Study of fatty acid profiles in cancer cells grown in culture using gas chromatography--mass spectrometry

The determination of long-chain fatty acids in the phospholipid, triglyceride and free fatty acid fractions of HT29/219 colon cancer cells grown in a medium containing either foetal calf serum or horse serum, was carried out using gas chromatography - mass spectrometry. Several bonded-phase capillary columns were tested for the separation of the fatty acid methyl esters, and a 30-m poly(ethylene glycol) column was found to give optimum separation. The mass spectrometer was set to the multiple ion detection mode to increase the sensitivity of the recording of the characteristic ions, consisting of the molecular ion and the base peak. The phospholipid and triglyceride compositions of the cells were different when the cells were grown in media containing different sera. Differences were also found in the turnover of the acids in the different lipid fractions, the phospholipids being the most important, when the cells were grown in different media. The cellular metabolism and turnover of certain fatty acids differed from others in the same cell. These differences emphasise the importance of a precise knowledge of the lipid composition of the culture medium in in vitro studies of cancer cells.     

Toward high sequence coverage of proteins in human breast cancer cells using on-line monolith-based HPLC-ESI-TOF MS compared to CE MS

A method is developed toward high sequence coverage of proteins isolated from human breast cancer MCF10 cell lines using a 2-D liquid separations. Monolithic-capillary columns prepared by copolymerizing styrene with divinylbenzene are used to achieve high-resolution separation of peptides from protein digests. This separation is performed with minimal sample preparation directly from the 2-D liquid fractionation of the cell lysate. The monolithic column separation is directly interfaced to ESI-TOF MS to obtain a peptide map. The protein digests were also analyzed by MALDI-TOF MS and an accurate M(r) of the intact protein was obtained using an HPLC-ESI-TOF MS. The result is that these techniques provide complementary information where nearly complete sequence coverage of the protein is obtained and can be compared to the experimental M(r) value. The high sequence coverage provides information on isoforms and other post-translational modifications that would not be available from methods that result in low sequence coverage. The results from the use of monolithic columns are compared to that obtained by CE-MS. The monolithic column separations provide a rugged and highly reproducible method for separating protein digests prior to MS analysis and is suited to confidently identify biomarkers associated with cancer progression.     

Monitoring of betulin nanoemulsion treatment and molecular changes in mouse skin cancer using surface enhanced Raman spectroscopy

The aim of this study was to obtain in vivo and ex vivo reproducible surface enhanced Raman signal from mouse skin and to use it for the differentiation of skin pathologies. We induced skin carcinoma in mice models using a chemical treatment and tested the chemopreventive activity of a new formulation based on natural compound betulin extracted from the bark of birch trees. Using surface enhanced Raman spectroscopy (SERS) we identified in vivo and ex vivo the spectral signatures characteristic to the healthy skin, melanoma skin induced in mouse models, and to the pathology evolution when the betulin nanoemulsion formulation was topically applied on the cancerous skin of mice. SERS markers associated to each pathology were identified and the signal was distinguished from the classical Raman signal of skin based on several biomarkers, such as the disappearance of the amide I band of proteins, the amplification of the 1574 cm⁻¹ band assigned to nucleic acid bases, and the appearance of the highly amplified band at 230 cm⁻¹ characteristic to the metal-biomolecules complex. The various skin pathologies were differentiated using principal components analysis and K-means clustering. The effectiveness of the betulin nanoemulsion treatment was validated by the histological examination and the chemometrics methods, which successfully confirmed the direct SERS differentiation between the cancerous and the betulin nanoemulsion treated skin.     

Global analysis of dynamic changes in lipid raft proteins during T-cell activation

Lipid rafts are considered as specialized microdomains within the plasma membrane with unique lipid compositions different from surrounding membranes. Following T-cell receptor (TCR) stimulation, lipid rafts assemble in T-cell/antigen-presenting cell (APC) contact site known as the immunological synapse, inner leaflets of which serve as activation or docking sites for downstream signaling components. To understand the signaling events occurring in lipid rafts, we globally analyzed dynamic changes in lipid raft proteins during TCR/CD28 costimulation using 2-D fluorescence difference gel electrophoresis. We detected multiple spots whose intensities were enhanced after costimulation, and identified proteins in these spots by PMF. Identified proteins include Src family tyrosine kinases, tyrosine phosphatase, phosphatidylinositol 3-kinase (PI3-kinase), actin-binding proteins, and regulators for small GTPases. Of particular interest, a number of pleckstrin homology (PH) domain-containing proteins were identified. Biochemical and histochemical analyses confirmed the translocation of these proteins from cytosol to lipid rafts. We also demonstrated that these proteins assembled at the T-cell/APC interface. These results indicate the efficacy of our system to systematically analyze dynamics of lipid raft proteins during extracellular stimulation.     

