We are grateful to current and former members of the Titorenko laboratory for discussions. We acknowledge the Centre for Biological Applications of Mass Spectrometry, the Centre for Structural and Functional Genomics, and the Centre for Microscopy and Cellular Imaging (all at Concordia University) for outstanding services. This study was supported by grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada (RGPIN 2014-04482 and CRDPJ 515900 - 17). K.M. was supported by the Concordia University Armand C. Archambault Fellowship and the Concordia University Dean of Arts and Sciences Award of Excellence.

1. Making and sterilizing a medium for growing yeast
<ol>
	<li>Make 180 mL of a complete yeast extract with bactopeptone (YP) medium. The complete YP medium contains 1% (w/v) yeast extract and 2% (w/v) bactopeptone.</li>
	<li>Distribute 180 mL of the YP medium equally into four 250 mL Erlenmeyer flasks. Each of these flasks contains 45 mL of the YP medium.</li>
	<li>Sterilize the flasks with YP medium by autoclaving at 15 psi/121 &#176;C for 45 min.</li>
</ol>
2. Wild-type yeast strain
<ol>
	<li>Use the BY4742 (MAT&#945; his3&#916;1 leu2&#916;0 lys2&#916;0 ura3&#916;0) strain.</li>
</ol>
3. Growing yeast in the YP medium containing 2% glucose
<ol>
	<li>Sterilize a 20% (w/v) stock solution of glucose by autoclaving at 15 psi/121 &#176;C for 45 min.</li>
	<li>Add 5 mL of the autoclaved 20% (w/v) stock solution of glucose to each of the two Erlenmeyer flasks with 45 mL of the sterilized YP medium. The final concentration glucose in the YP medium is 2% (w/v).</li>
	<li>Use a microbiological loop to inoculate yeast cells into each of the two Erlenmeyer flasks with the YP medium containing 2% glucose.</li>
	<li>Grow the yeast cells overnight at 30 &#176;C in a rotational shaker set at 200 rpm.</li>
	<li>Take an aliquot of yeast culture. Determine the total number of yeast cells per mL of culture. Count cells using a hemocytometer.</li>
</ol>
4. Cell transfer to and cell grows in the YP medium with 2% glucose
<ol>
	<li>Add 5 mL of the sterilized 20% (w/v) stock solution of glucose to each of the remaining two Erlenmeyer flasks with the autoclaved YP medium. The final concentration of glucose is 2% (w/v).</li>
	<li>Transfer a volume of the overnight yeast culture in YP medium with 2% glucose that contains the total number of 5.0 x 107 cells into each of the two Erlenmeyer flasks with the YP medium containing 2% glucose. Use a sterile pipette for the cell transfer.</li>
	<li>Grow the yeast cells for at least 24 h (or more, if the experiment requires) at 30 &#176;C in a rotational shaker set at 200 rpm.</li>
</ol>
5. Making reagents, preparing labware, and setting up equipment for cell quenching
<ol>
	<li>Prepare the following: 1) a quenching solution (60% high-grade (&#62;99.9%) methanol in 155 mM ammonium bicarbonate (ABC) buffer, pH = 8.0); 2) an ice-cold ABC solution (pH = 8.0); 3) a digital thermometer capable of measuring up to -20 &#176;C; 4) 500 mL large centrifuge bottles; 5) a pre-cooled high-speed centrifuge with a pre-cooled rotor and pre-cooled 500 mL centrifuge bottles for this rotor, all at -5 &#176;C; 6) metabolite extraction tubes (15 mL high-speed glass centrifuge tubes with polytetrafluoroethylene lined caps); and 7) dry ice.</li>
</ol>
6. Cell quenching
<ol>
	<li>Use a hemocytometer to determine the number of yeast cells per mL of YP with 2% glucose culture.</li>
	<li>Transfer a volume of the culture in YP medium with 2% glucose that contains the total number of 5.0 x 108 cells into pre-cooled 500 mL centrifuge bottles.</li>
	<li>Quickly fill the centrifuge bottle containing the cells up to the volume of 200 mL with a quenching solution stored at -20 &#176;C.</li>
