Targeted Proteomic Analysis of Central Metabolic Enzymes Using Nano-LC-Triple Quadrupole Mass Spectrometry

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Targeted proteomics is an analytical technique that measures the expression levels of pre-selected target proteins. Nano- liquid chromatography (nano-LC) is used in targeted proteomics because its low flow rates allow greater analytical sensitivity when dealing with samples that contain very small amounts of peptides. Mass spectrometers capable of high-speed operations, such as those with ultra-fast mass spectrometry (UFMS) technology, are also needed in targeted proteomics to perform the simultaneous analysis of large numbers of targeted proteins. This article describes applying targeted proteomics with nano-LC and ultra-fast mass spectrometry to the analysis of central metabolic enzymes in yeast and cultured human cells.

Introduction

A central metabolism is a basic feature shared by a wide range of organisms. For example, baker’s yeast (Saccharomyces cerevisiae) uses central metabolic pathways to perform a type of fermentation that converts glucose to ethanol and carbon dioxide. The outstanding fermentation performance of S. cerevisiae has been a feature of food production since ancient times, such as in baking and the brewing of Japanese sake and wine, and in more recent times for the production of bioethanol. Metabolic engineering of S. cerevisiae is also becoming more prevalent, with gene recombination and gene editing techniques used to modify the central metabolic pathways of S. cerevisiae to produce a variety of useful compounds. Central metabolic pathways also play a role in the ability of the oleaginous yeast Lipomyces starkeyi to accumulate large amounts of lipids. In the field of cancer research, associations between a central metabolism and mechanisms of disease are a focus of interest, with cancer cells known to specifically activate metabolic pathways that create lactate from glucose. The central metabolism functions thanks to some 100 enzymes, which catalyze the reactions on central metabolic pathways. Measuring the expression levels of enzymes on central metabolic pathways offers a shortcut to understanding how the production of useful substances can be enhanced through central metabolism regulation and the associations between disease and central metabolic functions. Targeted proteomics offers a method for measuring protein expression levels based on mass analysis, which involves obtaining a crude enzyme extract from a biological sample, digesting the enzyme extract with trypsin, and analyzing the resulting mixture of trypsin-digested peptides using the liquid chromatograph-triple quadrupole mass spectrometer. Before performing this analysis, trypsin-digested peptides that allow quantitative analysis of the target protein must be selected in advance for each targeted protein, after which a list of multiple reaction monitoring (MRM) transitions is used to selectively detect and quantify these peptides. Nano-LC is also used in targeted proteomics because its low flow rates allow greater analytical sensitivity when dealing with samples that contain very small amounts of peptides. Mass spectrometers capable of high- speed operations, such as those with ultra-fast mass spectrometry (UFMS) technology, are also needed in targeted proteomics to perform the simultaneous analysis of large numbers of targeted proteins. This article applies targeted proteomics using nano-LC and ultra-fast mass spectrometry to analyze central metabolic enzymes in yeast and cultured human cells.

December 11, 2023 GMT