Name: semi-targeted ultra-high-performance chromatography coupled to mass spectrometry analysis of phenolic metabolites in plasma of elderly adults
Uploaded: 2022-04-22T14:30:00
Description: Watch this Scientific Journal Video about Semi-Targeted Ultra-High-Performance Chromatography Coupled to Mass Spectrometry Analysis of Phenolic Metabolites in Plasma of Elderly Adults at JoVE.com

The authors are grateful for the financial support from CONACYT, Mexico (CB- 2016-01-286449), and UACJ-PIVA (Projects 313-17-16 and 335-18-13). OAMB wishes to thank CONACYT for his Ph.D. scholarship. Technical support from the Multimedia Production office from UACJ is gratefully acknowledged.

The plasma samples used in this protocol were collected in a previous study following all the ethical guidelines and approved by the Institutional Ethics and Bioethics Committee (CIEB-2018-1-37) from the Universidad Aut&#243;noma de Ciudad Ju&#225;rez. The complete protocol for the extraction and identification of the phenolic compounds and metabolites in plasma by UPLC-MS/MS is represented in Figure 1.
<img alt="Figure 1...

<ol>
	<li>Morley, J. E., Anker, S. D., von Haehling, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence,+incidence,+and+clinical+impact+of+sarcopenia:+facts,+numbers,+and+epidemiology-update+2014.">Prevalence, incidence, and clinical impact of sarcopenia: facts, numbers, and epidemiology-update 2014.</a> Journal of Cachexia, Sarcopenia and Muscle. 5 (4), 253-259 (2014).</li><li>Cruz-Jentoft, A. J., Sayer, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sarcopenia.">Sarcopenia.</a> The Lancet. 393 (10191), 2636-2646 (2019).</li><li>Beaudart, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutrition+and+physical+activity+in+the+prevention+and+treatment+of+sarcopenia:+systematic+review.">Nutrition and physical activity in the prevention and treatment of sarcopenia: systematic review.</a> Osteoporosis International. 28 (6), 1817-1833 (2017).</li><li>Ozer, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenolic+compositions+and+antioxidant+activities+of+Maya+nut+(Brosimum+alicastrum):+Comparison+with+commercial+nuts.">Phenolic compositions and antioxidant activities of Maya nut (Brosimum alicastrum): Comparison with commercial nuts.</a> International Journal of Food Properties. 20 (11), 2772-2781 (2017).</li><li>Subiria-Cueto, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brosimum+alicastrum+Sw.+(Ram&#243;n):+An+alternative+to+improve+the+nutritional+properties+and+functional+potential+of+the+wheat+flour+tortilla.">Brosimum alicastrum Sw. (Ram&#243;n): An alternative to improve the nutritional properties and functional potential of the wheat flour tortilla.</a> Foods. 8 (12), 1-18 (2019).</li><li>Mart&#237;nez-Ruiz, N., Torres, L. E. J., del Hierro-Ochoa, J. C., Larqu&#233;-Saavedra, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bebida+adicionada+con+Brosimum+alicastrum+sw.:+Una+alternativa+para+requerimientos+dietarios+especiales.">Bebida adicionada con Brosimum alicastrum sw.: Una alternativa para requerimientos dietarios especiales.</a> Revista Salud P&#250;blica y Nutrici&#243;n. 18 (3), 1-10 (2019).</li><li>Rodr&#237;guez-Tadeo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functionality+of+bread+and+beverage+added+with+brosimum+alicastrum+sw.+Seed+flour+on+the+nutritional+and+health+status+of+the+elderly.">Functionality of bread and beverage added with brosimum alicastrum sw. Seed flour on the nutritional and health status of the elderly.</a> Foods. 10 (8), 1-21 (2021).</li><li>Mu&#241;oz-Bernal, &. #. 2. 1. 1. ;. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuevo+acercamiento+a+la+interacci&#243;n+del+reactivo+de+Folin-Ciocalteu+con+az&#250;cares+durante+la+cuantificaci&#243;n+de+polifenoles+totales.">Nuevo acercamiento a la interacci&#243;n del reactivo de Folin-Ciocalteu con az&#250;cares durante la cuantificaci&#243;n de polifenoles totales.