We are grateful to current and former members of the Titorenko laboratory for discussions. We acknowledge the Centre for Biological Applications of Mass Spectrometry and the Centre for Structural and Functional Genomics (both 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 Concordia University Chair Fund (CC0113). K.M. was supported by the Concordia University Merit Award.

1. Preparation of sterile media for culturing yeast
<ol>
	<li>Prepare 90 mL of a complete YP medium that contains 1% (w/v) yeast extract and 2% (w/v) bactopeptone.</li>
	<li>Prepare 90 mL of a synthetic minimal YNB medium containing 0.67% (w/v) yeast nitrogen base without amino acids, 20 mg/L L-histidine, 30 mg/L L-leucine, 30 mg/L L-lysine, and 20 mg/L uracil.</li>
	<li>Divide 90 mL of the complete YP medium equally into two 250 mL Erlenmeyer flasks (i.e., 45 mL each).</li>
	<li>Divide 90 mL of the synthetic minimal YNB medium into two 250 mL Erlenmeyer flasks (i.e., 45 mL each).</li>
	<li>Autoclave the flasks with YP and YNB media at 15 psi/121 &#176;C for 45 min prior to use.</li>
</ol>
2. Yeast strain
<ol>
	<li>Use the wild type strain BY4742 (MAT&#945; his3&#916;1 leu2&#916;0 lys2&#916;0 ura3&#916;0).</li>
</ol>
3. Culturing yeast in the complete YP medium with glucose
<ol>
	<li>Autoclave a 20% (w/v) stock solution of glucose at 15 psi/121 &#176;C for 45 min prior to use.</li>
	<li>Add 5 mL of the sterile 20% (w/v) stock solution of glucose to each of the two Erlenmeyer flasks containing the sterile YP medium for a final concentration of 2% glucose (w/v).</li>
	<li>Use a microbiological loop to inoculate cells of the wild type strain BY4742 into each of the two Erlenmeyer flasks containing the YP medium with glucose.</li>
	<li>Culture the cells overnight at 30 &#176;C with rotational shaking at 200 rpm.</li>
	<li>Take an aliquot of yeast culture. Use a hemocytometer to determine the total number of yeast cells per mL of culture.</li>
</ol>
4. Transferring yeast to and culturing them in the synthetic minimal YNB medium with glucose
<ol>
	<li>Add 5 mL of the sterile 20% (w/v) stock solution of glucose to each of the two Erlenmeyer flasks containing the sterile YNB medium to a final concentration of 2% glucose (w/v).</li>
	<li>Use a sterile pipette to transfer a volume of the overnight yeast culture that contains the total number of 5.0 x 107 cells into each of the two Erlenmeyer flasks containing the YNB medium with glucose.</li>
	<li>Culture the cells for at least 24 h (or more, if the experiment requires) at 30 &#176;C with rotational shaking at 200 rpm.</li>
</ol>
5. Preparation of reagents, labware and equipment for lipid extraction
<ol>
	<li>Prepare the following: 1) high grade (&#62;99.9%) chloroform; 2) high grade (&#62;99.9%) methanol; 3) 28% (v/v) ammonium hydroxide solution in nano-pure water; 4) glass beads (acid-washed, 425-600 &#181;M); 5) a vortex with appropriate adapter; 6) 15 mL high-speed glass centrifuge tubes with polytetrafluoroethylene lined caps; 7) 17:1 and 2:1 mixtures of chloroform and methanol; 8) a chloroform/methanol (2:1) mixture with 0.1% ammonium hydroxide (v/v); 9) ABC solution (155 mM ammonium bicarbonate, pH = 8.0); 10) a mixture of internal lipid standards prepared in a 2:1 mixture of chloroform and methanol as indicated in Table 1; and 11) 2 mL glass sample vials with polytetrafluoroethylene lined caps for the extraction of cellular lipids.</li>
</ol>
6. Preparation of reagents, labware, and equipment for LC
<ol>
	<li>Prepare the following: 1) acetonitrile/2-propanol/nano-pure water (65:35:5) mixture; 2) a vortex with appropriate adapter; 3) an ultrasonic sonicator; 4) glass vials with inserts for a wellplate; 5) an LC system equipped with a binary pump, degasser, and an autosampler; 6) a C18 reverse-phase column (2.1 mm; 75 mm; pore size 130 &#197;; pH range of 1-11) coupled to a pre-column system; 7) mixture A: acetonitrile/water (60:40); and 8) mixture B: isopropanol/acetonitrile (90:10).</li>
</ol>
7. Lipid extraction from yeast cells
<ol>
	<li>Take an aliquot of yeast culture. Use a hemocytometer or measure OD600 to determine the total number of yeast cells per mL of culture.</li>
	<li>Take a volume of yeast culture that contains a total number of 5.0 x 107 cells (3.3 units OD600). Place this volume of culture into a prechilled 1.5 mL microcentrifuge tube.</li>
	<li>Harvest the cells by centrifugation at 16,000 x g for 1 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Add 1.5 mL of ice-cold nano-pure water and wash the cells by centrifugation at 16,000 x g for 1 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Add 1.5 mL of ice-cold ABC solution and wash the cells by centrifugation at 16,000 x g for 1 min at 4 &#176;C. Discard the supernatant. The cell pellet can be stored at -80 &#176;C prior to lipid extraction.</li>
	<li>To begin the lipid extraction, thaw the cell pellet on ice.</li>
	<li>Resuspend the cell pellet in 200 &#181;L of ice-cold nano-pure water. Transfer the cell suspension to a 15 mL high-strength glass screw top centrifuge tube with a polytetrafluoroethylene lined cap. Add the following to this tube: 1) 25 &#181;L of the mixture of internal lipid standards prepared in chloroform/methanol (2:1) mixture; 2) 100 &#181;L of 425-600 &#181;M acid-washed glass beads; and 3) 600 &#181;L of chloroform/methanol (17:1) mixture.</li>
	<li>Vortex the tube at high speed for 5 min at room temperature (RT) to disrupt the cells.</li>
	<li>Vortex the tube at low speed for 1 h at RT to facilitate the extraction of lipids.</li>
	<li>Incubate the sample for 15 min on ice to promote protein precipitation and the separation of the aqueous and organic phases from each other.</li>