	<li>Centrifuge the bottles in a high-speed centrifuge at 11,325 x g for 3 min at -5 &#176;C.</li>
	<li>Quickly and tenderly recover the bottle from the centrifuge; gently unscrew the lid and remove the supernatant without disturbing the pellet.</li>
	<li>Quickly resuspend the cell pellet in 10 mL of ice-cold ABC buffer and transfer the suspension into a 15 mL high-speed glass centrifuge tube with a polytetrafluoroethylene-lined cap for metabolite extraction.</li>
	<li>Collect cells by centrifugation in a clinical centrifuge at 3,000 x g for 3 min at 0 &#176;C.</li>
	<li>Quickly remove the supernatant and place the tube on dry ice to begin metabolite extraction or store the tube at -80 &#176;C until extraction.</li>
</ol>
7. Preparation of reagents, labware and equipment for metabolite extraction
<ol>
	<li>Prepare the following: 1) LC-MS grade chloroform; 2) LC-MS grade methanol; 3) LC grade nano-pure water; 4) LC-MS grade (ACN); 5) glass beads (acid-washed, 425-600 &#181;m); 6) a vortex with a foam tube holder kit with retainer; 7) 15 mL high-speed glass centrifuge tubes with polytetrafluoroethylene-lined caps; 8) MS vials; 9) dry ice; and 10) 1.5 mL tubes washed once with ethanol, once with ACN and once with nano-pure water. 
	&#8203;NOTE: Use only micropipette tips and tubes made of polypropylene that is resistant to organic solvents.</li>
</ol>
8. Metabolite extraction
<ol>
	<li>To the metabolites kept on dry ice or stored at -80 &#176;C tube from step 6.7, add the following: 1) 2 mL of chloroform stored at -20 &#176;C; 2) 1 mL of methanol stored at -20 &#176;C; 3) 1 mL of ice-cold nano-pure water; and 4) 200 &#181;L of 425-600 &#181;m acid-washed glass beads.</li>
	<li>Cover and close the mouth of a tube with aluminum foil. Place tubes in a foam tube holder kit with retainer and vortex them for 30 min at medium speed (i.e., at a speed that is set at 6, with 12 being the maximum speed of a vortex) at 4 &#176;C to facilitate metabolite extraction.</li>
	<li>Incubate the tube for 15 min on ice (NOT dry ice!) to promote protein precipitation and the separation of the upper aqueous from the lower organic phase.</li>
	<li>Centrifuge the tube in a clinical centrifuge at 3,000 x g for 10 min at 4 &#176;C. This centrifugation step allows separating the upper aqueous phase (which contains water-soluble metabolites) from the middle layer (which contains cell debris and proteins) and from the lower organic phase (which contains mostly lipids).</li>
	<li>Use a micropipette to transfer the upper aqueous phase (400 &#181;L) to a washed and labeled 1.5 mL tube containing 800 &#181;L of ACN that was stored at -20 &#176;C. 
	NOTE: There will be white cloud precipitation after adding the upper aqueous phase to ACN kept at -20 &#176;C.</li>
	<li>Centrifuge the tube with the sample in a tabletop centrifuge at 13,400 x g for 10 min at 4 &#176;C. 
	&#8203;NOTE: The white cloud precipitation will disappear after centrifugation.</li>
	<li>Transfer 800 &#181;L from the upper portion of a liquid in the tube to a labeled MS vial. Store the sample at 0 &#176;C until it is analyzed by LC-MS/MS.</li>
</ol>
9. Preparation of reagents, labware, and equipment for LC
<ol>
	<li>Prepare the following: 1) a vortex; 2) an ultrasonic sonicator; 3) MS glass vials; 4) an LC system equipped with a binary pump, degasser, and autosampler; 5) a zwitterionic-phase chromatography column (5 &#181;m polymer, 150 x 2.1 mm) named in Table of Materials; 6) a column heater; and 7) mobile phases, including phase A (5:95 ACN:water (v/v) with 20 mM ammonium acetate, pH = 8.0) and phase B (100% ACN).</li>
</ol>
10. Separation of extracted metabolites by LC
