Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.     

Separation of Organoarsenicals by Means of Zwitterionic Hydrophilic Interaction Chromatography (ZIC®‐HILIC) and Parallel ICP‐MS/ESI‐MS Detection

An analytical scheme was developed for the separation and detection of organoarsenicals using a zwitterionic stationary phase of hydrophilic interaction chromatography (ZIC^®‐HILIC) coupled in parallel to electrospray ionization mass spectrometry (ESI‐MS) and to inductively coupled plasma mass spectroscopy (ICP‐MS). The optimization of separation and detection for organoarsenicals was mainly focused on the influence of the percentage of acetonitrile (MeCN) used as a major component of the mobile phase. Isocratic and gradient elution was applied by varying the MeCN percentage from 78 % to 70 % MeCN and 22 % to 30 % of an aqueous solution of ammonium acetate (125 mM NH₄Ac; pH 8.3) on a ZIC^®‐HILIC column (150 × 2.1 mm id, 3.5 μm), to allow for the separation and successful detection of nine organoarsenicals (i.e., 3‐nitro‐4‐hydroxyphenylarsonic acid (roxarsone, Rox), phenylarsonic acid (PAA), p‐arsanilic acid (p‐ASA), phenylarsine oxide (PAO), dimethylarsinate (DMA), methylarsonate (MMA), arsenobetaine (AsB), arsenocholine (AsC) and trimethylarsine oxide (TMAO)) within 45 min. All analytes were prepared in the mobile phase. The flow rate of the mobile phase, the splitting ratio between ICP‐MS and ESI‐MS detection, and the oxygen addition were adapted to ensure that there appeared a stably burning inductively coupled plasma. Furthermore, the analytical method was evaluated by the identification and quantification of AsB in the reference material DORM‐2 (dogfish muscle) resulting in a 95‐% recovery with respect to the AsB concentration in the extract.     

Phenol Soluble Modulin (PSM) Variants of Community-Associated Methicillin-Resistant Staphylococcus aureus (MRSA) Captured Using Mass Spectrometry-Based Molecular Networking