Quantitation of ribavirin in human plasma and red blood cells using LC–MS/MS

LC–MS/MS has been applied for the rapid determination of the nucleoside analogue ribavirin in human plasma and red blood cells. The incorporation of ribavirin to the erythrocytes has been assayed after in vitro incubation of the cells at different concentrations of the antiviral drug. After protein precipitation, samples were injected into a C₈ column, achieving a complete separation of ribavirin from the endogenous isobaric compound uridine. Calibration ranges varied from 10 to 10 000 ng/mL in plasma and from 0.2 to 200 ng/cell pellet in red blood cells. Precision and accuracy values were always below 10 and 13%, respectively, in all assayed matrices. Ribavirin was demonstrated to remain unchanged after short and long time storage. No matrix effects could be assessed for the analyzed matrices. The developed method has been fully validated. Monitoring of ribavirin concentration in red blood cells in addition to the classic plasma monitoring of the drug could help to explain its efficacy and safety profiles in patients.     

Direct Visualization of Neurotransmitters in Rat Brain Slices by Desorption Electrospray Ionization Mass Spectrometry Imaging (DESI - MS)

Mass spectrometry imaging (MSI) of neurotransmitters has so far been mainly performed by matrix-assisted laser desorption/ionization (MALDI) where derivatization reagents, deuterated matrix and/or high resolution, or tandem MS have been applied to circumvent problems with interfering ion peaks from matrix and from isobaric species. We herein describe the application of desorption electrospray ionization mass spectrometry imaging (DESI)-MSI in rat brain coronal and sagittal slices for direct spatial monitoring of neurotransmitters and choline with no need of derivatization reagents and/or deuterated materials. The amino acids γ-aminobutyric (GABA), glutamate, aspartate, serine, as well as acetylcholine, dopamine, and choline were successfully imaged using a commercial DESI source coupled to a hybrid quadrupole-Orbitrap mass spectrometer. The spatial distribution of the analyzed compounds in different brain regions was determined. We conclude that the ambient matrix-free DESI-MSI is suitable for neurotransmitter imaging and could be applied in studies that involve evaluation of imbalances in neurotransmitters levels.

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Monitoring glycosylation pattern changes of glycoproteins using multi-lectin affinity chromatography

Previously, we reported that the distribution of glycoproteins into the lectin displacement fractions of a multi-lectin affinity column was determined by the glycosylation patterns of the proteins. This distribution was observed by the sequential use of displacers specific to the lectins in the column. In this study we have evaluated the multi-lectin column (containing Concanavalin A, Wheat germ agglutinin and Jacalin lectin) to screen glycoproteins with known glycosylation pattern changes. The presence of a glycoprotein in a given displacer fraction was determined by LC-MS/MS analysis of a tryptic digest. We have used the enzyme neuraminidase to modify the oligosaccharide chains present in human transferrin, and used the enzymes, neuraminidase and fucosidase, to modify glycoproteins present in human serum. Then, by comparison with the untreated samples, we demonstrated a distribution shift of the enzyme-treated serum glycoproteins in the displacement fractions isolated from the multi-lectin column. The fractions were analyzed by a protein assay, Sequest rank comparison and peak area measurement from the extracted ion chromatogram. The results indicated that the multi-lectin affinity column (M-LAC) is sensitive to changes in the content of sialic acid and fucosyl residues present in serum glycoproteins, and has the potential to be used to screen serum proteins for glycosylation changes due to disease. In addition, the use of a glycosidase to induce specific structural changes in glycoproteins can support the development of multi-lectin column formats specific for detecting changes in the glycoproteome of certain diagnostic fluids and types of disease.     