</a> TIP Revista Especializada en Ciencias Qu&#237;mico-Biol&#243;gicas. 20 (2), 28-33 (2017).</li><li>Luca, S. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioactivity+of+dietary+polyphenols:+The+role+of+metabolites.">Bioactivity of dietary polyphenols: The role of metabolites.</a> Critical Reviews in Food Science and Nutrition. 60 (4), 626-659 (2020).</li><li>Kawabata, K., Yoshioka, Y., Terao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+intestinal+microbiota+in+the+bioavailability+and+physiological+functions+of+dietary+polyphenols.">Role of intestinal microbiota in the bioavailability and physiological functions of dietary polyphenols.</a> Molecules. 24 (2), (2019).</li><li>de Llano, D. G., Moreno-Arribas, M. V., Bartolom&#233;, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cranberry+polyphenols+and+prevention+against+urinary+tract+Infections:+Relevant+considerations.">Cranberry polyphenols and prevention against urinary tract Infections: Relevant considerations.</a> Molecules. 25 (15), (2020).</li><li>Alsaleh, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry:+A+guide+for+the+clinician.">Mass spectrometry: A guide for the clinician.</a> Journal of Clinical and Experimental Hepatology. 9 (5), 597-606 (2019).</li><li>Wang, X., Sun, H., Zhang, A., Wang, P., Han, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-performance+liquid+chromatography+coupled+to+mass+spectrometry+as+a+sensitive+and+powerful+technology+for+metabolomic+studies.">Ultra-performance liquid chromatography coupled to mass spectrometry as a sensitive and powerful technology for metabolomic studies.</a> Journal of Separation Science. 34 (24), 3451-3459 (2011).</li><li>Feliciano, R. P., Mills, C. E., Istas, G., Heiss, C., Rodriguez-Mateos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absorption,+metabolism+and+excretion+of+cranberry+(poly)phenols+in+humans:+A+dose+response+study+and+assessment+of+inter-individual+variability.">Absorption, metabolism and excretion of cranberry (poly)phenols in humans: A dose response study and assessment of inter-individual variability.</a> Nutrients. 9 (3), (2017).</li><li>Mateos, R., Goya, L., Bravo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+and+metabolism+of+hydroxycinnamic+acids+(chlorogenic,+caffeic,+and+ferulic+acids)+by+HepG2+cells+as+a+model+of+the+human+liver.">Uptake and metabolism of hydroxycinnamic acids (chlorogenic, caffeic, and ferulic acids) by HepG2 cells as a model of the human liver.</a> Journal of Agricultural and Food Chemistry. 54 (23), 8724-8732 (2006).</li><li>Rodriguez Lanzi, ., Perdicaro, C., Antoniolli, D. J., Piccoli, A., Vazquez Prieto, M. A., Fontana, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenolic+metabolites+in+plasma+and+tissues+of+rats+fed+with+a+grape+pomace+extract+as+assessed+by+liquid+chromatography-tandem+mass+spectrometry.">Phenolic metabolites in plasma and tissues of rats fed with a grape pomace extract as assessed by liquid chromatography-tandem mass spectrometry.</a> Archives of Biochemistry and Biophysics. , 28-33 (2018).</li><li>Hou, Y., He, D., Ye, L., Wang, G., Zheng, Q., Hao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+detection+and+identification+strategy+for+untargeted+metabolomics+based+on+UPLC-MS.">An improved detection and identification strategy for untargeted metabolomics based on UPLC-MS.</a> Journal of Pharmaceutical and Biomedical Analysis. 191, 113531 (2020).</li><li>Nagy, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+identification+of+dimethoxycinnamic+acids+in+human+plasma+after+coffee+intake+by+liquid+chromatography-mass+spectrometry.">First identification of dimethoxycinnamic acids in human plasma after coffee intake by liquid chromatography-mass spectrometry.