<ol>
	<li>Subject the content of the MS vial to ultrasonic sonication for 15 min.</li>
	<li>Vortex the MS vial 3x for 10 sec at room temperature (RT).</li>
	<li>Place the MS vial into the well plate.</li>
	<li>During chromatograph, maintain the column at 45 &#176;C and a flow rate of 0.250 mL/min. Keep the sample in the well plate at 0 &#176;C. Refer to Table 1 for the LC gradients that need to be used during chromatography. 
	NOTE: A representative total ion chromatogram of water-soluble metabolites that were extracted from cells of the wild-type strain BY4742 is shown in Figure 1. The metabolites separated by LC were identified and quantified by mass spectrometric analysis that was performed in positive ionization [ESI (+)] mode, as described for step 11.</li>
</ol>
11. Mass spectrometric analysis of metabolites separated by LC
<ol>
	<li>Use a mass spectrometer equipped with heated electrospray ionization (HESI) for the identification and quantitation of water-soluble metabolites that were separated by LC. Use the mass spectrometer&#39;s analyzer for MS1 ions and the mass spectrometer&#39;s detector for MS2 ions. Use the settings provided in Table 2 and Table 3 for the data-dependent acquisition of MS1 and MS2 ions, respectively.</li>
	<li>Use a sample volume of 10 &#181;L for the injection in both the ESI (+) and ESI (-) modes.</li>
</ol>
12. Identification and quantitation of different metabolites by the processing of raw data from LC-MS/MS
<ol>
	<li>Use the software named in Table of Materials to conduct the identification and quantitation of different water-soluble metabolites from raw LC-MS/MS files. This software uses MS1 for metabolite quantitation and MS2 for metabolite identification. The software exploits the most extensively curated mass spectral fragmentation library to annotate the metabolites using the LC-MS/MS raw data by matching MS spectra. This software also uses the exact mass of MS1 and isotope pattern match to annotate metabolites using online databases. See Figure 2 for details.</li>
	<li>Use the library of databases and spectra, which is freely available online (https://www.mzcloud.org), to search for MS2 spectra of the raw data.</li>
</ol>
13. Membrane integrity assay by propidium Iodide (PI) staining and fluorescence microscopy
<ol>
	<li>After cell quenching performed as described for step 6, wash the quenched cells thoroughly with 15 mL of ABC buffer to remove the quenching solution. Collect cells by centrifugation at 3,000 x g for 5 min at 0 &#176;C.</li>
	<li>Resuspend the cell pellet in 1 mL of ABC buffer and add 0.5 mL of the PI solution (0.5 mg/mL).</li>
	<li>Vortex a tube with the sample 3x for 10 s and incubate it for 10 min in the dark and on ice.</li>
	<li>Centrifuge the tube with the sample in a tabletop centrifuge at 13,400 x g for 10 min at 4 &#176;C.</li>
	<li>Remove the supernatant and resuspend the pellet in 1 mL of ABC buffer.</li>
	<li>Centrifuge the tube at 13,400 x g for 10 min at 4 &#176;C and remove the supernatant. Repeat this step 2 more times to remove the PI bound to the cell surface.</li>
	<li>Resuspend the pellet in 300 &#181;L of ABC buffer. Place 10 &#181;L of the suspension on the surface of a microscope slide.</li>
	<li>Capture the differential interference contrast (DIC) and fluorescence microscopy images with a fluorescence microscope. Use a filter set up at the excitation and emission wavelengths of 593 nm and 636 nm (respectively).</li>
	<li>Use a software to count the total cell number (in the DIC mode) and the number of fluorescently stained cells. Also, use this software to determine the intensity of staining for individual cells.</li>
</ol>