Optimization of photodynamic therapy response by survivin gene knockdown in human metastatic breast cancer T47D cells

Photodynamic therapy (PDT) leads to the generation of cytotoxic oxygen species that appears to stimulate several different signaling pathways, some of which lead to cell death, whereas others mediate cell survival. In this context, we observed that PDT mediated by methyl-5-aminolevulinic acid as the photosensitizer resulted in over-expression of survivin, a member of the inhibitor of apoptosis (IAP) family that correlates inversely with patient prognosis. The role of survivin in resistance to anti-cancer therapies has become an area of intensive investigation. In this study, we demonstrate a specific role for survivin in modulating PDT-mediated apoptotic response. In our experimental system, we use a DNA vector-based siRNA, which targets exon-1 of the human survivin mRNA (pSil_1) to silence survivin expression. Metastatic T47D cells treated with both pSil_1 and PDT exhibited increased apoptotic indexes and cytotoxicity when compared to single-agent treated cells. The treatment resulted in increased PARP and caspase-3 cleavage, a decrease in the Bcl-2/Bak ratio and no participation of heat shock proteins. In contrast, the overexpression of survivin by a survivin-expressed vector increased cell viability and reduced cell death in breast cancer cells treated with PDT. Therefore, our data suggest that combining PDT with a survivin inhibitor may attribute to a more favorable clinical outcome than the use of single-modality PDT.     

基于鸟枪法脂质组学研究环氧酶-2抑制剂对关节炎模型血清脂代谢的干预作用

心血管疾病(CVD)是类风湿关节炎(RA)患者死亡的重要原因之一,脂代谢紊乱与CVD发生有密切关联,因而有必要对RA和药物治疗导致的脂代谢改变进行探讨。该研究采用胶原诱导法(CIA)构建关节炎模型,引入多维质谱鸟枪法开展血清脂质分析,检测了血清中105种脂质分子,发现模型大鼠体内7种磷脂酰肌醇、15种鞘磷脂、5种神经酰胺、10种磷脂酰胆碱和2种溶血磷脂酰胆碱异常上调,环氧酶-2(COX-2)抑制剂可部分修复紊乱的脂代谢,但对5种磷脂酰胆碱和1种溶血磷脂酰胆碱具有异常调控作用。该研究从脂质分子水平探讨了RA及COX-2抑制剂对脂代谢的干预作用,可以为RA的心血管风险研究提供信息。     

Nuclear protein profiling of Jurkat cells during heat stress-induced apoptosis by 2-DE and MS/MS

Heat stress causes severe constraints on numerous physiological functions of cells, such as the repression of splicing of mRNA precursors. In this study, we performed proteomic profiling of a nuclear fraction of Jurkat cells during heat stress using 2-DE and LC-MS/MS. We found 10 protein spots whose expression had changed after heat stress at 43 degrees C for 30 min. Seven of those protein spots, periodic tryptophan protein 1 homolog (PWP1), importin beta-1 subunit, sumoylated protein, splicing factor 3a subunit 3 (SF3a3), TAR DNA-binding protein 43, U2 small nuclear ribonucleoprotein auxiliary factor 35 kDa subunit (U2AF35) and small ubiquitin-related modifier-1 (SUMO-1) were downregulated, while three other protein spots, Protein SET, 40S ribosomal protein SA and 60S acidic ribosomal protein P0 were upregulated by the heat stress. We focused on the downregulation of two splicing factors, which might participate in the repression of pre-mRNA processing by heat stress, leading to cell apoptosis.     

Determination of reduced folates in tumor and adjacent mucosa of colorectal cancer patients using LC‐MS/MS

