Liquid Chromatography Mass Spectrometry (LC-MS)

To determine whether metabolic signatures associated with lameness could be discovered with untargeted metabolomics, we developed a novel workflow using direct infusion-tandem mass spectrometry to rapidly analyse (2 min per sample) dried milk spots (DMS) that were stored on commercially available Whatman® FTA® DMPK cards for a prolonged period (8 and 16 days). An orthogonal partial least squares-discriminant analysis (OPLS-DA) method validated by triangulation of multiple machine learning (ML) models and stability selection was employed to reliably identify important discriminative metabolites. With this approach, we were able to differentiate between lame and healthy cows based on a set of lipid molecules and several small metabolites. 

Wenshi He, Ana S. Cardoso, Robert M. Hyde, Martin J. Green, David J. Scurr, Rian L. Griffiths, Laura V. Randall and Dong-Hyun Kim, Analyst, 2022, 147, 5537

The use of the Droplet Microarray (DMA) provides a surface-assisted LESA-MS method delivering significant improvement of the surface extraction repeatability leading to the acquisition of more robust and higher quality data. Such a method shows potential to be used for LESA-MS for controlled and reproducible surface extraction and for acquisition of high quality, qualitative data in a high-throughput manner.

Meurs J, Alexander MR, Levkin PA, Widmaier S, Bunch J, Barrett DA and Kim D-H, Anal. Chem. 2018, 90, 10, 6001-6005

Here, we present a model system for growing biofilms on discs before utilizing rapid and direct surface sampling MS, namely, liquid extraction surface analysis, to study the microbial exometabolome. One of the benefits of this approach is its surface-specific nature, enabling mimicking biofilm formation in a way that the study of planktonic liquid cultures cannot imitate. Our model system provides a route to investigate changes in the exometabolome, such as metabolites that become circulatory in the presence of multiple pathogens, and provides a rapid analytical approach to gaining a mechanistic understanding of bacterial signalling.

Shaun N. Robertson, Fadi Soukarieh, Thomas M. White, Miguel Camara, Manuel Romero, and Rian L. Griffiths, Anal. Chem. 2023, 95, 11, 5079–5086

We report a liquid chromatography–isotope dilution mass spectrometry method for the simultaneous quantification of 131 intracellular bacterial metabolites of Clostridium autoethanogenum. A comprehensive mixture of uniformly ¹³C-labeled internal standards (U-¹³C IS) was biosynthesized from the closely related bacterium Clostridium pasteurianum using 4% ¹³C–glucose as a carbon source. The U-¹³C IS mixture combined with ¹²C authentic standards was used to validate the linearity, precision, accuracy, repeatability, limits of detection, and quantification for each metabolite. The method was subsequently applied for the daily monitoring of the intracellular metabolites of C. autoethanogenum during a CO gas fermentation over 40 days as part of a study to optimize biofuel production. The concentrations of the metabolites were estimated at steady states of different pH levels using the robust-fitting mathematical approach, and we demonstrate improved accuracy of results compared to conventional regression.

Laudina Safo, Salah Abdelrazig, Alexander Grosse-Honebrink, Thomas Millat, Anne M. Henstra, Rupert Norman, Neil R. Thomas, Klaus Winzer, Nigel P. Minton, Dong-Hyun Kim, and David A. Barrett, ACS Omega 2021, 6, 21, 13518–13526

In this study, an in vivo13C labelling strategy was employed to explore four species (Escherichia coli, Arthrospira platensis, Saccharomyces cerevisiae and Pichia pastoris) as a source of 13C-labelled internal standards (13C-ISs) for more accurate and quantitative liquid chromatography (LC)-mass spectrometry (MS)-based lipidomics. Overall, use of a biologically generated 13C-IS lipid mixture of 357 identified lipid ions resulted in significant reduction in the lipid CV% of normalisation compared with other normalisation methods using total ion counts or a commercially available deuterated internal standard mixture. This improved normalisation using 13C-IS was confirmed in a typical lipidomics analysis using a large number of samples (>100+) and long analysis time (>70 h).

Malak A. Jaber, Bruna de Falco, Salah Abdelrazig, Catharine A. Ortori, David A. Barrett and Dong-Hyun Kim, Anal. Methods, 2023, 15, 2925

This study represents successful application of LC–MS metabolomics approach to characterise M1 and M2 macrophages providing functional readouts that show unique metabolic signature for each phenotype. This data could contribute to a better understanding of M1 and M2 functional properties and could pave the way for developing new therapeutics targeting different immune diseases.

Alaa Abuawad, Chidimma Mbadugha, Amir M. Ghaemmaghami & Dong-Hyun Kim, Metabolomics, 2020, 16:33

• Equipped with HPLC and UHPLC.
• Quantitative and qualitative targeted metabolite profiling, and for ID confirmation. Used where sensitivity is required.
• Advanced MS scanning options and information dependant acquisition offer unique approaches to metabolite profiling and identification.

• Equipped with NanoLC, UHPLC and TriVersa NanoMate..
• Small molecules profiling and identification.
• Quantitative and qualitative measurements.
• Metabolomics and lipidomics with high resolution measurements.

• Resolution > 500,000 FWHM.
• MSn capabilities.

• Surface extraction and analysis under ambient conditions.
• Direct infusion high throughput analysis.
• Advantages where UHV analysis may not be suitable; alternative to ToF-SIMS and MALDI.
• Coupled to QExactive MS to achieve a high resolution and confidence in identification using data dependant MS/MS.
• The new LESAPLUS allows for automated LESA experiments plus additional nano-LC separation. 

• Surface analysis/imaging under ambient conditions.
• Unlike regular MALDI, it does not require a vacuum chamber, which means the analysis is faster and the samples can be kept in their native statuss.