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1. Electroblotting through a tryptic membrane for LC-MS/MS analysis of proteins separated in electrophoretic gels

   https://doi.org/10.1039/D0AN01380C  

   Bickner, A. N. ; Champion, M. M. ; Hummon, A. B. ; Bruening, M. L. ( November 2020 , The Analyst)

   Digestion of proteins separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) remains a popular method for protein identification using mass-spectrometry based proteomics. Although robust and routine, the in-gel digestion procedure is laborious and time-consuming. Electroblotting to a capture membrane prior to digestion reduces preparation steps but requires on-membrane digestion that yields fewer peptides than in-gel digestion. This paper develops direct electroblotting through a trypsin-containing membrane to a capture membrane to simplify extraction and digestion of proteins separated by SDS-PAGE. Subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) identifies the extracted peptides. Analysis of peptides from different capture membrane pieces shows that electrodigestion does not greatly disturb the spatial resolution of a standard protein mixture separated by SDS-PAGE. Electrodigestion of an Escherichia coli ( E. coli ) cell lysate requires four hours of total sample preparation and results in only 13% fewer protein identifications than in-gel digestion, which can take 24 h. Compared to simple electroblotting and protein digestion on a poly(vinylidene difluoride) (PVDF) capture membrane, adding a trypsin membrane to the electroblot increases the number of protein identifications by 22%. Additionally, electrodigestion experiments using capture membranes coated with polyelectrolyte layers identify a higher fraction of small proteolytic peptides than capture onmore »PVDF or in-gel digestion.« less
2. Understanding the structural complexity of dissolved organic matter: isomeric diversity

   https://doi.org/10.1039/c8fd00221e  

   Leyva, Dennys ; Tose, Lilian V. ; Porter, Jacob ; Wolff, Jeremy ; Jaffé, Rudolf ; Fernandez-Lima, Francisco ( August 2019 , Faraday Discussions)

   In the present work, the advantages of ESI-TIMS-FT-ICR MS to address the isomeric content of dissolved organic matter are studied. While the MS spectra allowed the observation of a high number of peaks ( e.g. , PAN-L: 5004 and PAN-S: 4660), over 4× features were observed in the IMS-MS domain ( e.g. , PAN-L: 22 015 and PAN-S: 20 954). Assuming a total general formula of C x H y N 0–3 O 0–19 S 0–1 , 3066 and 2830 chemical assignments were made in a single infusion experiment for PAN-L and PAN-S, respectively. Most of the identified chemical compounds (∼80%) corresponded to highly conjugated oxygen compounds (O 1 –O 20 ). ESI-TIMS-FT-ICR MS provided a lower estimate of the number of structural and conformational isomers ( e.g. , an average of 6–10 isomers per chemical formula were observed). Moreover, ESI-q-FT-ICR MS/MS at the level of nominal mass ( i.e. , 1 Da isolation) allowed for further estimation of the number of isomers based on unique fragmentation patterns and core fragments; the later suggested that multiple structural isomers could have very closely related CCS. These studies demonstrate the need for ultrahigh resolution TIMS mobility scan functions ( e.g. , R = 200–500)more »in addition to tandem MS/MS isolation strategies.« less
3. MetaboDirect: an analytical pipeline for the processing of FT-ICR MS-based metabolomic data

   https://doi.org/10.1186/s40168-023-01476-3  

   Ayala-Ortiz, Christian ; Graf-Grachet, Nathalia ; Freire-Zapata, Viviana ; Fudyma, Jane ; Hildebrand, Gina ; AminiTabrizi, Roya ; Howard-Varona, Cristina ; Corilo, Yuri E. ; Hess, Nancy ; Duhaime, Melissa B. ; et al ( February 2023 , Microbiome)

   Microbiomes are now recognized as the main drivers of ecosystem function ranging from the oceans and soils to humans and bioreactors. However, a grand challenge in microbiome science is to characterize and quantify the chemical currencies of organic matter (i.e., metabolites) that microbes respond to and alter. Critical to this has been the development of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), which has drastically increased molecular characterization of complex organic matter samples, but challenges users with hundreds of millions of data points where readily available, user-friendly, and customizable software tools are lacking.