</a> Journal of Chromatography A. 1218 (3), 491-497 (2011).</li><li>Marmet, C., Actis-Goretta, L., Renouf, M., Giuffrida, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+phenolic+acids+and+their+methylates,+glucuronides,+sulfates+and+lactones+metabolites+in+human+plasma+by+LC-MS/MS+after+oral+ingestion+of+soluble+coffee.">Quantification of phenolic acids and their methylates, glucuronides, sulfates and lactones metabolites in human plasma by LC-MS/MS after oral ingestion of soluble coffee.</a> Journal of Pharmaceutical and Biomedical Analysis. 88, 617-625 (2014).</li><li>McCord, J., Strynar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+per-and+polyfluorinated+chemical+species+with+a+combined+targeted+and+non-targeted-screening+high-resolution+mass+spectrometry+workflow.">Identifying per-and polyfluorinated chemical species with a combined targeted and non-targeted-screening high-resolution mass spectrometry workflow.</a> Journal of Visualized Experiments. 2019 (146), 1-15 (2019).</li><li>Mu&#241;oz-Bernal, &. #. 2. 1. 1. ;. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phytochemical+characterization+and+antiplatelet+activity+of+Mexican+red+wines+and+their+by-products.">Phytochemical characterization and antiplatelet activity of Mexican red wines and their by-products.</a> South African Journal of Enology and Viticulture. 42 (1), 77-90 (2021).</li><li>Mu&#241;oz-Bernal, &. #. 2. 1. 1. ;. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enriquecimiento+de+un+vino+tinto+con+un+extracto+de+compuestos+fen&#243;licos+provenientes+de+orujo+de+uva:+bioaccesibilidad,+an&#225;lisis+sensorial+y+respuesta+biol&#243;gica.">Enriquecimiento de un vino tinto con un extracto de compuestos fen&#243;licos provenientes de orujo de uva: bioaccesibilidad, an&#225;lisis sensorial y respuesta biol&#243;gica.</a> Universidad Aut&#243;noma de Ciudad Ju&#225;rez. , (2021).</li><li>Low, D. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Data+sharing+in+PredRet+for+accurate+prediction+of+retention+time:+Application+to+plant+food+bioactive+compounds.">Data sharing in PredRet for accurate prediction of retention time: Application to plant food bioactive compounds.</a> Food Chemistry. , 357 (2021).</li><li>S&#225;nchez-Pat&#225;n, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gut+microbial+catabolism+of+grape+seed+flavan-3-ols+by+human+faecal+microbiota.+Targeted+analysis+of+precursor+compounds,+intermediate+metabolites+and+end-products.">Gut microbial catabolism of grape seed flavan-3-ols by human faecal microbiota. Targeted analysis of precursor compounds, intermediate metabolites and end-products.</a> Food Chemistry. 131 (1), 337-347 (2012).</li><li>Zhang, X., Sandhu, A., Edirisinghe, I., Burton-Freeman, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+and+urinary+(poly)phenolic+profiles+after+4-week+red+raspberry+(Rubus+idaeus+L.)+intake+with+or+without+fructo-oligosaccharide+supplementation.">Plasma and urinary (poly)phenolic profiles after 4-week red raspberry (Rubus idaeus L.) intake with or without fructo-oligosaccharide supplementation.</a> Molecules. 25 (20), (2020).</li></ol>

The identification and quantification of the bioactive phytochemicals that are absorbed after consumption of a food or food supplement are crucial for demonstrating and understanding the health benefits of these compounds and the foods containing them. In the present work, the UPLC-MS/MS method was developed, aimed only at the identification of the main phenolic compounds and their metabolites that increased in concentration in plasma after a 30-day nutritional intervention with two food products specially formulated for...

