Project description:Nano-flow chromatography-tandem mass spectrometer (nLC-MS/MS) is a popular choice in proteome research as it provided high-sensitivity analysis with minimal sample usage, but its quantitative proteomics applications become restricted by low analytical throughput, and low robustness. To circumvent this limitation, we describe the performance of the new Vacuum Insulated Probe Heated ElectroSpray Ionization source (VIP-HESI) coupled to the Bruker timsTOF mass spectrometer and show it enhances micro-spray flow rate chromatography signals in comparison to nanospray source conditions using the CaptiveSpray (CS) and ElectroSpray Ionization (ESI) sources. In addition, using a 0.5 x 200mm and 1 × 150 mm columns, achieved excellent reproducibility of chromatographic retention time, coefficient of variation, and precision quantification using data from un-depleted mouse plasma, HeLa and K562 digest peptide samples.
Project description:A novel one-dimensional on-line pH gradient-eluted strong cation exchange (SCX)-nano-ESI-MS/MS method was developed for protein identification and tested with mixture of six standard proteins, total lysate of HuH7 and N2a cells, as well as membrane fraction of N2a cells. This method utilized an on-line nano-flow SCX column in a nano-LC system coupled with a nano-electrospray high-resolution mass spectrometer. Protein digests were separated on a nano-flow SCX column with a pH gradient and directly introduced into a mass spectrometer through nano-electrospray ionization. SCXLC-MS/MS showed identification capability for higher proportion of basic peptides compared to RPLC-MS/MS method, especially for histidine-containing peptides. Our SCXLC-MS/MS method is an excellent alternative method to the RPLC-MS/MS method for analysis of standard proteins, total cell and membrane proteomes.
Project description:Eriocitrin, found in lemon fruit, has shown a wide range of biological properties. Herein, to evaluate the intestinal metabolic profile of eriocitrin in colon, the flavonoids in mice colon contents were identified by ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS), and a total of 136 flavonoids were found, including eriocitrin and its six metabolites (eriodictyol, homoeriodictyol, hesperetin, eriodictyol-3'-O-glucoside, hesperetin-7-O-glucoside and eriodictyol-7-O-(6''-O-galloyl) glucoside). Mice colon contents were used for 16S rDNA gene sequencing and gas chromatography-mass (GC-MS). Resultu showed that eriocitrin significantly alters the beta diversity of the gut microbiota, the probiotics such as Lachnospiraceae_UCG_006 were significantly enriched, and the production of butyrate, valerate and hexanoate in the colon pool of short-chain fatty acids (SCFAs) were significant increased. The spearman's association analysis performed some intestinal bacteria may be involved in the metabolism of eriocitrin. Collectively, our results preliminarily suggesting the metabolism of eriocitrin in the gut, demonstrate alterations of eriocitrin on gut microbiota, which warrants further investigation to determine its potential use in food and biomedical applications.
Project description:Retinoblastoma (RB) is an intraocular childhood tumor which, if left untreated, leads to blindness and mortality. Nucleolin (NCL) protein which is differentially expressed on the tumor cell surface, binds ligands and regulates carcinogenesis and angiogenesis. We found that NCL is over expressed in RB tumor tissues and cell lines compared to normal retina. We studied the effect of nucleolin-aptamer (NCL-APT) to reduce proliferation in RB tumor cells. Aptamer treatment on the RB cell lines (Y79 and WERI-Rb1) led to significant inhibition of cell proliferation. Locked nucleic acid (LNA) modified NCL-APT administered subcutaneously (s.c.) near tumor or intraperitoneally (i.p.) in Y79 xenografted nude mice resulted in 26 and 65% of tumor growth inhibition, respectively. Downregulation of inhibitor of apoptosis proteins, tumor miRNA-18a, altered serum cytokines, and serum miRNA-18a levels were observed upon NCL-APT treatment. Desorption electrospray ionization mass spectrometry (DESI MS)-based imaging of cell lines and tumor tissues revealed changes in phosphatidylcholines levels upon treatment. Thus, our study provides proof of concept illustrating NCL-APT-based targeted therapeutic strategy and use of DESI MS-based lipid imaging in monitoring therapeutic responses in RB.
Project description:Oral submucous fibrosis (OSF) is one of the most common precancerous malignant orders (PMO) with high rates exacerbating into oral squamous cell carcinoma (OSCC), mostly occurred in patients with chewing betel-nut habits in Southeast Asia and Pacific region. However, the spatial characteristics and heteogeneities of tumor microenvironment (TME) in OSF-associated OSCC still remains unclear. Here, we characterized the spatiotemporal changes of OSF-associated OSCC at different malignant states by performing 10x Visium Spatial Transcriptomics (ST) sequencing and airflow-assisted desorption electrospray ionization-mass spectrometry imaging (AFADESI-MSI) analysis.