A liquid chromatography electrospray ionization tandem mass spectrometry (LC‐MS/MS) method has been developed for the determination of 5,10‐methylenetetrahydrofolate (methyleneTHF), tetrahydrofolate (THF) and 5‐methyltetrahydrofolate (methylTHF) in colorectal mucosa and tumor tissues. The folate extraction method includes homogenization, heat and folate conjugase treatment to hydrolyze polyglutamyl folate to monoglutamyl folate. Before analysis on LC‐MS/MS, simple and fast sample purification with ultrafiltration (molecular weight cut‐off membrane, 10 kDa) was performed. Folates were detected and quantified using positive electrospray. The method described in the present paper was successfully applied to determine the level of three folate monoglutamates in mucosa and tumor samples from 77 colorectal cancer patients, starting from a limited amount of tissue. The results showed that the LC‐MS/MS method has a great advantage over other previously used methods because of its high sensitivity and selectivity. Significantly higher levels of methyleneTHF and THF were found in tumor compared with matched mucosa tissues. Folate levels in adjacent mucosa were associated with tumor location, age and gender. The correlation between folate levels and tumor site further strengthens the fact that development of right‐ and left‐sided tumors follows different pathways. Copyright © 2012 John Wiley & Sons, Ltd.     

Monitoring of protein profiles for the optimization of recombinant fermentation processes using public domain databases

The expression of human superoxide dismutase in fed-batch fermentation of E. coli HMS174(DE3)(pET3ahSOD) was studied as model system. Due to the frequently used strong T7 promoter system a high metabolic load is exerted, which triggers stress response mechanisms and finally leads to the differentiation of the host cell. As a consequence, host cell metabolism is partly shifted from growth to survival accompanied by significant alterations of the protein pattern. In terms of process optimization two-dimensional electrophoresis deserves as a powerful tool to monitor these changes on protein level. For the analysis of samples derived from different states of recombinant protein production wide-range Immobiline Dry Strips pH 3-10 were used. In order to establish an efficient procedure for accelerated process optimization and to avoid costly and time-consuming analysis like mass spectrometry (MS), a database approach for the identification of significant changes of the protein pattern was evaluated. On average, 935 spots per gel were detected, whereby 50 are presumably stress-relevant. Out of these, 24 proteins could be identified by using the SWISS-2DPAGE database (www.expasy.ch/ch2d/). The identified proteins are involved in regulatory networks, energy metabolism, purine and pyrimidine nucleotide synthesis and translation. By this database approach, significant fluctuations of individual proteins in relation to recombinant protein production could be identified. Seven proteins show strong alterations (>100%) directly after induction and can therefore be stated as reliable marker proteins for the assessment of stress response. For distinctive interpretation of this highly specific information, a bioinformatic and statistic tool would be essential in order to perceive the role and contribution of individual proteins in stress response.     

Studies on the influence of esterase inhibitor to the pharmacokinetic profiles of oseltamivir and oseltamivir carboxylate in rats using an improved LC/MS/MS method

Oseltamivir (O), an ethyl ester prodrug of oseltamivir carboxylate (OC), is currently the drug of choice for avian influenza. Previous studies have found that the addition of esterase inhibitor can inhibit the metabolism of O to OC in plasma samples. The current study aims to evaluate the impact of dichlorvos on the rat plasma concentrations of O and OC and subsequent effect on their pharmacokinetics. The plasma samples of rats after oral administration of O were divided into two equal portions for treatment with/without dichlorvos. O and OC plasma concentrations were analyzed by a sensitive and specific LC/MS/MS method, using cephalexin as internal standard for both two analytes. The samples were extracted with an MCX cartridge and separated on a Nova‐Pak CN HP column eluted with a mobile phase of 0.15% formic acid in 50% methanol. The results showed that dichlorvos significantly inhibited further hydrolysis of O to OC during the period of rat plasma sample treatment. A significant difference in the pharmacokinetic parameters of O (except for T_max and t_1/2,λz) was found when the plasma samples were treated with dichlorvos. The use of dichlorvos is recommended in all rat studies which require plasma concentration determination of O and OC. Copyright © 2009 John Wiley & Sons, Ltd.     