The authors declare that they have no competing financial interests.

To successfully use the protocol described here, follow the preventive measures described below. Chloroform and methanol extract various substances from laboratory plasticware. Therefore, handle them with caution. Avoid the use of plastics in steps that involve contact with any of these two organic solvents. Use borosilicate glass pipettes for these steps. Rise these pipettes with chloroform and methanol before use. Use only micropipette tips and tubes made of polypropylene that is resistant to organic solvents. During s...

Water-soluble metabolites are low-molecular-weight intermediates and products of metabolism that contribute to essential cellular processes. These evolutionarily conserved processes include the conversion of nutrients into usable energy, synthesis of macromolecules, cellular growth and signaling, cell cycle control, regulation of gene expression, stress response, post-translational regulation of metabolism, maintenance of mitochondrial functionality, vesicular cellular trafficking, autophagy, cellular aging, and regulated cell death1,2,3.
Many of these essential roles of water-soluble metabolites have been discovered by studies in the budding yeast S. cerevisiae1,3,4,7,9,14,15,16,17,18,19,20,21,22. This unicellular eukaryote is a useful model organism for dissecting mechanisms through which water-soluble metabolites contribute to cellular processes due to its amenability to advanced biochemical, genetic, and molecular biological analyses23,24,25,26. Although the LC-MS/MS methods of non-targeted metabolomics have been used for studying the roles of water-soluble metabolites in budding yeast3,18,22,27, this type of analysis requires the improvement of its versatility, robustness, sensitivity, and ability to distinguish between different structural isomers and stereoisomeric forms of these metabolites.
Recent years are marked by significant advances in applying the LC-MS/MS methods of non-targeted metabolomics to the profiling of water-soluble metabolites in vivo. However, many challenges in using this methodology remain2,28,29,30,31,32,33,34,35,36. These challenges include the following. First, the intracellular concentrations of many water-soluble metabolites are below a threshold of sensitivity for the presently used methods. Second, the efficiency of metabolic activity quenching is too low, and the extent of quenching-associated cell leakage of intracellular metabolites is too high for current methods; hence, the presently used methods under-estimate the intracellular concentrations of water-soluble metabolites. Third, the existing methods cannot differentiate the structural isomers (i.e., molecules with the same chemical formula but different atomic connectivity) or stereoisomers (i.e., molecules with the same chemical formula and atomic connectivity, but with the different atomic arrangement in space) of specific metabolites; this prevents the correct annotation of certain metabolites by the presently used methods. Fourth, the existing mass spectral online databases of parent ions (MS1) and secondary ions (MS2) are incomplete; this affects the correct identification and quantitation of specific metabolites using the raw LC-MS/MS data produced with the help of the current methods. Fifth, the existing methods cannot use a single type of metabolite extraction to recover all or most classes of water-soluble metabolites. Sixth, the existing methods cannot use a single type of the LC column to separate from each other all or most classes of water-soluble metabolites.
Here, we optimized conditions for quenching of metabolic activity within S. cerevisiae cells, maintaining most of the water-soluble metabolites within these cells before extraction, and extracting most classes of water-soluble metabolites from yeast cells. We developed a versatile, robust, and sensitive method for the LC-MS/MS-based identification and quantification of more than 370 water-soluble metabolites extracted from S. cerevisiae cells. This method of non-targeted metabolomics enables to assess the intracellular concentrations of various energy carrier molecules, nucleotides, amino acids, monosaccharides, intermediates of glycolysis, and tricarboxylic cycle intermediates. The developed LC-MS/MS method permits the identification and quantification of different structural isomers and stereoisomeric forms of water-soluble metabolites with diverse structural, physical, and chemical properties.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Chemicals</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Fisher Scientific</td>
			<td>A9554</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium acetate</td>
			<td>Fisher Scientific</td>
			<td>A11450</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Sigma</td>
			<td>9830</td>
			<td></td>
		</tr>
		<tr>
			<td>Bactopeptone</td>
			<td>Fisher Scientific</td>
			<td>BP1420-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific</td>
			<td>C297-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Fisher Scientific</td>
			<td>D16-10</td>
			<td></td>
		</tr>
		<tr>
			<td>L-histidine</td>
			<td>Sigma</td>
			<td>H8125</td>
			<td></td>
		</tr>
		<tr>
			<td>L-leucine</td>
			<td>Sigma</td>
			<td>L8912</td>
			<td></td>
		</tr>
		<tr>
			<td>L-lysine</td>
			<td>Sigma</td>
			<td>L5501</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A4564</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A4564</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Thermo Scientific</td>
			<td>R37108</td>
			<td></td>
		</tr>
		<tr>
			<td>Uracil</td>
			<td>Sigma</td>
			<td>U0750</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Fisher Scientific</td>
			<td>BP1422-2</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Hardware equipment</td>
		</tr>
		<tr>
			<td>500 ml centrifuge bottles</td>
			<td>Beckman</td>
			<td>355664</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 1100 series LC system</td>
			<td>Agilent Technologies</td>
			<td>G1312A</td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman Coulter Centrifuge</td>
			<td>Beckman</td>
			<td>6254249</td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman Coulter Centrifuge Rotor</td>
			<td>Beckman</td>
			<td>JA-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Centra CL2 clinical centrifuge</td>
			<td>Thermo Scientific</td>
			<td>004260F</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital thermometer</td>
			<td>Omega</td>
			<td>HH509</td>
			<td></td>
		</tr>
		<tr>
			<td>Foam Tube Holder Kit with Retainer</td>
			<td>Thermo Scientific</td>
			<td>02-215-388</td>
			<td></td>
		</tr>
		<tr>
			<td>SeQuant ZIC-pHILIC zwitterionic-phase column (5&#181;m polymer 150 x 2.1 mm)</td>
			<td>Sigma Milipore</td>
			<td>150460</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Orbitrap Velos MS</td>
			<td>Fisher Scientific</td>
			<td>ETD-10600</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic sonicator</td>
			<td>Fisher Scientific</td>
			<td>15337416</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Fisher Scientific</td>
			<td>2215365</td>
			<td></td>
		</tr>
		<tr>
			<td>ZORBAX Bonus-RP, 80&#197;, 2.1 x 150 mm, 5 &#181;m</td>
			<td>Agilent Technologies</td>
			<td>883725-901</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Laboratory materials</td>
		</tr>
		<tr>
			<td>2-mL Glass sample vials with Teflon lined caps</td>
			<td>Fisher Scientific</td>
			<td>60180A-SV9-1P</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beads (acid-washed, 425-600 &#956;m)</td>
			<td>Sigma-Aldrich</td>
			<td>G8772</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Fisher Scientific</td>
			<td>267110</td>
			<td></td>
		</tr>
		<tr>
			<td>15-mL High-speed glass centrifuge tubes with Teflon lined caps</td>
			<td>PYREX</td>
			<td>05-550</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Software</td>
		</tr>
		<tr>
			<td>Compound Discoverer 3.1</td>
			<td>Fisher Scientific</td>
			<td>V3.1</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Yeast strain</td>
		</tr>
		<tr>
			<td>Yeast strain BY4742</td>
			<td>Dharmacon</td>
			<td>YSC1049</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative metabolomics of saccharomyces cerevisiae using liquid chromatography coupled with tandem mass spectrometry