Molecular genetic analysis indicates that the problematic human bacterial pathogen methicillin-resistant Staphylococcus aureus possesses more than 2000 open reading frames in its genome. This number of potential gene products, coupled with intrinsic mechanisms of posttranslational modification, endows methicillin-resistant Staphylococcus aureus with a highly complex biochemical repertoire. Recent proteomic and metabolomic advances have provided methodologies to better understand and characterize the biosynthetic factors released by microbial organisms. Here, the emerging tool of mass spectrometry-based molecular networking was used to visualize and map the repertoire of biosynthetic factors produced by a community-associated methicillin-resistant Staphylococcus aureus strain representative of the epidemic USA300 clone. In particular, the study focused on elucidating the complexity of the recently discovered phenol soluble modulin family of peptides when placed under various antibiotic treatment stresses. Novel PSM truncated variant peptides were captured, and the type of variants that were clustered by the molecular networks platform changed in response to the different antibiotic treatment conditions. After discovery, a group of the peptides were selected for functional analysis in vitro. The peptides displayed bioactive properties including the ability to induce proinflammatory responses in human THP-1 monocytes. Additionally, the tested peptides did not display antimicrobial activity as previously reported for other phenol soluble modulin truncated variants. Our findings reveal that the PSM family of peptides are quite structurally diverse, and suggest a single phenol soluble modulin parent peptide can functionally spawn differential bioactivities in response to various external stimuli.A significant percentage of the human population harbors Staphylococcus aureus as a natural resident of their microbial flora (1). In non-infected individuals, S. aureus maintains a homeostatic coexistence with neighboring microbes and with the host (2). However, when equilibrium is disrupted, S. aureus is capable of establishing diseases ranging from minor superficial infections to potentially life-threatening invasive conditions (3, 4). Through the discovery and administration of antibiotics, clinicians have significantly reduced the morbidity and mortality associated with S. aureus (5, 6). In recent years, the consequences of persistent antibiotic exposure have become increasingly manifest through the emergence of resistant strains, including those left unperturbed by methicillin and related β-lactam antibiotics (MRSA)¹ or those with intermediate or full resistance to vancomycin (VISA/VRSA) (7). Certain drug-resistant S. aureus strains, such as the USA300 clone, have spread beyond the hospital setting and into the community, producing serious infections even in previously healthy individuals (8, 9). Both community-associated (CA) and hospital-associated (HA) multi-drug resistant S. aureus are highly evolved, multifaceted pathogens that pose an increasing threat to the health of not only our own, but future generations as well.The pathogenic mechanisms of S. aureus have been examined extensively through different stages of infection (10, 11), with an improved understanding of specific functions that various staphylococcal components contribute to virulence (12). The ability to outcompete other microflora for colonization, or to circumvent the host immune response during infectious spread, largely reflects a complex network of proteins and non-protein molecules that S. aureus secretes into its surroundings-which may collectively be termed biosynthetic release factors. One component of this network is the newly studied and multifunctional phenol soluble modulin (PSM) family of peptides. Associated with biofilm formation and possessing bioactivity toward host cells, PSMs are produced more abundantly by CA-MRSA compared with HA-MRSA strains (13, 14). PSMs are biosynthesized and released with formylated initiator methionine, allowing binding and activation of host neutrophils via the formyl peptide receptor 2 (FPR2) (15). A number of PSMs show cytolytic properties toward host cell lines, and supporting this, isogenic PSM-knockout mutants have decreased virulence in murine models of invasive infection (13). Recently, it was found that many in vitro PSMs phenotypes (e.g. neutrophil lysis, FPR2 binding) were strongly inhibited by serum lipoproteins (16). The same study showed up-regulation of PSM biosynthesis following phagocytosis, suggesting an important role in intracellular survival. Additionally, N-terminal truncated derivative forms of PSMs (dPSMs) with antibacterial properties were identified (17, 18). The finding that dPSMs possess antibacterial activities that are absent in the corresponding full-length parent PSMs, inspired a hypothesis that dPSMs endow CA-MRSA strains with a competitive advantage against resident microbiota upon colonizing host epithelia (i.e. once truncated PSMs gain antimicrobial properties).The invaluable tool of MS has played a major role in characterizing biosynthetic release factors for a diverse number of organisms (19). Over the past decade, the field has spawned several innovative methods to optimize data collection, processing, and organization. The advancement of MS processing methods has improved the capabilities for extracting significant information regarding an organism''s biochemical repertoire. One emerging tool to study biosynthetic release factor potential is the tandem MS data processing program, Spectral Networks. In conjunction with Cytoscape graphical mapping, Spectral Networks allows for the construction of a molecular network map that gives a visualization of tandem mass spectra grouping, based on similar scoring of overlapping ion fragmentation patterns (20, 21). The matching of spectra, termed spectral pairing, is unlike the conventional mode of processing MS data by matching against a reference library or specifying specific posttranslational modifications for identification (22). Instead, within a molecular network map, ion fragmentation patterns can be represented by correlation points, displayed as circular nodes, connected with a straight line to other nodes containing high similarity scores. The power of molecular networking is the information provided on tandem MS subpopulation clusters, which represent structurally matching but variant protein or non-protein biosynthetic release factors.In this work, the MS-based molecular networking platform was applied to a representative strain of the epidemic CA-MRSA USA300 clone. The study sought to provide insight into the biosynthetic release factor map of this pre-eminent human bacterial pathogen under normal growth and antibiotic challenged conditions. Biologically relevant pharmacological stimuli studied included sub-inhibitory concentrations of commonly prescribed commercial antibiotics as well as the innate human defense peptide LL-37 (23–25). In particular, our study goal was to monitor any variations in the type of dPSMs clustered within the molecular network map in response to these stimuli. We show an enriched extract derived from CA-MRSA alone or under these specific challenge conditions provides a highly complex biosynthetic release factor map. Analysis of subpopulation node clusters associated with PSMs prompted the discovery of several previously unreported α, β, and γ type dPSMs. The antibiotic stimuli modulated the type of dPSMs clustered, allowing for differential subclusters to be visualized that were absent in the untreated sample; including nodes that did not sequence to proteogenic PSMs. A group of the newly discovered PSM variants were assessed for functional properties and showed the induction of proinflammatory responses in THP-1 cells. In addition, in contrast to previous reports (17, 18), the tested dPSMs did not display antimicrobial activity. We conclude MS-based molecular networking can be used as a targeted discovery tool with the ability to identify previously unreported bioactive peptides.     

