代謝産物分析のためのタンデム液体クロマトグラフィー - 質量分析に基づくアプローチ

The overall goal of this sampling method is to harvest metabolite samples from Staphyococcus aureus for subsequent quantification by Tandem Liquid Chromatography-Mass Spectrometry. Our lab is interested in studying the intersection of metabolism and pathogenesis in bacteria relevant to human health. Currently, we're using the human opportunistic pathogen Staphylococcus aureus as a model organism.

One key question that we wish to answer is how bacteria adjust their physiology and pathogenicity when faced with nutrient depletion during host infection. This technique is advantageous because it provides a snapshot into the distribution of specific metabolites amongst the many metabolic pathways in actively growing cells, with minimal perturbation of their physiological state. To begin, streak the S.aureus strains of interest for isolation on tryptic soy agar from the frozen glycerol stock.

Incubate the cells at 37 degrees Celsius for 16-24 hours. The next day, inoculate 4 mLs of tryptic soy broth, or TSB, in sterile glass incubation tubes with single colonies of each strain. Incubate the cultures, inclined with rotation, at 37 degrees Celsius for 16-20 hours.

Use a spectrophotometer to measure the optical density of the cultures at 600 nanometers, abbreviated as OD600. Using this measurement, dilute the cells to an OD600 of 0.05 in 50 mLs of sterile TSB medium, in 250 mL Delong flasks. Incubate the cultures at 37 degrees Celsius in a warm bath with shaking at 280 rpm.

Every 30 minutes, take OD600 measurements. As the optical densities increase, it may become necessary to dilute the cultures with TSB so that they remain within the linear absorbance range of the spectrophotometer. While the cells are growing, freshly prepare the extraction buffer solutions using the highest purity reagents available as described in the text protocol.

When the cultures achieve an OD600 between 0.8 and 1.0, subculture them into 50 mLs of fresh pre-warmed TSB to an OD600 of 0.01 to 0.05, and repeat the incubation and measurement steps. Prepare a bed of crushed dry ice in an appropriate vessel, such as a glass dish, ice bucket, or cooler. As the optical densities of the cultures approach the desired harvest point, add 1 mL of quenching solution to a 35 mm untreated petri dish, and pre-cool on dry ice for at least 5 minutes.

For our purposes, the desired harvest point is an optical density of about 0.4 to 0.5. But this can be varied, depending on experimental goals. Next, place a stainless steel filter frit in a rubber stopper, and place it atop a vacuum flask attached to a house vacuum, or vacuum pump.

Apply the vacuum, and place a mixed cellulose ester membrane on top. Use a filter with the same diameter as the frit to ensure that all cells are collected. It's critical to collect the cells and halt metabolic activity as quickly as possible, so as to provide a snapshot of metabolic abundances under a given condition.

Even small delays can affect the steady-state physiology of the cells. At an OD600 of between 0.4 and 0.5, use a serological pipette to remove 13 mLs of culture from the flask. Immediately apply the sample to the filter.

After the entire sample has been filtered, immediately wash the filter with no more than 5 mL of ice cold PBS to wash away medium-associated metabolites. Then disconnect the vacuum and use a pair of sterile tweezers to remove the filter from the frit. Invert the filter into the pre-chilled quench solution.

Incubate the filter in quench solution on dry ice for at least 20 minutes. Using the sterile tweezers, invert the filter in the petri dish and use a micropipette to rinse the cells off of the membrane into the quench solution. Resuspend the cells in quench solution, and then transfer the cell suspension to a sterile 2 mL impact-resistant tube containing approximately 100 microliters of 0.1 mm silica beads.

Store this on dry ice or at 80 degrees Celsius. To begin the metabolite extraction, first thaw the samples on wet ice. Then disrupt the cells in a homogenizer with four 30-second bursts at 6000 rpm, with two minute cooling periods on dry ice between cycles.

Clarify the lysates for 15 minutes in a pre-chilled refrigerated microcentrifuge tube at maximum speed. Following centrifugation, transfer the supernatant to a clean microcentrifuge tube. Using a micropipette, transfer a small portion of the sample to a microcentrifuge tube for quantification of residual peptide content as described in the text protocol.

Store the remainder of the sample at 80 degrees Celsius. Refer to the text protocol for analysis of the metabolites via liquid chromatography and mass spectrometry. The CodY null mutant was used to investigate branched-chain amino acid depletion as the CodY protein adjusts the expression of dozens of genes in response to the drop in concentration of these nutrients.

The codY mutation has little effect on the growth rate of S.aureus cells. The drop in optical density at the 3-hour mark indicates the back-dilution step, which increases the number of cell doublings before harvest, and dilutes out various molecules that accumulate during overnight growth. An RNA sequence analysis of the transcriptomes of the two strains show significant changes in the expression of genes involved in the synthesis of the aspartate family of amino acids.

Here, the numbers below gene names indicate the fold change in expression in a codY null mutant, when compared to the wild type strain. Following LC-MS quantification of the extracted metabolites, the abundances between the two strains were compared. Focusing on some compounds related to the aspartate family of amino acids, the abundances of precursors, such as aspartate, and O-acetylhomoserine, are decreased in the codY mutant.

Conversely, end products like threonine and isoleucine are increased. Here, the dashed line indicates the cutoff for changes in abundance for this experiment. After watching this video, you should have a good understanding of how to harvest metabolites from Staphylococcus aureus for quantification via liquid chromatography mass spectrometry.

This protocol is also suitable with modification for metabolite harvest from other bacterial species. Because bacteria respond to dynamic environments, it's important to perform the filtering and wash steps quickly to minimize potential physiological changes induced by sampling. Changes can also be minimized by keeping the frit and PBS cold during sampling.