	<li>Centrifuge the tube in a clinical centrifuge at 3,000 x g for 5 min at RT. This centrifugation step allows to separate the upper aqueous phase from the lower organic phase, which contains all lipid classes.</li>
	<li>Use a borosilicate glass pipette to transfer the lower organic phase (~400 &#181;L) to another 15 mL high-strength glass screw top centrifuge tube with a polytetrafluoroethylene lined cap. Do not disrupt the glass beads or upper aqueous phase during such transfer. Keep the lower organic phase under the flow of nitrogen gas.</li>
	<li>Add 300 &#181;L of chloroform-methanol (2:1) mixture to the remaining upper aqueous phase to allow the extraction of sphingolipids and PA, PS, PI, and CL. Vortex the tube vigorously for 5 min at RT.</li>
	<li>Centrifuge the tube in a clinical centrifuge at 3,000 x g for 5 min at RT.</li>
	<li>Use a borosilicate glass pipette to transfer the lower organic phase (~ 200 &#181;L) formed after centrifugation to the organic phase collected at step 7.13.</li>
	<li>Use the flow of nitrogen gas to evaporate the solvent in the combined organic phases. Close the tubes containing the lipid film under the flow of nitrogen gas. Store these tubes at -80 &#176;C.</li>
</ol>
8. Separation of extracted lipids by LC
<ol>
	<li>Add 500 &#181;L of acetonitrile/2-propanol/nano-pure water (65:35:5) mixture to a tube containing the lipid film obtained at step 7.16. Vortex the tube 3x for 10 s at RT.</li>
	<li>Subject the content of the tube to ultrasonic sonication for 15 min. Vortex the tube 3x for 10 s at RT.</li>
	<li>Take 100 &#181;L of a sample from the tube and add it to a glass vial with an insert used for a wellplate. Eliminate air bubbles in the insert before pacing it into the wellplate.</li>
	<li>Use an LC system to separate different lipid species on a reverse-phase C18 column CSH coupled to a pre-column system (see Table of Materials). During the separation, maintain the column at 55 &#176;C and at a flow rate of 0.3 mL/min. Keep the sample in the wellplate at RT.</li>
	<li>Use the mobile phases that consist of mixture A (acetonitrile/water [60:40 (v/v)]) and mixture B (isopropanol/acetonitrile [90:10 (v/v)]). For a positive mode of the detection of parent ions created using the electrospray ionization (ESI) ion source, the ESI (+) mode, the mobile phases A and B contain ammonium formate at the final concentration of 10 mM. For a negative mode of parent ions detection, the ESI (-) mode, the mobile phases A and B contain ammonium acetate at the final concentration of 10 mM.</li>
	<li>Use a sample volume of 10 &#181;L for the injection into both the ESI (+) and ESI (-) mode.</li>
	<li>Separate different lipid species by LC using the following LC gradient: 0-1 min 10% (phase B); 1-4 min 60% (phase B); 4-10 min 68% (phase B); 10-21 97% (phase B); 21-24 min 97% (phase B); 24-33 min 10% (phase B).</li>
	<li>Run extraction blanks as the first sample, between every four samples, and as the last sample. Subtract the background to normalize data.</li>
	<li>A representative total ion chromatogram from LC/MS data of lipids extracted from cells of the wild type strain BY4742 is shown in Figure 1.</li>
</ol>
9. Mass spectrometric analysis of lipids separated by LC
<ol>
	<li>Use a mass spectrometer equipped with a HESI (heated electrospray ionization) ion source to analyze lipids that were separated by LC. Use the settings provided in Table 2.</li>
	<li>Use the Fourier transform analyzer to detect parent ions (MS1) at a resolution of 60,000 and within the mass range of 150-2,000 Da.</li>
	<li>Use the settings provided in Table 3 to detect secondary ions (MS2).</li>
</ol>
10. Identification and quantitation of different lipid classes and species by processing of raw data from LC-MS/MS
<ol>
	<li>See the Table of Materials for software to carry out the identification and quantitation of different lipids from raw LC-MS/MS files. This software uses the largest lipid database, containing more than 1.5 million lipid ion precursors (MS1) and their predicted fragment ions (MS2). The software also uses MS1 peaks for lipid quantitation and MS2 for lipid identification. A representative chromatogram of two isomeric phosphatidylserine forms (34:0) that have the same m/z value but different retention times, as well as their MS1 and MS2 spectra, are shown in Figure 2, Figure 3, and Figure 4, respectively.</li>
	<li>Search LC-MS raw files containing full-scan MS1 data and data-dependent MS2 data for free (unesterified) fatty acids (FFA), cardiolipin (CL), phytoceramide (PHC), phytosphingosine (PHS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), and triacylglycerol (TAG) lipid classes using an m/z tolerance of 5 ppm for precursor ions and 10 ppm for product ions. Other search parameters are shown in Table 4. Follow the instructions provided in the software user manual. The identities of internal lipid standards and lipid species with unusual fatty acid composition need to be verified manually.</li>
	<li>To identify and quantitate different lipid classes and species with the help of freely available open-source alternatives for the Lipid Search software, use the Lipid Data Analyzer (<a href="http://genome.tugraz.at/lda2/lda_download.shtml" target="_blank">http://genome.tugraz.at/lda2/lda_download.shtml</a>), MZmine 2 (<a href="http://mzmine.github.io/" target="_blank">http://mzmine.github.io/</a>), or XCMS (<a href="https://bioconductor.org/packages/release/bioc/html/xcms.html" target="_blank">https://bioconductor.org/packages/release/bioc/html/xcms.html</a>) software to process raw data from LC-MS/MS.</li>
</ol>