Purification of Monoclonal Antibodies Using High Performance Liquid Chromatography (HPLC)

Recent increases in the ability to detect low levels of immunofluorescence have shown the need for highly purified primary immunoreagents. There are now reports of purification of monoclonal antibodies using HPLC with reverse phase columns. In this study we have utilized standard size exclusion HPLC to purify both biotinylated and non-biotinylated monoclonal antibodies from hybridoma culture supernatants. Results indicated that both biotinylated and non-biotinylated monoclonal antibodies retained their antigen binding capacity after purification, and were not different in this capacity from commercially available, affinity purified reagents. These findings indicate that size exclusion HPLC may be used in the purification of biologically active monoclonal antibodies, and suggest that this technique may be used in the large scale production of antibodies and their fragments, in antibody purification from ascites fluid, and in antisera quality control.     

Investigations on Aberrant Glycosylation of Glycosphingolipids in Colorectal Cancer Tissues Using Liquid Chromatography and Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF-MS)

Cancer is a leading cause of death and alterations of glycosylation are characteristic features of malignant cells. Colorectal cancer is one of the most common cancers and its exact causes and biology are not yet well understood. Here, we compared glycosylation profiles of colorectal tumor tissues and corresponding control tissues of 13 colorectal cancer patients to contribute to the understanding of this cancer. Using MALDI-TOF(/TOF)-MS and 2-dimensional LC-MS/MS we characterized enzymatically released and 2-aminobenzoic acid labeled glycans from glycosphingolipids. Multivariate data analysis revealed significant differences between tumor and corresponding control tissues. Main discriminators were obtained, which represent the overall alteration in glycosylation of glycosphingolipids during colorectal cancer progression, and these were found to be characterized by (1) increased fucosylation, (2) decreased acetylation, (3) decreased sulfation, (4) reduced expression of globo-type glycans, as well as (5) disialyl gangliosides. The findings of our current research confirm former reports, and in addition expand the knowledge of glycosphingolipid glycosylation in colorectal cancer by revealing new glycans with discriminative power and characteristic, cancer-associated glycosylation alterations. The obtained discriminating glycans can contribute to progress the discovery of biomarkers to improve diagnostics and patient treatment.Worldwide, cancer is a leading cause of death. With estimated 1.2 million diagnoses in 2008, colorectal cancer is the third most common cancer in the world and the fourth most common cause of death with an annual mortality of ∼600 000 (1). The exact causes of colorectal cancer are unknown, but different risk factors such as age, polyps, personal and family history, ulcerative colitis, or Crohn''s colitis have been proposed (2). Standard screening procedures include flexible sigmoidoscopy, colonoscopy, and immunological fecal occult blood testing. Each of them has its advantages and drawbacks such as invasiveness or low sensitivity and specificity (3). The method of choice for the treatment of colorectal cancer is surgery and therapeutic decisions are based on the tumor, lymph node, and metastasis staging-system as a prognostic factor (4). Current research has led to improved treatment strategies of colorectal cancer, however, the clinical outcome, the progression of the disease, and the response to the treatment remain variable among individuals. The heterogeneity of colorectal cancer at the molecular level—caused by accumulation of multiple genetic changes—may be one of the main reasons for this variability (5). Genetic factors such as instabilities, but also expression levels (6) can explain part of the cancer biology, but glycomics is gaining importance to complement the overall picture as aberrant glycosylation of proteins and lipids has been shown to be correlated with disease and malignancy (7, 8).Glycosylation is involved in many biological processes and especially its functional role in cellular interaction with respect to adhesion, cell growth, and signaling is prone to be affected in cancer progression, invasion, and metastasis (9). Several cancer-associated alterations in protein glycosylation have been reported: (1) increased branching of N-glycans, (2) higher density of O-glycans, and (3) incomplete synthesis of glycans. More particularly, an increased or induced expression of GlcNAc transferase V resulting in N-glycan structures with β1–6GlcNAc antennae (5, 10), and the expression of (sialyl) Tn-antigens (11) as aberrant O-glycosylation have been reported (10).Altered glycosphingolipid (GSL)¹ glycosylation of the cell surface membrane during malignancy can affect cell recognition, adhesion, and signal transduction (12) and is found to reflect: (1) incomplete synthesis with or without precursor accumulation, (2) neosynthesis (9), (3) increased sialylation, and (4) increased fucosylation (13). In many cancers, including colorectal cancer, an overexpression of the (sialyl) Lewis X antigen (10, 14) and the expression of (sialyl) Lewis A (15) are considered to be related to malignant transformation—reflecting incomplete synthesis of sialyl 6-sulfo Lewis X and disialyl Lewis A (16) as well as neosynthesis (17). Studies on gangliosides showed an overexpression of these sialylated GSLs in human malignant melanoma (18). Furthermore, the involvement of gangliosides in cell adhesion and motility was reported, which contributes to tumor metastasis (19). Specifically, the gangliosides GD3 (Hex2NeuAc2ceramide) and GM2 (Hex2HexNAc1NeuAc1ceramide) have been found to be associated with tumor-angiogenesis (19). The up-regulation of fucosyltransferases in cancer was shown to cause a higher degree of fucosylation in malignant tissues (20) and Moriwaki et al. proposed that the increase in the fucosylation for GSLs was an early event in cancer (21). Misonou et al. investigated glycans derived from GSLs in colorectal cancer tissues showing aberrant glycan structures based on linkage differences as well as increased sialylation and fucosylation compared with control tissue (22), which is in line with observed changes in GSL glycosylation with regard to cancer progression (9, 13).Recently, we investigated the N-glycosylation profiles of colorectal tumors and correlating control tissues for biomarker discovery. Statistical analyses revealed an increase of sulfated glycan structures as well as paucimannosidic glycans and glycans containing sialylated Lewis type epitopes in the tumor tissue, whereas structures with bisecting GlcNAc were found to be decreased in malignancy (23). To further progress the understanding of colorectal cancer biology and the improvement of diagnostic tools and patient treatment, we complemented this recent study on N-glycosylation by an investigation of the glycosphingolipid-derived glycans (named GSL-glycans in the following) from frozen tumor tissues and corresponding control tissues from the same 13 colorectal cancer patients. GSL-glycans were enzymatically released, labeled with 2-aminobenzoic acid (AA) and analyzed by hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection as well as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Employing multivariate statistical analysis, this approach revealed an intricate GSL-glycosylation pattern of tumor tissues and specific glycosylation differences in comparison to the corresponding control tissue.     