   Here, we build on years of analytical experience with diverse sample types to develop MetaboDirect, an open-source, command-line-based pipeline for the analysis (e.g., chemodiversity analysis, multivariate statistics), visualization (e.g., Van Krevelen diagrams, elemental and molecular class composition plots), and presentation of direct injection high-resolution FT-ICR MS data sets after molecular formula assignment has been performed. When compared to other available FT-ICR MS software, MetaboDirect is superior in that it requires a single line of code to launch a fully automated framework for the generation and visualization of a wide range of plots, with minimal coding experience required. Among the tools evaluated, MetaboDirect is alsomore »uniquely able to automatically generate biochemical transformation networks (ab initio) based on mass differences (mass difference network-based approach) that provide an experimental assessment of metabolite connections within a given sample or a complex metabolic system, thereby providing important information about the nature of the samples and the set of microbial reactions or pathways that gave rise to them. Finally, for more experienced users, MetaboDirect allows users to customize plots, outputs, and analyses.

   Application of MetaboDirect to FT-ICR MS-based metabolomic data sets from a marine phage-bacterial infection experiment and aSphagnumleachate microbiome incubation experiment showcase the exploration capabilities of the pipeline that will enable the research community to evaluate and interpret their data in greater depth and in less time. It will further advance our knowledge of how microbial communities influence and are influenced by the chemical makeup of the surrounding system. The source code and User’s guide of MetaboDirect are freely available through (https://github.com/Coayala/MetaboDirect) and (https://metabodirect.readthedocs.io/en/latest/), respectively.

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4. Three-in-One Simultaneous Extraction of Proteins, Metabolites and Lipids for Multi-Omics

   https://doi.org/10.3389/fgene.2021.635971  

   Kang, Jianing ; David, Lisa ; Li, Yangyang ; Cang, Jing ; Chen, Sixue ( April 2021 , Frontiers in Genetics)

   Elucidation of complex molecular networks requires integrative analysis of molecular features and changes at different levels of information flow and regulation. Accordingly, high throughput functional genomics tools such as transcriptomics, proteomics, metabolomics, and lipidomics have emerged to provide system-wide investigations. Unfortunately, analysis of different types of biomolecules requires specific sample extraction procedures in combination with specific analytical instrumentation. The most efficient extraction protocols often only cover a restricted type of biomolecules due to their different physicochemical properties. Therefore, several sets/aliquots of samples are needed for extracting different molecules. Here we adapted a biphasic fractionation method to extract proteins, metabolites, and lipids from the same sample (3-in-1) for liquid chromatography-tandem mass spectrometry (LC-MS/MS) multi-omics. To demonstrate utility of the improved method, we used bacteria-primed Arabidopsis leaves to generate multi-omics datasets from the same sample. In total, we were able to analyze 1849 proteins, 1967 metabolites, and 424 lipid species in single samples. The molecules cover a wide range of biological and molecular processes, and allow quantitative analyses of different molecules and pathways. Our results have shown the clear advantages of the multi-omics method, including sample conservation, high reproducibility, and tight correlation between different types of biomolecules.
5. Distinct classes of multi-subunit heterogeneity: analysis using Fourier Transform methods and native mass spectrometry

   https://doi.org/10.1039/D0AN00726A  

   Cleary, Sean P. ; Prell, James S. ( June 2020 , The Analyst)

   Native electrospray mass spectrometry is a powerful method for determining the native stoichiometry of many polydisperse multi-subunit biological complexes, including multi-subunit protein complexes and lipid-bound transmembrane proteins. However, when polydispersity results from incorporation of multiple copies of two or more different subunits, it can be difficult to analyze subunit stoichiometry using conventional mass spectrometry analysis methods, especially when m / z distributions for different charge states overlap in the mass spectrum. It was recently demonstrated by Marty and co-workers (K. K. Hoi, et al. , Anal. Chem. , 2016, 88 , 6199–6204) that Fourier Transform (FT)-based methods can determine the bulk average lipid composition of protein-lipid Nanodiscs assembled with two different lipids, but a detailed statistical description of the composition of more general polydisperse two-subunit populations is still difficult to achieve. This results from the vast number of ways in which the two types of subunit can be distributed within the analyte ensemble. Here, we present a theoretical description of three common classes of heterogeneity for mixed-subunit analytes and demonstrate how to differentiate and analyze them using mass spectrometry and FT methods. First, we first describe FT-based analysis of mass spectra corresponding to simple superpositions, convolutions, and multinomial distributions formore »two or more different subunit types using model data sets. We then apply these principles with real samples, including mixtures of single-lipid Nanodiscs in the same solution (superposition), mixed-lipid Nanodiscs and copolymers (convolutions), and isotope distribution for ubiquitin (multinomial distribution). This classification scheme and the FT method used to study these analyte classes should be broadly useful in mass spectrometry as well as other techniques where overlapping, periodic signals arising from analyte mixtures are common.« less