Sarcopenia is a progressive skeletal disorder related to an accelerated loss of muscle in the elderly population. This condition increases the risk of falls and leads to limited activities of daily living. Sarcopenia is present in about 5%-10% of persons over 65 years old and about 50% of persons aged 80 years or older1. No specific drugs have been approved for the treatment of sarcopenia, so prevention with physical activity and a well-balanced diet is important1,2. Nutritional interventions with specially formulated foods enriched with dairy protein and essential amino acids have shown positive results in preventing sarcopenia2. In other studies, authors have included vitamins and antioxidants, like vitamin E and isoflavones, in the diet, increasing the benefits for muscle gain on the waist and hips3.
Brosimum alicastrum Sw. (Ram&#243;n) is a tree that grows in the Mexican tropical regions; it has been consumed by Mayan cultures due to its high nutritional value4. It is a good source of protein, fiber, minerals, and phenolic antioxidants, such as chlorogenic acid5. Since it can be ground into powder and used in baking products or consumed in beverages, recent studies have evaluated the incorporation of Ram&#243;n seed flour (RSF) into different foods to improve their nutritional value. An RSF-supplemented cappuccino-flavored beverage was formulated, which was high in dietary fiber and had more than 6 g of protein per serving, and was highly accepted by consumers; thus, it was considered a potential alternative for meeting special dietary requirements6. In a follow-up study, RSF was also used to formulate a muffin and a new beverage rich in protein, dietary fiber, micronutrients, and phenolic antioxidants. The muffin and beverage were used in a dietary intervention for elderly individuals, who consumed both products twice per day for 30 days. After this period, the nutritional and sarcopenic status of the participants improved, and the total phenolic content of plasma increased7. However, the determination of total phenolic compounds in plasma was carried out by a spectrophotometric method, so identification of the actual phenolic compounds that were absorbed was not possible; moreover, this method is not completely specific for phenolic compounds, so some overestimation may occur8.
Identification and quantification of the phenolic compounds that are absorbed after consumption of foods rich in these antioxidants is a difficult task but is necessary to demonstrate the biological activity of these phytochemicals. The bioavailability of most phenolic compounds is low; less than 5% of them can be found without structural transformation in plasma. Phenolic compounds undergo several biotransformations, such as methylation, sulfonation, or glucuronidation, which are carried out by enterocytes and hepatocytes9. Phenolic compounds are also biotransformed by the microbiota into bacterial catabolites that may exert their beneficial effects in the body after being absorbed into the plasma10. For example, phenylacetic acid is a product of the bacterial transformation of flavonoids and oligomeric proanthocyanidins, which can inhibit up to 40% of bacteria (Escherichia coli) adhesion in the urinary tract after cranberry consumption11.
