Dataset Information

Cell Data-Indenpendent acquisition LC-MS/MS

ABSTRACT: 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.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Cervix Carcinoma

SUBMITTER: Yuanyuan Li

LAB HEAD: Weijie Qin

PROVIDER: PXD025634 | Pride | 2021-11-08

REPOSITORIES: Pride

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Publications

An Integrated Strategy for Mass Spectrometry-Based Multiomics Analysis of Single Cells.

Li Yuanyuan Y Li Hang H Xie Yuping Y Chen Shuo S Qin Ritian R Dong Hangyan H Yu Yongliang Y Wang Jianhua J Qian Xiaohong X Qin Weijie W

Analytical chemistry 20211013 42

Single-cell-based genomics and transcriptomics analysis have revealed substantial cellular heterogeneity among seemingly identical cells. Knowledge of the cellular heterogeneity at multiomics levels is vital for a better understanding of tumor metastasis and drug resistance, stem cell differentiation, and embryonic development. However, unlike genomics and transcriptomics studies, single-cell characterization of metabolites, proteins, and post-translational modifications at the omics level remai ...[more]

PMID: 34643370

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Project description:During the colonization of hosts, bacterial pathogens are presented with many challenges that must be overcome for colonization to successfully occur. This requires bacterial sensing of the surroundings and adaptation to the conditions encountered. One of the major impediments to pathogen colonization of the mammalian gastrointestinal tract is the antibacterial action of bile. Salmonella enterica serovar Typhimurium has specific mechanisms involved in resistance to bile. Besides being resistant to it, Salmonella can also successfully multiply in bile, using it as a source of nutrients. This accomplishment is highly relevant to pathogenesis, as Salmonella colonizes the gallbladder of hosts, where it can be carried asymptomatically and promote further host spread and transmission. In order to gain insights into the mechanisms used by Salmonella to grow in bile, we studied the changes elicited by Salmonella in the chemical composition of bile during growth in vitro and in vivo through a metabolomics approach. Our data suggest that phospholipids are an important source of carbon and energy for Salmonella during growth in the laboratory as well as during gallbladder infections of mice. Further studies in this area will generate a better understanding of how Salmonella exploits this generally hostile environment for its own benefit. For in vitro studies, bile was extracted from C57BL/6 mice and used immediately. An overnight culture of Salmonella enterica serovar Typhimurium SL1344 was used to inoculate 10 ?L of bile at an approximate density of 5x10^6 cells/mL. The experiment was performed in duplicate, and a total of 2 uninfected and 2 infected bile samples were studied. Samples were incubated for 24 hours at 37 oC with shaking. After incubation, samples were centrifuged to remove bacteria and the supernatant was saved for metabolomic analyses. For in vivo studies, C57BL/6 mice were infected with approximately 10^8 bacterial cells by oral gavage. Four groups of three to four mice each were either infected with Salmonella or kept uninfected (total of 11 mice per treatment group). Five days after infection, all mice were sacrificed and bile was collected. Samples were centrifuged and supernatants saved for metabolomic analyses. Samples were prepared by mixing equal volumes of bile from three to four mice, generating three samples per treatment (uninfected and infected), which were used in the subsequent steps. Seven microliters of each sample was evaporated and the residue resuspended in 50% acetonitrine. Extracts were infused into a 12-T Apex-Qe hybrid quadrupole-FT-ICR mass spectrometer equipped with an Apollo II electrospray ionization source, a quadrupole mass filter and a hexapole collision cell. Raw mass spectrometry data were processed as described elsewhere (Han et al. 2008. Metabolomics. 4:128-140). To identify differences in metabolite composition between different groups of samples, we filtered the list of masses for metabolites which were present on one set of samples but not the other. Additionally, we calculated the ratios between averaged intensities of metabolites from each group of mice. To assign possible metabolite identities to the masses selected as described above, the monoisotopic neutral masses of interest were queried against the Human Metabolome Database (HMDB, http://www.hmdb.ca), with a tolerance of 0.001 Da.

Dataset Information

Cell Data-Indenpendent acquisition LC-MS/MS

Publications

An Integrated Strategy for Mass Spectrometry-Based Multiomics Analysis of Single Cells.

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