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within a cell, tissue, organ, biological fluid, or organism. Metabolomics traditionally focuses on water-soluble metabolites. The water-soluble metabolome is the final product of a complex cellular network that integrates various genomic, epigenomic, transcriptomic, proteomic, and environmental factors. Hence, the metabolomic analysis directly assesses the outcome of the action for all these factors in a plethora of biological processes within various organisms. One of these organisms is the budding yeast Saccharomyces cerevisiae, a unicellular eukaryote with the fully sequenced genome. Because S. cerevisiae is amenable to comprehensive molecular analyses, it is used as a model for dissecting mechanisms underlying many biological processes within the eukaryotic cell. A versatile analytical method for the robust, sensitive, and accurate quantitative assessment of the water-soluble metabolome would provide the essential methodology for dissecting these mechanisms. Here we present a protocol for the optimized conditions of metabolic activity quenching in and water-soluble metabolite extraction from S. cerevisiae cells. The protocol also describes the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of the extracted water-soluble metabolites. The LC-MS/MS method of non-targeted metabolomics described here is versatile and robust. It enables the identification and quantification of more than 370 water-soluble metabolites with diverse structural, physical, and chemical properties, including different structural isomers and stereoisomeric forms of these metabolites. These metabolites include various energy carrier molecules, nucleotides, amino acids, monosaccharides, intermediates of glycolysis, and tricarboxylic cycle intermediates. The LC-MS/MS method of non-targeted metabolomics is sensitive and allows the identification and quantitation of some water-soluble metabolites at concentrations as low as 0.05 pmol/&#181;L. The method has been successfully used for assessing water-soluble metabolomes of wild-type and mutant yeast cells cultured under different conditions.

To improve a quantitative assessment of water-soluble metabolites within a yeast cell, we optimized the conditions of cell quenching for metabolite detection. Cell quenching for this purpose involves a rapid arrest of all enzymatic reactions within a cell31,33,37,38. Such an arrest of cellular metabolic activity is an essential step of any method for the quantit...

Watch this Scientific Journal Video about Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry at JoVE.com

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol for the identification and quantitation of major classes of water-soluble metabolites in the yeast Saccharomyces cerevisiae. The described method is versatile, robust, and sensitive. It allows the separation of structural isomers and stereoisomeric forms of water-soluble metabolites from each other.

