, which produced a homogenous layer of matrix crystal on the sample. The detection results and reproducibility are satisfactory. With this method, a profile of abundant membrane lipids is generated, which is characteristic of cell type. In conclusion, this in situ MALDI-MS cellular lipid analysis method provides a platform for sensitive and robust molecular profiling of mammalian cells.     

Characterization and classification of rat neural stem cells and differentiated cells by comparative metabolic and lipidomic profiling

It is necessary to characterize and classify neural stem cells (NSCs) and differentiated cells (DCs) for potential use of NSC to treat neurodegenerative diseases. We therefore performed an analysis of NSCs and DCs using gas chromatography mass spectrometry (GC-MS) and direct infusion mass spectrometry (DI-MS) with elaborate multivariate statistical analysis for the characterization and classification of rat NSCs and DCs. GC-MS and DI-MS detected a total of 92 metabolites and lipids in NSCs and DCs, and the levels of 72 of them differed significantly between NSCs and DCs. The optimal model for partial least squares (PLS) discriminant analysis was constructed by applying 3 and 2 PLS components with a unit-variance scaling method for classifying NSCs and DCs based on the data obtained in the GC-MS and DI-MS analyses, respectively. The obtained results from PCA and PLS-DA suggest that creatinine, lactic acid, lysine, glutamine, glycine, pyroglutamic acid, PG 18:1/20:2, PS 18:0/20:2, PI 18:0/20:3, PC 16:0/20:4, PI 16:0/20:4, and PI 18:1/20:4 were the main contributors that provided distinct characteristics of NSCs and DCs. The results of this study suggest objective and complementary criteria for the characterization and classification of NSCs and DCs for potential clinical applications.

Graphical abstract

     

Mass spectrometry of quaternary ammoniohexanoates: Intermolecular alkyl transfer during field desorption

Intermolecular alkyl transfer occurs during field desorption of quaternary ammoniohexanoates, resulting in mass spectra containing structurally diagnostic adduct ions. Methyl, ethyl and propyl groups attached to nitrogen readily undergo intermolecular transfer to give [M+CH₃]⁺, [M+C₂H₅]⁺ and [M+C₃H₇]⁺ ions, respectively. Evidence is presented that alkyl groups even as large as C₁₀H₂₁ can transfer intermolecularly at high emitter temperatures. In addition to the alkyl ion adducts, the field desorption spectra of \documentclass{article}\pagestyle{empty}\begin{document}$ {\rm C}_{10} {\rm H}_{21} \mathop {\rm N}\limits^ + \left({{\rm CH}_3 } \right)_2 \left({{\rm CH}_2 } \right)_5 {\rm COO}^ - $\end{document} show several other adduct and fragment ions whose relative intensities depend strongly on emitter current. The field desorption results are compared with earlier pyrolysis electron impact results on similar compounds.     

Mass spectrometry as a tool for the selective profiling of destruxins; their first identification in Lecanicillium longisporum

Mass spectrometry was applied to the identification of the destruxins (dtxs), cyclic peptides that are commonly produced by the fungal insect‐pathogen, Metarhizium anisopliae. The aim of the study was to optimise a methodology in order to firstly determine whether these compounds were present in other species and to determine the effect of differing growth conditions upon the dtx content detected. Matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry (MALDI‐ToF‐MS) was initially used to analyse the dtxs, but limitations were indicated. Nano‐scale high‐performance liquid chromatography/electrospray ionisation mass spectrometry (HPLC/ESI‐MS) and automated ‘data‐dependent’ tandem mass spectrometric (MS/MS) analysis were also applied, utilising characteristic neutral losses during fragmentation to confirm the presence of the dtxs. This latter approach distinguished the dtx E and B isoforms by retention time and diagnostic neutral losses during fragmentation allowing extraction of the destruxin data from a complex dataset. This process revealed the presence of a number of dtxs in the fungal species Lecanicillium longisporum, a species previously not known to produce dtxs, and dtx production in this species was shown to be significantly higher in aerated cultures compared with still cultures. Copyright © 2009 John Wiley & Sons, Ltd.     

