Center for Innovative Technology /cit Integrated ‘omics’ capabilities utilizing state-of-the-art multi-dimensional mass spectrometry and automated high content microscopy Mon, 08 Jun 2020 13:45:01 +0000 en-US hourly 1 https://wordpress.org/?v=5.8 https://cdn.vanderbilt.edu/vu-wp0/wp-content/uploads/sites/166/2019/02/27124736/WholeGear_Transparent-32x32.png Center for Innovative Technology /cit 32 32 A Guide to Metabolite Annotation: A Mini-Series (Part II) /cit/a-guide-to-metabolite-annotation-a-mini-series-part-ii/ Mon, 15 Jun 2020 07:37:09 +0000 /cit/?p=1509 The post A Guide to Metabolite Annotation: A Mini-Series (Part II) appeared first on Center for Innovative Technology.

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A Guide to Metabolite Annotation: A Mini-Series (Part II)


Collision Cross Section Data

Confident MS-based metabolite annotation requires experimental data that supports a specific molecule or class of compound. A high-resolution high-mass accuracy measurement is the first filter to determine metabolite candidates, and fragmentation (MS/MS) measurements are necessary to provide product ion knowledge that can help assign structural information.

For some isomeric small molecules, however, fragmentation data can be non-diagnostic. Retention time measurements obtained from liquid chromatography may resolve isomeric species, but in some cases, co-elution remains a concern. Ion mobility collision cross sections often provide additional confidence for isomeric, co-eluting species, by rendering orthogonal data to support annotations.

As such, members of the McLean Research Group and CIT have developed a collision cross section compendium to increase confidence in identifications and narrow the chemical search space for the small molecule omics community: 

Go to the publication

Collision Cross Section Data

Figure adapted from .



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Collision Cross Section Data
Metabolite Annotations
Katrina Leaptrot, Post Doc in the McLean Lab
Large Metabolomics Studies
High-Throughput Multi-Omic Analyses
qualitative amino acid panel

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A Guide to Metabolite Annotation: A Mini-Series /cit/a-guide-to-metabolite-annotation-a-mini-series/ Mon, 08 Jun 2020 07:33:40 +0000 /cit/?p=1505 The post A Guide to Metabolite Annotation: A Mini-Series appeared first on Center for Innovative Technology.

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A Guide to Metabolite Annotation: A Mini-Series (Part I)


Annotation can be a major hurdle for untargeted metabolomics workflows. In these types of analyses, the experimental mass for each compound of interest is compared against databases of known compound masses to generate a list of candidate matches. High-resolution high-mass accuracy instruments such as those housed in the CIT can minimize a list of candidates by applying a narrow mass tolerance window, thus minimizing false positive hits (as shown below). MS measurements alone are often only capable of determining molecular formula. In our next Molecular Omics update, you’ll see how additional orthogonal data (i.e., retention time, fragmentation data, and collision cross sections) can guide structure elucidation and increase metabolite annotation confidence.

Metabolite Annotations


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Small Molecule Omics and Cancer Biology /cit/small-molecule-omics-cancer-biology/ Tue, 14 Jan 2020 15:07:10 +0000 /cit/?p=1474 The post Small Molecule Omics and Cancer Biology appeared first on Center for Innovative Technology.

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Small Molecule Omics and Cancer Biology


“Oncometabolites” initiate or sustain cancer tumor growth and metastasis. The term oncometabolite was first used for 2- hydroxyglutarate, as it was found to activate hypoxiainduced, oncogenic pathways and affect DNA methylation, specifically in gliomas and acute myeloid leukemia.1 Understanding metabolic factors that enable cancer cell proliferation and finding reprogrammed metabolic pathways has led to several other oncometabolic targets (or metabolites) to be discovered.2-4 Of the metabolites involved in cancer biology, lipids have not only been shown to be involved in intracellular signaling of cancer cells, but de novo lipid synthesis has been found to be a key metabolic activity in cancer cell growth and survival and targeting lipid metabolic pathways has been shown to affect chemosensitivity.3,5 Previous data has also shown that branched-chain amino acid (BCAA) can serve as metabolic substrates in several types of cancer.6



The CIT has developed a conformational lipid atlas containing highly-accurate structural measurements in support of high-confidence lipidomic annotations. This lipid atlas is available in the CIT to help us and our collaborators better understand the role of lipids in cancer. Please contact us if you are interested in performing analyses to determine which lipids may be contributing to or reprogramming cancer cell proliferation in your specific biological system.

In addition, the CIT has also developed a qualitative amino acids MS-based assay that can be applied to diverse biological sample types (e.g. plasma, serum, urine, cells or tissue) without the need for tagging. These analyses are performed with high precision (≤ 5 ppm) and low variability (≤ 10% RSD).