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The authors declare that they have no competing financial interests.

The following precautions are important for the successful implementation of the protocol described here:
1. Chloroform and methanol are toxic. They efficiently extract various substances from surfaces, including laboratory plasticware and your skin. Therefore, handle these organic solvents with caution by avoiding the use of plastics in steps that involve contact with chloroform and/or methanol, using borosilicate glass pipettes for these steps, and rinsing these pipettes with chloroform and ...

A body of evidence indicates that lipids, one of the major classes of biomolecules, play essential roles in many vital processes within a eukaryotic cell. These processes include the assembly of lipid bilayers that constitute the plasma membrane and membranes surrounding cellular organelles, transport of small molecules across cell membranes, response to changes in the extracellular environment and intracellular signal transduction, generation and storage of energy, import and export of proteins confined to different organelles, vesicular trafficking of proteins within the endomembrane system and protein secretion, and several modes of regulated cell death1,2,3,4,5,6,7,8,9,10.
The budding yeast S. cerevisiae, a unicellular eukaryotic organism, has been successfully used to uncover some of the mechanisms underlying the essential roles of lipids in these vital cellular processes4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20. S. cerevisiae is a valuable model organism for uncovering these mechanisms because it is amenable to comprehensive biochemical, genetic, cell biological, chemical biological, system biological, and microfluidic dissection analyses21,22,23,24,25. Further progress in understanding mechanisms through which lipid metabolism and intracellular transport contribute to these vital cellular processes requires sensitive mass spectrometry technologies for the quantitative characterization of the cellular lipidome, understanding the lipidome molecular complexity, and integrating quantitative lipidomics into a multidisciplinary platform of systems biology1,2,3,26,27,28,29,30.
Current methods for the mass spectrometry-assisted quantitative lipidomics of yeast cells and cells of other eukaryotic organisms are not sufficiently versatile, robust, or sensitive. Moreover, these currently used methods are unable to differentiate various isobaric or isomeric lipid species from each other. Here we describe a versatile, robust, and sensitive method that allows use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for quantitative analysis of major cellular lipids of S. cerevisiae.