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within ...

quantitative-metabolomics-saccharomyces-cerevisiae-using-liquid

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JoVE Journal

Biochemistry

4.2K Views.  Concordia University. We present a protocol for the identification and quantitation of major classes of water-soluble metabolites in the yeast Saccharomyces cerevisiae. The described method is versatile, robust, and sensitive. It allows the separation of structural isomers and stereoisomeric forms of water-soluble metabolites from each other.

Video: Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

A Tandem Liquid Chromatography&#8211;Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the abundances of key metabolites to which regulatory factors respond, adjusting cellular metabolism. In recent years, a number of proteins and RNA have emerged as important regulators of virulence gene expression. For example, the CodY protein responds to levels of branched-chain amino acids and GTP and is widely conserved in low G+C Gram-positive bacteria. As a global regulator in Staphylococcus aureus, CodY controls the expression of dozens of virulence and metabolic genes. We hypothesize that S. aureus uses CodY, in part, to alter its metabolic state in an effort to adapt to nutrient-limiting conditions potentially encountered in the host environment. This manuscript describes a method for extracting and analyzing metabolites from S. aureus using liquid chromatography coupled with mass spectrometry, a protocol that was developed to test this hypothesis. The method also highlights best practices that will ensure rigor and reproducibility, such as maintaining biological steady state and constant aeration without the use of continuous chemostat cultures. Relative to the USA200 methicillin-susceptible S. aureus isolate UAMS-1 parental strain, the isogenic codY mutant exhibited significant increases in amino acids derived from aspartate (e.g., threonine and isoleucine) and decreases in their precursors (e.g., aspartate and O-acetylhomoserine). These findings correlate well with transcriptional data obtained with RNA-seq analysis: genes in these pathways were up-regulated between 10- and 800-fold in the codY null mutant. Coupling global analyses of the transcriptome and the metabolome can reveal how bacteria alter their metabolism when faced with environmental or nutritional stress, providing potential insight into the physiological changes associated with nutrient depletion experienced during infection. Such discoveries may pave the way for the development of novel anti-infectives and therapeutics.

A Tandem Liquid Chromatography&amp;#8211;Mass Spectrometry-based Approach for Metabolite Analysis of &lt;em&gt;Staphylococcus aureus&lt;/em&gt;

Here we describe a protocol for the extraction of metabolites from Staphylococcus aureus and their subsequent analysis via liquid chromatography and mass spectrometry.

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the ...

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal transduction, metabolism, and development. These metabolites perform this regulatory activity by binding to proteins, thereby changing protein conformation, catalytic activity, and/or interactions. The method described here uses affinity chromatography coupled to mass spectrometry or Western blotting to identify proteins that interact with inositol phosphates or phosphoinositides. Inositol phosphates or phosphoinositides are chemically tagged with biotin, which is then captured via streptavidin conjugated to agarose or magnetic beads. Proteins are isolated by their affinity of binding to the metabolite, then eluted and identified by mass spectrometry or Western blotting. The method has a simple workflow that is sensitive, non-radioactive, liposome-free, and customizable, supporting the analysis of protein and metabolite interaction with precision. This approach can be used in label-free or in amino acid-labelled quantitative mass spectrometry methods to identify protein-metabolite interactions in complex biological samples or using purified proteins. This protocol is optimized for the analysis of proteins from Trypanosoma brucei, but it can be adapted to related protozoan parasites, yeast or mammalian cells.

This protocol focuses on the identification of proteins that bind to inositol phosphates or phosphoinositides. It uses affinity chromatography with biotinylated inositol phosphates or phosphoinositides that are immobilized via streptavidin to agarose or magnetic beads. Inositol phosphate or phosphoinositide binding proteins are identified by Western blotting or mass spectrometry.

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal ...

Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is going to move beyond the laboratory and become a widespread and standard just in time manufacturing technology, we must understand the performance limits of these systems. Toward this question, we developed a robust protocol to quantify 40 compounds involved in glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, energy metabolism and cofactor regeneration in CFPS reactions. The method uses internal standards tagged with 13C-aniline, while compounds in the sample are derivatized with 12C-aniline. The internal standards and sample were mixed and analyzed by reversed-phase liquid chromatography-mass spectrometry (LC/MS). The co-elution of compounds eliminated ion suppression, allowing the accurate quantification of metabolite concentrations over 2-3 orders of magnitude where the average correlation coefficient was 0.988. Five of the forty compounds were untagged with aniline, however, they were still detected in the CFPS sample and quantified with a standard curve method. The chromatographic run takes approximately 10 min to complete. Taken together, we developed a fast, robust method to separate and accurately quantify 40 compounds involved in CFPS in a single LC/MS run. The method is a comprehensive and accurate approach to characterize cell-free metabolism, so that ultimately, we can understand and improve the yield, productivity and energy efficiency of cell-free systems.