References:

  1. Wishart DS, Emerging applications of metabolomics in drug discovery and precision medicine, Nat Rev Drug Discov 2016, 15, 473-484.
  2. Luengo A et al., Targeting Metabolism for Cancer Therapy, Cell Chem Biol 2017, 24, 1161-1180.
  3. Röhrig F et al., The multifaceted roles of fatty acid synthesis in cancer, Nat Rev Cancer 2016, 16, 732-749.
  4. Adams JL et al., Big opportunities for small molecules in immuno-oncology, Nat Rev Drug Discov 2015, 14, 603-622.
  5. Beloribi-Djefaflia S, Vasseur S, Guillaumond F, Lipid metabolic reprogramming in cancer cells, Oncogenesis 2016, 5, e189.
  6. Mayers JR, et al., Tissue of origin dictates branched-chain amino acid metabolism in mutant Kras-driven cancers, Science 2016, 353, 1161-1165.

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The Human Microbiome’s Metabolome /cit/the-human-microbiomes-metabolome/ Wed, 31 Jul 2019 08:58:20 +0000 /cit/?p=1398 The post The Human Microbiome’s Metabolome appeared first on Center for Innovative Technology.

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The CIT is Excited to be a Part of the ý Microbiome Initiative


Microbiota are diverse and changing dynamically. The human microbiome plays an important role in our health and its composition and functionality is influenced by a variety of factors such as nutrition, environmental and pharmaceutical exposures, stress, and lifestyle. Several specific interactions between the microbiome’s and host’s metabolomes have been observed and described as co-metabolism via global, untargeted mass spectrometry analysis.1-4

Several microbiome-host interactions currently under investigation are shown in Figure 2 below. Examples for such interactions include bidirectional communication between liver and gut microbiota, in which liver metabolites influence gut microbiota composition thereby affecting intestinal barrier integrity. At the same time, gut microbiota also affect regulation of bile acid synthesis, glucose and lipid metabolism in the liver.5Microbiome metabolites have also been shown to affect depression, anxiety, and cognition in bidirectional communication across the gut-brain axis via neurocrine, endocrine, and inflammatory signals.6

The CIT is proud to be a member of the newly established Vanderbilt Microbiome Initiative and looks forward to support collaborators in further investigating the interesting relationship and interactions between host and microbiome metabolomes. We offer full-service, streamlined workflows, cutting edge untargeted and targeted molecular omics (incl. metabolomics, lipidomics, amino acids) resources for data acquisition and analysis, and a collaborative approach that enables our collaborators to focus on what matters most to them.

For a consultation, funding application support, or custom method development, please contact us today.

To read more about the ý Microbiome Initiative, please visit their website:

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Advancing Lipidomics /cit/lipidomics/ Wed, 17 Apr 2019 12:16:10 +0000 /cit/?p=1349 The post Advancing Lipidomics appeared first on Center for Innovative Technology.

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High Confidence Lipidomics via a Lipid Structure Atlas Enables the CIT to Predict Dysregulation

Lipids play an important role in health and disease, such as de novo lipid synthesis in cancer cell growth and survival or dysregulation of lipid metabolism in neurodegenerative diseases. Yet lipidomics research, the study of lipid species, pathways and their interactions with other metabolites inside an organism, is considered an emerging discipline. To advance the field, high-precision ion mobility-mass spectrometry resources in the CIT were utilized to develop a conformational database for lipids.

The atlas includes collision cross section information for numerous lipid classes, including: sphingomyelin, cerebroside, ceramide, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidic acid. The figure below outlines the important correlations that were discovered for glycerophospholipids (PS) and sphingolipids (GlcCer). This lipid atlas is available in the CIT to help collaborators better understand the role of lipids in their specific disease.

Katrina Leaptrot, Post Doc in the McLean Lab

Read the publication:
Read the ý News article:
Read the ý Institute of Chemical Biology’s summary article: /vicb/discovery_featured3.html



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Liquid Handling Platform Added for Large Metabolomics Studies /cit/large-metabolomics-studies/ Thu, 28 Feb 2019 13:14:58 +0000 /cit/?p=1069 The post Liquid Handling Platform Added for Large Metabolomics Studies appeared first on Center for Innovative Technology.

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Liquid Handling Platform Will Enable Increased Throughput for up to 3,000 Samples/Setup


Manual sample handling is labor-intensive and can contributes to the technical variability of analytical results. In comparison, automated liquid sample handling provides increased overall sample throughput, enabling larger sample sets to be analyzed more efficiently and with minimal technical variation.