High-Sensitivity Detection of 4-(3-Pyridinylmethylamino-carboxypropyl) Phenylthiohydantoins by Capillary Liquid Chromatography–Microelectrospray Ion Trap Mass Spectrometry

We describe the separation and detection at the low-femtomole level of 4-(3-pyridinylmethylaminocarboxypropyl) phenylthiohydantoins (311-PTHs) by capillary liquid chromatography–microelectrospray ion trap mass spectrometry. Highest sensitivity was obtained in the multiple-ion monitoring operating mode in which we detected 311-PTHs at the 5-fmol level with a signal-to-noise ratio of approximately 10. We investigated the fragmentation patterns of the isobaric 311-PTH isoleucine and 311-PTH leucine by electrospray ionization ion trap tandem mass spectrometry. The compounds could be differentiated by a fragment ion of mass m/z=366.1 which was specific for the breakdown of 311-PTH leucine, thus allowing for the unambiguous identification of the 311-PTH derivatives of all 20 naturally occurring amino acids by their masses and fragmentation patterns.     

Pharmacokinetic Study of 14-(3-Methylbenzyl)matrine and 14-(4-Methylbenzyl)matrine in Rat Plasma Using Liquid Chromatography-Tandem Mass Spectrometry

A rapid, sensitive and selective high-performance liquid chromatography-tandem mass spectrometric method (HPLC-MS) was developed and validated to determine the 14-(3-methylbenzyl)matrine (3MBM) and 14-(4-methylbenzyl)matrine (4MBM) levels in rat plasma in the present study. The analytes were separated using a C18 column (1.9 μm, 2.1 mm × 100 mm) equipped with a Security Guard C18 column (5 μm, 2.1 mm × 10 mm), followed by detection via triple-quadrupole mass spectrometry using an electrospray ionization (ESI) source. Sample pretreatment involved one-step protein precipitation with isopropanol:ethyl acetate (v/v, 25:75), and pseudoephedrine hydrochloride was used as an internal standard. The method was linear in the concentration range of 5–2000 ng/ml for both compounds. The intra-day and inter-day relative standard deviations (RSDs) were less than 15%, and all relative errors (REs) were within 15%. The proposed method enables the unambiguous identification and quantification of these two compounds in vivo. This study is the first to determine the 3MBM and 4MBM levels in rat plasma after oral administration of these compounds. These results provide a meaningful basis for evaluating the clinical applications of these medicines.     