The structural diversity of naturally occurring phenolic compounds, added to the diversity of their metabolites and their low bioavailability, makes their identification in plasma even more challenging. Metabolomic profiling, using spectroscopic analysis platforms like nuclear magnetic resonance (NMR) and tandem mass spectroscopy (MS/MS), is probably the best approach to achieve this goal; unfortunately, the equipment is not easily accessible, and the development of analysis protocols is still limited12. Several studies have reported MS/MS coupled with a separation system (such as liquid chromatography) as a strategy for reducing the complexity of mass spectra in metabolomic studies. The recent introduction of ultra-high-performance liquid chromatography (UPLC) separation methods has reduced the time of analysis and increased the resolution and sensitivity compared with conventional high-performance liquid protocols, so UPLC-MS/MS systems have rapidly been widely accepted by the analytical metabolomics community13. In this way, some studies have investigated phenolic metabolites and detected glucuronidated derivatives from caffeic acid, quercetin, and ferulic acid, as well as sulfonated derivatives from syringic and vanillic acid in the plasma of individuals after cranberry intake14. Previous protocols have intended to find phenolic compounds and phenolic metabolites in biofluids such as plasma. These protocols were based on identification and quantification by high-performance liquid chromatography (HPLC) coupled to a UV-vis detector15. Nevertheless, such protocols require the use of authentic standards to assess absolute identification and accurate quantification. A wide range of studies have identified the most common metabolites in biofluids (sulphonated, glucuronidated, and methylated forms) by UPLC-MS and UPLC-MS/MS; however, a large part of the bacterial metabolites has not been reported due to the lack of databases that contain their complete information16. Metabolite identification is complicated by the cost and commercial availability of metabolite standards. Therefore, the best strategy may be untargeted or semi-targeted MS/MS metabolite analysis, which relies on the use of molecular feature information (m/z, monoisotopic exact mass, isotopic distribution, and fragmentation pattern) to determine the chemical identity and compares it with freely available online databases that contain polyphenol metabolites identified in biofluids after the consumption of polypolyphenol-richts12. The most important databases used in UPLC-MS/MS studies for the identification of phenolic compounds and their metabolites are the Human Metabolome Database (HMDB), LipidBlast Library, METLIN Library, and other complementary databases, such as PubChem, ChemSpider, and Phenol Explorer17.
In the present study, a semi-targeted UPLC-MS/MS method was developed to analyze the plasma samples of the group of elderly persons involved in the RSF-containing muffin and beverage consumption study7. Data from different free online databases of plasma metabolites were collected and integrated into a specialized database. This database can be accessed automatically by the equipment software to identify the polyphenolic metabolites in the five plasma samples before and after the 30-day nutritional intervention. This is done to identify the main phenolic compounds, or their metabolites, that are absorbed from the specially formulated functional foods designed for the prevention of sarcopenia.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetonitrile</td>
			<td>Tedia</td>
			<td>Al1129-001</td>
			<td>LC Mass spectrometry</td>
		</tr>
		<tr>
			<td>Autosampler</td>
			<td>Agilent Technologies</td>
			<td>G4226A</td>
			<td>1290 Infinity series</td>
		</tr>
		<tr>
			<td>C18 reverse phase column</td>
			<td>Agilent Technologies</td>
			<td>959757-902</td>
			<td>Zorbax Eclipse plus C18 2.1x50 mm, 1.8 &#956;m; Rapid resolution HD</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5452000018</td>
			<td>Mini Spin; Rotor F-45-12-11</td>
		</tr>
		<tr>
			<td>Column compartment with thermostat</td>
			<td>Agilent Technologies</td>
			<td>G1316C</td>
			<td>1290 Infinity series</td>
		</tr>