Project description:In this work, a microwell-chip was prepared and modified. The microwell-chip was used for extraction of metabolites and subsequent protein digestion. Next, direct electrospray ionization mass spectrometry (ESI-MS) was adopted for metabolome identification and a data independent acquisition (DIA)-MS approach was established for simultaneous proteome profiling and phosphoproteome analysis. In particular, application of this strategy provides a multi-omics view of cellular changes.
Project description:The clinical outcomes of M2 subtype Acute Myeloid Leukemia (AML-M2) with t(8;21) are poor. Here we report that FTY720 (Fingolimod), a sphingosine analogue and an FDA approved drug for treatment of multiple sclerosis, showed great antitumorigenic activity against Kasumi-1 cell line, xenograft mouse model and leukemic blasts isolated from AML-M2 with t(8;21) patients. Primary investigation indicated that FTY720 caused cell apoptosis through caspase and protein phosphatase 2A (PP2A) activation. Transcriptomic profiling further revealed that FTY720 treatment could upregulate AML1 target genes and interfere with genes involved in ceramide synthesis. FTY720 treatment led to the elimination of AML1-ETO oncoprotein and caused cell cycle arrest. More importantly, FTY720 treatment resulted in rapid and significant increment of pro-apoptotic ceramide levels, determined by HPLC-ESI-MS/MS (high-performance liquid chromatography-electrospray ionization tandem mass spectrometry) based lipidomic approaches. Additionally, structural simulation model indicated that the directly binding of ceramide to inhibitor 2 of PP2A (I2PP2A) could reactivate PP2A and cause cell death. This study demonstrates, for the first time, that accumulation of ceramide plays a central role in FTY720 induced cell death of AML-M2 with t(8;21). Targeting sphingolipid metabolism by using FTY720 may provide novel insight into drug development for AML-M2 treatment.
Project description:The discovery of dynamic and reversible modifications in messenger RNA (mRNA) is opening new directions in RNA modification-mediated regulation of biological processes. Methylation is the most prevalent modification occurring in mRNA and the methylation group is mainly decorated in the adenine, cytosine, and guanine base, or in the 2’-hydroxyl group of ribose. However, methylation of the uracil base (5-methyluridine, m5U) hasn’t been discovered in mRNA of eukaryotes. In the current study, we established a method by N-cyclohexyl-N’-β-(4-methylmorpholinium) ethylcarbodiimide p-toluenesulfonate (CMCT) labelling coupled with liquid chromatography-electrospray ionization-mass spectrometry analysis (LC-ESI-MS/MS) for the sensitive determination of uridine modifications in RNA. Our results demonstrated that the detection sensitivities of uridine modifications in RNA increased up to 1543 folds upon CMCT labelling. Using the developed method, we identified the distinct existence of m5U in mRNA of various mammalian cells and tissues. In addition, the stable isotope tracing monitored by mass spectrometry revealed that the methylation group of m5U originated from S-Adenosyl-L-methionine (SAM). Our study expanded the list of modifications occurring in mRNA of mammals. Future work on transcriptome-wide mapping of m5U will further uncover the functional roles of m5U in mRNA of mammals.
Project description:The clinical outcomes of M2 subtype Acute Myeloid Leukemia (AML-M2) with t(8;21) are poor. Here we report that FTY720 (Fingolimod), a sphingosine analogue and an FDA approved drug for treatment of multiple sclerosis, showed great antitumorigenic activity against Kasumi-1 cell line, xenograft mouse model and leukemic blasts isolated from AML-M2 with t(8;21) patients. Primary investigation indicated that FTY720 caused cell apoptosis through caspase and protein phosphatase 2A (PP2A) activation. Transcriptomic profiling further revealed that FTY720 treatment could upregulate AML1 target genes and interfere with genes involved in ceramide synthesis. FTY720 treatment led to the elimination of AML1-ETO oncoprotein and caused cell cycle arrest. More importantly, FTY720 treatment resulted in rapid and significant increment of pro-apoptotic ceramide levels, determined by HPLC-ESI-MS/MS (high-performance liquid chromatography-electrospray ionization tandem mass spectrometry) based lipidomic approaches. Additionally, structural simulation model indicated that the directly binding of ceramide to inhibitor 2 of PP2A (I2PP2A) could reactivate PP2A and cause cell death. This study demonstrates, for the first time, that accumulation of ceramide plays a central role in FTY720 induced cell death of AML-M2 with t(8;21). Targeting sphingolipid metabolism by using FTY720 may provide novel insight into drug development for AML-M2 treatment. A six chip study using total RNA recovered from three separate cultures of DMSO-treated Kasumi-1 cells and three separate cultures of FTY720-treated Kasumi-1 cells.Each chip measures the expression level of genes from DMSO-/FTY720-treated Kasumi-1 cells.
Project description:The 17.62 kDa vitellogenin B derived yolk protein was purified by a specific cation exchange chromatography, subjected to a liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and finally identified using short MS/MS protein signatures similar to vitellogenin of a near species from the same family, red seabream, Pagrus major, (Temminck and Schlegel, 1843).