Generation of Genomic Deletions (of Rig-I GENE) in Goat Primary Cell Culture Using CRISPR/CAS9 Method

CRISPR/Cas9 system is a natural immune system in prokaryotes protecting them from infectious viral or plasmid DNA invading the cells. This RNA-guided system can act as powerful tool for introducing genomic alterations in eukaryotic cells with high efficiency. In the present study, Rig-Igene is taken as model gene to study the efficiency of CRISPR/Cas9 system induced gene deletion in primary fibroblast cell culture. Rig-I(retinoic acid-inducible gene-1) is involved in regulating immune response in mammals. In this study, we optimized the CRISPR/Cas9 method for knocking out Rig-Igene in Goat primary fibroblasts by using a NHEJ pathway. Cells were screened for inactivation of the Rig-Igene and two positive clones were found out of thirty colonies screened. Thus, cells containing Rig-Igene inactivation could be achieved by CRISPR/Cas9 in goat fibroblast cells.     

The Ectomesenchymal-endodermal Interaction System (EEIS) of Triturus alpestris in Tissue Culture

Differentiation of visceral cartilage was studied in the Ectomesenchymal-endodermal Interaction System (EEIS), a hanging-drop culture system containing a piece of head neural fold tissue from behind the prospective ear region of a Triturus alpestris neurula together with a piece of ventrolateral pharynx endoderm from the same embryo. Cartilage cells differentiated only from those neural crest cells which have been in contact with the pharynx endoderm. Cartilage differentiation occurred neither in cultures containing neural fold or pharynx endoderm alone nor in distance cultures in which both explants were separated from each other at a greater distance than usual. However, in some critical cultures of the EEIS containing pharynx endoderm, excised more ventrally from the regular site, accumulations of ectomesenchymal cells were observed near the endoderm which were not transformed into cartilage.
The differentiation of cartilage was classified into three stages: (1) prechondroblasts (day 1 to 5), (2) chondroblasts (about day 6) and (3) chondrocytes (about day 10).
The data obtained suggested that cellular contact between the two inductively correlated tissues was a prerequisite for the transmission mechanism while mitotic cell divisions were mainly responsible for the spread of the inductive message within the responding ectomesenchymal cells.     

繁茂膜海绵原细胞富集细胞团培养过程中的细胞迁移规律

海绵是重要的生物活性物质来源, 近10年来, 从海绵中发现的具有生物活性的新化合物占海洋生物来源的30%以上, 并且大多具有显著的抗肿瘤, 抗艾滋病病毒的活性。但是, 由于海绵生物量不能满足这些活性物质进一步研究和商业化的需求, 目前仅有一种活性物质被成功的商业化, 这不仅是商业开发的损失, 也是提高人类生活质量活动的一种损失。为了解决海绵供给不足的问题, 人们进行了包括化学合成、海绵养殖以及海绵细胞培养在内的多种尝试,目前的研究结果表明, 海绵细胞离体培养技术是最有可能彻底解决海绵供给不足的途径之一。但是由于海绵自身的特殊性, 还没有人成功的建立起海绵细胞系以满足生产需要。人们发现, 海绵细胞的相互接触对于离体海绵细胞长期培养至关重要。经过多年的探索, 大连化物所海洋生物产品工程组建立了开发出了海绵原细胞富集细胞团培养技术, 通过对海绵组织内的原细胞进行富集来获得可长期培养的海绵细胞。海绵原细胞是海绵组织内的“干细胞”, 具有很强的分化、增殖潜力, 同时也是海绵组织内负责消化的主要细胞类型。为了探索海绵原细胞的增殖、分化规律, 本研究基于海绵原细胞富集细胞团培养体系, 构建了海绵细胞培养实时观测平台, 对繁茂膜海绵原细胞、领细胞、上皮细胞3类主要海绵细胞类型在海绵细胞团形成及生长的全过程进行观察, 了解不同类型细胞迁移规律的变化。通过对视频记录进行分析,发现离散的海绵细胞与细胞团内的海绵细胞具有截然相反的运动规律, 海绵细胞的运动具有很强的协同性。伴随原细胞在细胞团内不停息的迁移, 还观察到海绵细胞团内新生骨针的迁移以及细胞间进行颗粒物质的传递。这些信息的获得, 将有助于进一步了解不同细胞的功能与作用, 也有助于在此基础上探索海绵细胞的增殖、分化控制规律。