In collaboration with Prof. Renã Robinson’s lab, the Center for Innovative Technology (CIT) has acquired a Beckman Coulter Biomek i-7 Automated Liquid Handling Workstation that will be available as a resource for molecular omics studies at the CIT. Center staff are completing all necessary hands-on training in order to successfully integrate this new resource into our workflows.

The workstation will enable the CIT to serve large-scale projects with several hundred or thousand samples in an efficient and highly reproducible manner.



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Quantity of Identified Metabolites in Untargeted Experiments /cit/quantity-identified-metabolites-untargeted-experiments/ Thu, 21 Feb 2019 14:01:19 +0000 /cit/?p=1058 The post Quantity of Identified Metabolites in Untargeted Experiments appeared first on Center for Innovative Technology.

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How Many Metabolites will be Identified in a Global, Untargeted Metabolomics Study?


This is a difficult question to answer!

The CIT typically observes between 500 and 10,000 compounds, depending on sample type. It is important to note that there is a range of annotation confidences assigned to each detected compound. The CIT uses a classification system to describe annotation confidence. Briefly, more experimental evidence (e.g., diagnostic fragmentation data, retention time, collision cross section, reference standard, etc.) enables a higher confidence metabolite identification assignment. While some compounds can be unambiguously identified (Level 1), others may be reported in groups (multiple candidate annotations; Level 3) owing to the fact that numerous metabolites are isomeric and metabolite databases are inherently incomplete. We search our extensive in-house library to reduce the number of candidates and/or increase the confidence of candidates when possible.

This classification scheme is widely adopted by the metabolomics community, see for more information.





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Metabolic Quenching /cit/metabolic-quenching/ Mon, 21 Jan 2019 13:13:22 +0000 /cit/?p=1002 The post Metabolic Quenching appeared first on Center for Innovative Technology.

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What is Metabolic Quenching?


Representative and valid cytoplasmic concentrations are essential for ensuring the significance of results in the field of metabolome analysis. One of the most crucial points in this respect is the sampling itself.

Metabolic quenching is a rapid and sudden stopping of the metabolism on a timescale that is much faster than the conversion rates of investigated metabolites.  The high turnover rates not only require a rapid quenching technique, but also the correct application. By applying robust techniques like rapid sampling in combination with reproducible sample processing, we can ensure fast and reliable metabolic inactivation during all steps.

To minimize metabolic turnover rates, we recommend to quickly freeze samples in liquid nitrogen immediately after collection and store all samples at -80°C prior to sample transfer to the CIT. Cold ammonium formate buffer (provided by CIT upon request) may be added to tissue and cell samples to remove residual enzymatic activity. Finally, freeze-thaw cycles should be avoided as much as possible.

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High-Throughput Multi-Omic Analyses /cit/high-throughput-multi-omics/ Mon, 21 Jan 2019 13:12:58 +0000 /cit/?p=996 The post High-Throughput Multi-Omic Analyses appeared first on Center for Innovative Technology.

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Multi-disciplinary collaboration across campus:

An Integrated, High-Throughput Strategy for Multi-Omic Analyses


In collaboration with the ý DARPA RTA Team (R. Caprioli, E. Skaar, J. Wikswo, J. McLean, B. Lacey, J. Norris), the CIT has contributed to developing an automated multi-omic sample preparation approach. The approach, sample preparation for multi-omics technologies (SPOT), provides equivalent performance to typical individual omic preparation methods but greatly enhances throughput and minimizes the resources required for multiomic experiments. In this manuscript, SPOT was used to understand the mechanism of action of Zn-treated HL-60 cells using transcriptomic, proteomic and metabolomic data generated from a common cell culture sample.

The details are available in the Journal of Proteome Research:



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Qualitative Amino Acid Panel /cit/qualitative-amino-acid-panels/ Mon, 21 Jan 2019 13:12:39 +0000 /cit/?p=998 The post Qualitative Amino Acid Panel appeared first on Center for Innovative Technology.

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New Service:

Qualitative Amino Acid Panel


Amino acids, often referred to as the building blocks of proteins, are compounds that play many critical roles in the body. They are needed for vital processes like the building of proteins and biosynthesis of hormones and neurotransmitters. In addition, they can be predictors of outcome in heart failure, hepatobiliary and some cancers, as well as markers for prediabetes (glycine), type 2 diabetes (valine, leucine, isoleucine, phenylalanine and tyrosine) or kidney function (tryptophan-kynurenine ratio).

The CIT has developed a 20 amino acids MS-based assay that can be applied to diverse biological sample types (e.g. plasma, serum, urine, cells or tissue). These analyses are performed with high precision (≤ 5 ppm) and low variability (≤ 10% RSD).

See image below for an extracted ion chromatogram of individual amino acids.


qualitative amino acid panel


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