Correlation of product ion profiles with molecular structures of androgenic and anabolic steroids in ESI MS/MS

Androgenic and anabolic steroids (AASs) are a class of chemical substances closely related to testosterone in molecular structure. They can be abused to enhance performances in human and equine athletes, and are banned by the sports authorities. To assist with method development for doping analyses of AASs, investigations were conducted to correlate their product ion profiles with the molecular structures. Although very similar in chemical structure, AASs generated noticeably different product ion profiles from collision‐induced dissociation (CID). On the basis of both outlines of the product ion profiles and molecular structures, AASs studied were classified into six subclasses. In each subclass, the product ion profiles were identical or similar. However, the product ion profiles in one subclass were remarkably different from those in another. The classification reveals that the position and number of double bond(s) in conjugation with the 3‐carbonyl group in the molecular structure of an AAS have significant effects on product ion profile. The presence or absence of the 19‐methyl group in an AAS also has a remarkable influence on its product ion profile. A substitution in the A‐, B‐ or D‐ring of an AAS may cause a shift in mass value of the product ions. The correlation of product ion profiles with molecular structures of AASs has the implication that each AAS can be characterized by a combination of its [M + H]⁺ ion and product ion profile and as a result be identified with specificity. The classified product ion pattern may be useful in the identification of unknown AASs. Copyright © 2010 John Wiley & Sons, Ltd.     

Hypoxia induces Wee1 expression and attenuates hydrogen peroxide-induced endothelial damage in MS1 cells

In an oxygen-depleted environment, endothelial cells initiate an adaptive pattern of synthesis, which may enable them to survive hypoxic crises. Using high-resolution two-dimensional gel electrophoresis in conjunction with mass spectroscopy, we obtained a 24 differential display of proteins in the pancreatic endothelial cell line, MS-1, at four time points following induction of hypoxia. The induction of Wee1 under hypoxia was confirmed both at the mRNA and protein levels. The phosphorylation of cell division cycle 2, which is downstream of Wee1, was also increased after hypoxic exposure. In addition, pre-exposure to hypoxia attenuated a decrease in hydrogen peroxide-induced cell number. The induction of bax (a pro-apoptotic protein) and reduction of bcl (an anti-apoptotic protein) after hypoxia stimulus were also attenuated by hypoxic pre-exposure. Moreover, hydrogen peroxide-induced morphologic damage did not appear in the wild-type Wee1-expressing cells. Taken together, our results suggest that Wee1 may have important role in hypoxia- induced pathophysiological situations in endothelial cells.     

Dissection of molecular and histological subtypes of papillary thyroid cancer using alternative splicing profiles

Despite growing evidence of the relevance of alternative splicing (AS) to cancer development and progression, the biological implications of AS for tumor behaviors, including papillary thyroid cancer (PTC), remain elusive. With the aim of further understanding the molecular and histological subtypes of PTC, we in this study explored whether AS events might act as new molecular determinants. For this purpose, AS profiles were analyzed in RNA-sequencing data from The Cancer Genome Atlas (TCGA) and from a Korean patient dataset. A total of 23 distinct exon-skipping (ES) events that correlated significantly with PTC oncogenic activity and differentiation scores were identified. The two top-ranked ES events, NUMA1_17515 in exon 18 of NUMA1 and TUBB3_38175 in exon 6 of TUBB3, showed high correlations with oncogenic activities and discriminated histological and molecular subtypes of PTC. Furthermore, two novel intron-retention (IR) events for TUBB3 were uncovered. All ES and IR events for the TUBB3 gene were predicted to induce nonsense-mediated mRNA decay. The relative abundances of intron reads in the PTC dataset from TCGA showed IR levels to differ significantly among PTC subtypes, possibly reflecting their different tumor behaviors. This study provides a landscape of AS changes among PTC subtypes and identified two significant AS events, NUMA1_17515 and TUBB3_38175, as potential AS biomarkers for PTC subclassification and characterization. The AS events identified in this study may be involved in the development of phenotypic differences underlying the functional characteristics and histological differentiation of PTCs.Subject terms: Cancer genomics, RNA splicing     

Targeting of cancer cells using click-functionalized polymer capsules

Targeted delivery of drugs to specific cells allows a high therapeutic dose to be delivered to the target site with minimal harmful side effects. Combining targeting molecules with nanoengineered drug carriers, such as polymer capsules, micelles and polymersomes, has significant potential to improve the therapeutic delivery and index of a range of drugs. We present a general approach for functionalization of low-fouling, nanoengineered polymer capsules with antibodies using click chemistry. We demonstrate that antibody (Ab)-functionalized capsules specifically bind to colorectal cancer cells even when the target cells constitute less than 0.1% of the total cell population. This precise targeting offers promise for drug delivery applications.