		<tr>
			<td>Diode Array Detector (UV-Vis)</td>
			<td>Agilent Technologies</td>
			<td>G4212B</td>
			<td>1260 Infinity series</td>
		</tr>
		<tr>
			<td>Electrospray ionnization source</td>
			<td>Agilent Technologies</td>
			<td>G3251B</td>
			<td>Dual sprayer ESI source</td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>J.T. Baker</td>
			<td>0128-02</td>
			<td>Baker reagent, ACS</td>
		</tr>
		<tr>
			<td>Mass Hunter Data Acquisition</td>
			<td>Agilent Technologies</td>
			<td>G3338AA</td>
			<td></td>
		</tr>
		<tr>
			<td>Mass Hunter Personal Compound Datbase and Library Manager</td>
			<td>Agilent Technologies</td>
			<td>G3338AA</td>
			<td></td>
		</tr>
		<tr>
			<td>Mass Hunter Qualitative Analysis</td>
			<td>Agilent Technologies</td>
			<td>G3338AA</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tube</td>
			<td>Brand</td>
			<td>BR780546</td>
			<td>Microcentrifuge tube, 2 mL with lid</td>
		</tr>
		<tr>
			<td>Pure ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023-1L</td>
			<td>200 proof, for molecular biology</td>
		</tr>
		<tr>
			<td>Q-TOF LC/MS</td>
			<td>Agilent Technologies</td>
			<td>G6530B</td>
			<td>6530 Accurate Mass</td>
		</tr>
		<tr>
			<td>Quaternary pump</td>
			<td>Agilent Technologies</td>
			<td>G4204A</td>
			<td>1290 Infinity series</td>
		</tr>
		<tr>
			<td>Syringe filter</td>
			<td>Thermo Scientific</td>
			<td>44514-NN</td>
			<td>17 mm, 0.45 &#956;m, nylon membrane</td>
		</tr>
		<tr>
			<td>Thermostat</td>
			<td>Agilent Technologies</td>
			<td>G1330B</td>
			<td>1290 Infinity series</td>
		</tr>
		<tr>
			<td>Vial</td>
			<td>Agilent Technologies</td>
			<td>8010-0199</td>
			<td>Amber, PFTE red silicone 2 mL with screw top and blue caps</td>
		</tr>
		<tr>
			<td>Vial insert</td>
			<td>Agilent Technologies</td>
			<td>5183-2089</td>
			<td>Vial insert 200 &#956;L for 2mL standard opening, conical</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Tedia</td>
			<td>WL2212-001</td>
			<td>LC Mass spectrometry</td>
		</tr>
	</tbody>
</table>

semi-targeted ultra-high-performance chromatography coupled to mass spectrometry analysis of phenolic metabolites in plasma of elderly adults

A group of 23 elderly persons was given functional meals (a beverage and a muffin) specially formulated for the prevention of sarcopenia (age-related loss of muscle mass). Plasma samples were taken at the beginning of the intervention and after 30 days of consuming the functional meals. A semi-targeted ultra-high-performance chromatography coupled with tandem mass (UPLC-MS/MS) analysis was carried out to identify phenolic compounds and their metabolites. Plasma proteins were precipitated with ethanol and the samples were concentrated and resuspended in the mobile phase (1:1 acetonitrile: water) before injection into the UPLC-MS/MS instrument. Separation was carried out with a C18 reverse-phase column, and compounds were identified using their experimental mass, isotopic distribution, and fragment pattern. Compounds of interest were compared to those of data banks and the internal semi-targeted library. Preliminary results showed that the major metabolites identified after the intervention were phenylacetic acid, glycitin, 3-hydroxyphenylvaleric acid, and gomisin M2.

The step-by-step process for the identification of phenolic metabolites through the semi-targeted UPLC-MS/MS analysis, in negative mode, of plasma samples is depicted in Figure 2. First, the total ion chromatogram (TIC) from the plasma phenolics extract (obtained after protein precipitation of the total plasma sample) was obtained through the instrument&#39;s qualitative software. Then, the extracted ion chromatogram was used, and the exact mass and fragmentation patter...

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Semi-Targeted Ultra-High-Performance Chromatography Coupled to Mass Spectrometry Analysis of Phenolic Metabolites in Plasma of Elderly Adults

The goal of this protocol is to detect phenolic metabolites in plasma using a semi-targeted chromatography-mass spectrometry method.

A group of 23 elderly persons was given functional meals (a beverage and a muffin) specially formulated for the prevention of sarcopenia (age-related loss ...