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	<tbody>
		<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>2 mL Glass sample vials with Teflon lined caps</td>
			<td>Fisher Scientific</td>
			<td>60180A-SV9-1P</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Fisher Scientific</td>
			<td>A461-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Fisher Scientific</td>
			<td>A9554</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 1100 series LC system</td>
			<td>Agilent Technologies</td>
			<td>G1312A</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent1100 Wellplate</td>
			<td>Agilent Technologies</td>
			<td>G1367A</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>Ammonium formate</td>
			<td>Fisher Scientific</td>
			<td>A11550</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium hydroxide</td>
			<td>Fisher Scientific</td>
			<td>A470-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Bactopeptone</td>
			<td>Fisher Scientific</td>
			<td>BP1420-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Cardiolipin</td>
			<td>Avanti Polar Lipids</td>
			<td>750332</td>
			<td></td>
		</tr>
		<tr>
			<td>Centra CL2 clinical centrifuge</td>
			<td>Thermo Scientific</td>
			<td>004260F</td>
			<td></td>
		</tr>
		<tr>
			<td>Ceramide</td>
			<td>Avanti Polar Lipids</td>
			<td>860517</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific</td>
			<td>C297-4</td>
			<td></td>
		</tr>
		<tr>
			<td>CSH C18 VanGuard</td>
			<td>Waters</td>
			<td>186006944</td>
			<td>Pre-column system</td>
		</tr>
		<tr>
			<td>Free fatty acid (19:0)</td>
			<td>Matreya</td>
			<td>1028</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>Glucose</td>
			<td>Fisher Scientific</td>
			<td>D16-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Fisher Scientific</td>
			<td>267110</td>
			<td></td>
		</tr>
		<tr>
			<td>L-histidine</td>
			<td>Sigma</td>
			<td>H8125</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipid Search software (V4.1)</td>
			<td>Fisher Scientific</td>
			<td>V4.1</td>
			<td>LC-MS/MS analysis software</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>Phosphatidylcholine</td>
			<td>Avanti Polar Lipids</td>
			<td>850340</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatidylethanolamine</td>
			<td>Avanti Polar Lipids</td>
			<td>850704</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatidylglycerol</td>
			<td>Avanti Polar Lipids</td>
			<td>840446</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatidylinositol</td>
			<td>Avanti Polar Lipids</td>
			<td>LM1502</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatidylserine</td>
			<td>Avanti Polar Lipids</td>
			<td>840028</td>
			<td></td>
		</tr>
		<tr>
			<td>Reverse-phase column CSH C18</td>
			<td>Waters</td>
			<td>186006102</td>
			<td></td>
		</tr>
		<tr>
			<td>Sphingosine</td>
			<td>Avanti Polar Lipids</td>
			<td>860669</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Orbitrap Velos MS</td>
			<td>Fisher Scientific</td>
			<td>ETD-10600</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricylglycerol</td>
			<td>Larodan, Malmo</td>
			<td>TAG Mixed FA</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic sonicator</td>
			<td>Fisher Scientific</td>
			<td>15337416</td>
			<td></td>
		</tr>
		<tr>
			<td>Uracil</td>
			<td>Sigma</td>
			<td>U0750</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Fisher Scientific</td>
			<td>2215365</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Fisher Scientific</td>
			<td>BP1422-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast nitrogen base without amino acids</td>
			<td>Fisher Scientific</td>
			<td>DF0919-15-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast strain BY4742</td>
			<td>Dharmacon</td>
			<td>YSC1049</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Our method for a quantitative assessment of major cellular lipids within a yeast cell with the help of LC-MS/MS was versatile and robust. It allowed us to identify and quantify 10 different lipid classes in S. cerevisiae cells cultured in the synthetic minimal YNB medium initially containing 2% glucose. These lipid classes include free (unesterified) fatty acids (FFA), CL, phytoceramide (PHC), phytosphingosine (PHS), PC, PE, PG, PI, PS, and TAG (Supplemental Table 1</stro...

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

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.

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 ...

quantitative-analysis-cellular-lipidome-saccharomyces-cerevisiae

Research

JoVE Journal

Biochemistry

7.2K Views.  Concordia University. 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.

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

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

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 ...

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 ...

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.

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.

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