A microfluidic processor for gene expression profiling of single human embryonic stem cells

The gene expression of human embryonic stem cells (hESC) is a critical aspect for understanding the normal and pathological development of human cells and tissues. Current bulk gene expression assays rely on RNA extracted from cell and tissue samples with various degree of cellular heterogeneity. These 'cell population averaging' data are difficult to interpret, especially for the purpose of understanding the regulatory relationship of genes in the earliest phases of development and differentiation of individual cells. Here, we report a microfluidic approach that can extract total mRNA from individual single-cells and synthesize cDNA on the same device with high mRNA-to-cDNA efficiency. This feature makes large-scale single-cell gene expression profiling possible. Using this microfluidic device, we measured the absolute numbers of mRNA molecules of three genes (B2M, Nodal and Fzd4) in a single hESC. Our results indicate that gene expression data measured from cDNA of a cell population is not a good representation of the expression levels in individual single cells. Within the G0/G1 phase pluripotent hESC population, some individual cells did not express all of the 3 interrogated genes in detectable levels. Consequently, the relative expression levels, which are broadly used in gene expression studies, are very different between measurements from population cDNA and single-cell cDNA. The results underscore the importance of discrete single-cell analysis, and the advantages of a microfluidic approach in stem cell gene expression studies.     

Development and validation of a gas chromatography/mass spectrometry method for the metabolic profiling of human colon tissue

In this study, a gas chromatography/mass spectrometry (GC/MS) method was developed and validated for the metabolic profiling of human colon tissue. Each colon tissue sample (20 mg) was ultra‐sonicated with 1 mL of a mixture of chloroform/methanol/water in the ratio of 20:50:20 (v/v/v), followed by centrifugation, collection of supernatant, drying, removal of moisture using anhydrous toluene and finally derivatization using N‐methyl‐N‐trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS). A volume of 1 µL of the derivatized mixture was injected into the GC/MS system. A total of 53 endogenous metabolites were separated and identified in the GC/MS chromatogram, all of which were selected to evaluate the sample stability and precision of the method. Of the identified endogenous metabolites 19 belonging to diverse chemical classes and covering a wide range of the GC retention times (Rt) were selected to investigate the quantitative linearity of the method. The developed GC/MS method demonstrated good reproducibility with intra‐ and inter‐day precision within relative standard deviation (RSD) of ±15%. The metabolic profiles of the intact tissue were determined to be stable (100 ± 15%) for up to 90 days at ?80°C. Satisfactory results were also obtained in the case of other stability‐indicating studies such as freeze/thaw cycle stability, bench‐top stability and autosampler stability. The developed method showed a good linear response for each of the 19 analytes tested (r² > 0.99). Our GC/MS metabolic profiling method was successfully applied to discriminate biopsied colorectal cancer (CRC) tissue from their matched normal tissue obtained from six CRC patients using orthogonal partial least‐squares discriminant analysis [two latent variables, R²Y = 0.977 and Q² (cumulative) = 0.877]. Copyright © 2009 John Wiley & Sons, Ltd.     

Evaluation of automated single mass spectrometry to tandem mass spectrometry function switching for comprehensive drug profiling analysis using a quadrupole time-of-flight mass spectrometer

A liquid chromatographic mass spectrometric strategy for systematic toxicological analysis (STA) is presented using the automatic 'on-the-fly' single mass spectrometry mode to tandem mass spectrometry mode (MS to MS/MS) switching abilities of a quadrupole time-of-flight (Q-TOF) instrument. During the chromatographic run, the quadrupole is initially set to transmit all masses until (an) ion(s) reaches a certain set threshold. Thereupon, the quadrupole automatically switches to the MS/MS mode, selecting the ion(s), which are subsequently fragmented in the high-efficiency hexapole collision cell, thus generating product ions that are further mass analyzed by the TOF. By limiting the TOF spectral accumulation time in the MS/MS mode to a statistically acceptable minimum, the quadrupole almost instantly switches back to the MS mode. Qualitative information, comprising the complementary MS ([M + H](+) ion mass) and MS/MS (informative product ion profile) data, as well as quantitative information obtained by integration of the MS extracted ion chromatogram(s), can be obtained in one single acquisition. Optimization of the automatic switching parameters, such as threshold, TOF spectral accumulation time, detection window and collision energy, was carried out by injection of a mix of 17 common drugs which were not necessarily baseline separated in the chromatographic system used. Indeed, the complete separation of the drugs is not deemed necessary since up to 8 different ions can 'simultaneously' be selected for MS/MS if they reach the preset criteria. In addition, the quantitative performance of the method was defined. In a second phase, the developed method was field-tested. To that end, the resulting data from extracts of urine samples were compared with and found to be in close concordance with those obtained by a standard toxicological analysis. This innovative approach clearly holds the potential for a substantial advance in the introduction of LC/MS in STA.