Improve Research Reproducibility A Bio-protocol resource

Submit a Protocol
Receive Our Alerts
Log In
/
Sign Up
- My Bio Page
- Edit My Profile
- Change Password
- Log Out
EN
- EN - English
- CN - 中文

Systems Biology

Categories

3D Genomics

Connectomics

Epigenomics

Genomics

Interactome

Mechanomics

Metabolomics

Biofluid

Polar Metabolite

Leaf

Lipidomics

Neurometabolite

Seed

Tissue

Whole organism

Microbiomics

Proteomics

Spatial transcriptomics

Transcriptomics

Protocols in Past Issues

Date Views

Automated Sequential Derivatization for Gas Chromatography-[Orbitrap] Mass Spectrometry-based Metabolite Profiling of Human Blood-based Samples

0 Q&A

2710 Views

Mar 5, 2025

Many small molecules require derivatization to increase their volatility and to be amenable to gas chromatographic (GC) separation. Derivatization is usually time-consuming, and typical batch-wise procedures increase sample variability. Sequential automation of derivatization via robotic liquid handling enables the overlapping of sample preparation and analysis, maximizing time efficiency and minimizing variability. Herein, a protocol for the fully automated, two-stage derivatization of human blood–based samples in line with GC–[Orbitrap] mass spectrometry (MS)-based metabolomics is described. The protocol delivers a sample-to-sample runtime of 31 min, being suitable for better throughput routine metabolomic analysis.

Metabolomic and Lipidomic Analysis of Bone Marrow Derived Macrophages

Gretchen L. Seim

Steven V. John

JF Jing Fan

1 Q&A

9366 Views

Jul 20, 2020

Macrophages are highly plastic immune cells that are capable of adopting a wide array of functional phenotypes in response to environmental stimuli. The changes in macrophage function are often supported and regulated by changes in cellular metabolism. Capturing a comprehensive picture of metabolism is vital for understanding the role of metabolic rewiring in the immune response. Here we present a method for systematically quantifying the abundance of metabolites and lipids in primary murine bone marrow derived macrophages (BMDMs). This method simultaneously extracts polar metabolites and lipids from BMDMs using a rapid two-phase extraction procedure. The polar metabolite fraction and lipid fraction are subsequently analyzed by separate liquid chromatography-mass spectrometry (LC-MS) methods for optimized coverage and quantification. This allows for a comprehensive characterization of cellular metabolism that can be used to understand the impact of a variety of environmental stimuli on macrophage metabolism and function.

Isolation and Quantification of Metabolite Levels in Murine Tumor Interstitial Fluid by LC/MS

MS Mark R Sullivan

CL Caroline A Lewis

AM Alexander Muir

1 Q&A

11664 Views

Nov 20, 2019

Cancer is a disease characterized by altered metabolism, and there has been renewed interest in understanding the metabolism of tumors. Even though nutrient availability is a critical determinant of tumor metabolism, there has been little systematic study of the nutrients directly available to cancer cells in the tumor microenvironment. Previous work characterizing the metabolites present in the tumor interstitial fluid has been restricted to the measurement of a small number of nutrients such as glucose and lactate in a limited number of samples. Here we adapt a centrifugation-based method of tumor interstitial fluid isolation readily applicable to a number of sample types and a mass spectrometry-based method for the absolute quantitation of many metabolites in interstitial fluid samples. In this method, tumor interstitial fluid (TIF) is analyzed by liquid chromatography-mass spectrometry (LC/MS) using both isotope dilution and external standard calibration to derive absolute concentrations of targeted metabolites present in interstitial fluid. The use of isotope dilution allows for accurate absolute quantitation of metabolites, as other methods of quantitation are inadequate for determining nutrient concentrations in biological fluids due to matrix effects that alter the apparent concentration of metabolites depending on the composition of the fluid in which they are contained. This method therefore can be applied to measure the absolute concentrations of many metabolites in interstitial fluid from diverse tumor types, as well as most other biological fluids, allowing for characterization of nutrient levels in the microenvironment of solid tumors.

Extraction and Analysis of Pan-metabolome Polar Metabolites by Ultra Performance Liquid Chromatography–Tandem Mass Spectrometry (UPLC-MS/MS)

DM Dania M. Malik

SR Seth Rhoades

AW Aalim Weljie

0 Q&A

10450 Views

Feb 5, 2018

Modern triple quadrupole mass spectrometers provide the ability to detect and quantify a large number of metabolites using tandem mass spectrometry (MS/MS). Liquid chromatography (LC) is advantageous, as it does not require derivatization procedures and a large diversity in physiochemical characteristics of analytes can be accommodated through a variety of column chemistries. Recently, the comprehensive optimization of LC-MS metabolomics using design of experiments (COLMeD) approach has been described and used by our group to develop robust LC-MS workflows (Rhoades and Weljie, 2016). The optimized LC-MS/MS method described here has been utilized extensively for metabolomics analysis of polar metabolites. Typically, tissue or biofluid samples are extracted using a modified Bligh-Dyer protocol (Bligh and Dyer, 1959; Tambellini et al., 2013). The protocol described herein describes this workflow using targeted polar metabolite multiple reaction monitoring (MRM) from tissues and biofluids via ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). This workflow has been utilized extensively for chronometabolic analysis (Krishnaiah et al., 2017), with applications generalized to other types of analyses as well (Sengupta et al., 2017; Sivanand et al., 2017).