Here, we present a robust protocol to quantify 40 compounds involved in central carbon and energy metabolism in cell-free protein synthesis reactions. The cell-free synthesis mixture is derivatized with aniline for effective separation using reversed-phase liquid chromatography and then quantified by mass spectrometry using isotopically labelled internal standards.

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management of product yield and quality. Metabolomics data provides a read-out of these interactions at a given moment in time and is informative of an organism&#39;s biochemical status. Further, individual metabolites or panels of metabolites can be used as precise biomarkers for yield and quality prediction and management. The plant metabolome is predicted to contain thousands of small molecules with varied physicochemical properties that provide an opportunity for a biochemical insight into physiological traits and biomarker discovery. To exploit this, a key aim for metabolomics researchers is to capture as much of the physicochemical diversity as possible within a single analysis. Here we present a liquid chromatography-mass spectrometry-based untargeted metabolomics method for the analysis of field-grown wheat grain. The method uses the liquid chromatograph quaternary solvent manager to introduce a third mobile phase and combines a traditional reversed-phase gradient with a lipid-amenable gradient. Grain preparation, metabolite extraction, instrumental analysis and data processing workflows are described in detail. Good mass accuracy and signal reproducibility were observed, and the method yielded approximately 500 biologically relevant features per ionization mode. Further, significantly different metabolite and lipid feature signals between wheat varieties were determined.

A method for the untargeted analysis of wheat grain metabolites and lipids is presented. The protocol includes an acetonitrile metabolite extraction method and reversed phase liquid chromatography-mass spectrometry methodology, with acquisition in positive and negative electrospray ionization modes.

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management ...

Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan structure are fairly conserved across all bacteria, there is also considerable variation between Gram-positives/negatives and between species. In addition, there are numerous known variations, modifications, or adaptations to the peptidoglycan that can occur within a bacterial species in response to growth phase and/or environmental stimuli. These variations produce a highly dynamic structure that is known to participate in many cellular functions, including growth/division, antibiotic resistance, and host defense avoidance. To understand the variation within peptidoglycan, the overall structure must be broken down into its constitutive parts (known as muropeptides) and assessed for overall cellular composition. Peptidoglycomics uses advanced mass spectrometry combined with high-powered bioinformatic data analysis to examine peptidoglycan composition in fine detail. The following protocol describes the purification of peptidoglycan from bacterial cultures, the acquisition of muropeptide intensity data through a liquid chromatograph&#8212;mass spectrometer, and the differential analysis of peptidoglycan composition using bioinformatics.

This protocol covers a detailed analysis of peptidoglycan composition using liquid chromatography mass spectrometry coupled with advanced feature extraction and bioinformatic analysis software.

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan ...

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de novo&#160;from acetyl-CoA and are primarily present in eukaryotes in the form of triglycerides (TGs) and sterol-esters (SEs). The enzymes responsible for the synthesis of NLs are highly conserved from Saccharomyces cerevisiae (yeast) to humans, making yeast a useful model organism to dissect the function and regulation of NL metabolism enzymes. While much is known about how acetyl-CoA is converted into a diverse set of NL species, mechanisms for regulating NL metabolism enzymes, and how mis-regulation can contribute to cellular pathologies, are still being discovered. Numerous methods for the isolation and characterization of NL species have been developed and used over decades of research; however, a quantitative and simple protocol for the comprehensive characterization of major NL species has not been discussed. Here, a simple and adaptable method to quantify the de novo&#160;synthesis of major NL species in yeast is presented. We apply 14C-acetic acid metabolic labeling coupled with thin layer chromatography to separate and quantify a diverse range of physiologically important NLs. Additionally, this method can be easily applied to study in vivo reaction rates of NL enzymes or degradation of NL species over time.

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Here, a protocol is presented for the metabolic labeling of yeast with 14C-acetic acid, which is coupled with thin layer chromatography for the separation of neutral lipids.

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de ...