The goal of this protocol is to detect phenolic metabolites in plasma by a semi-targeted UPLC-MS/MS method. Plasma proteins were precipitated with ethanol and samples concentrated, and re-suspended in the mobile phase, prior to injection in the UPLC equipment. Compounds were identified using a semi targeted library assembled in the lab, using PCDL manager for metabolomics, software and different free online databases.We implemented a nutritional intervention with a muffin and beverage formulate with brosimum alicastrum seed flour. At the end of the intervention, the nutritional and sarcopenic status of the participants were improved, and the total phenolic content of plasma was increased. But we need to identify which phenolic compounds had been absorbed and contributed the positive effect.Identification and quantification of the phenolic compounds that are absorbed after consumption of foods rich in these antioxidants is difficult task but necessary, if one is to demonstrate biological activity of these phytochemicals. The bioavailability of most phenolic components is low, moreover less than 5%of them enter without structural transformation into plasma. Most go through several biotransformation, such as methylation, sulfonation, or glucuronidation.A wide range of studies identified the most common metabolites in biofluids by UPLC-MS and UPLC-MS/MS. However, a large part of the bacteria metabolite is not reported to the lack of databases that contain their complete information. Metabolite identification is complicated by the cost and commercial availability of metabolite standards.Therefore, the best strategy may be untargeted or semi-targeted MS/MS metabolite analysis. This analysis rely on the use of molecular feature information, mass to charge ratio monoisotopic exact mass, isotopic distribution, and fragmentation pattern. To determine the chemical identity and compare it with free available online databases that contain polyphenol metabolites identified in biofluids after consumption of polyphenol rich diets.In the present study, we have developed a semi-targeted UPLC-MS/MS method to analyze the plasma samples of a group of elderly persons involved in a nutritional intervention. Store plasma samples at minus 80 degrees Celsius until analysis. Defrost plasma samples at room temperature for 15 minutes or at 37 Celsius for five minutes.Place 200 microliters of plasma sample in a two millimeter microtube and mix with 1000 microliters of pure ethanol. Vortex plasma sample for 30 seconds. Centrifuge sample at 6, 580 centrifugal force for five minutes.After centrifugation, collect the supernatant with a micropipette or Pasteur pipette and place it in a new microtube. Reserve the supernatant. Mix a pellet with 1000 microliters pure ethanol, vertex for 30 seconds, and then centrifuge at the same conditions.Collect the supernatant and mix with the first supernatant. Remove ethanol from the sample by using pure nitrogen at 135 psi. Keep the needle one centimeter away from the top of the microtube to prevent sample loss and flush until a sample is dry.Resuspend dry samples in 100 microliters of a one is to one mixture of acetonitrile with water. Filter through a 45 micro nylon syringe membrane directly into an HPLC vial micro insert Inject three microliters of sample onto UPLC equipped with a C 18 reverse-phase column. Set the autosampler temperature at 20 Celsius and the column thermostat at 25 Celsius.Inject each sample in triplicate. Use 0.1%formic acid in water as solvent A, and 100%acetonitrile as solvent B.Set the flow rate at 0.4 millimeters per minute and a gradient program as follows, zero to one minute 10%B, one to four minute 30%B, four to six minutes 38%B, six to eight minutes 60%B, eight to 8.5 minutes 60%B, 8.5 to nine minutes 10%B. Set the mass spectrometer in negative mode ionization.Use nitrogen as drying gas at 300 Celsius and flow rate at 13 liters per minute. Set the nebulizer pressure at 30 psi. Set a capillary voltage at 4, 000 volts, fragmentor voltage at 175 volts, and Skimmer voltage at 65 volts.Scan the masses between 100 and 1100 m/z and for the mass spectrometry, scan masses between 50 to 1000 m/z. Set data acquisition into Auto MS/MS mode. Use the following reference mass, 119.036 and 966.0007.Search for phenolic compounds, phenolic metabolites and other compounds of interest in scientific literature. Open the database management software included in the UPLC system. Select file, then select new personal database compound library, PCDL.Then select, create new PCDL. Select the type of PCDL LC/MS metabolomics. Set the name for PCDL.Then select create. In the toolbar, select PCDL and then the allow editing option. Then click the find compounds button.Compounds can be added to the personal library two ways, first by copying them from the instrument's general library. To do this, open the instruments existing database included in the data management so far. Click the button find compounds.In the single search option, enter the compound search criteria to find the compound of interest. In the compound results table, select the compound of interest. To select more than one compound, click the first compound, then hold down the CTRL key, and then click each compound of interest.Then, right click on all the highlighted compounds and select Append to PCDL. In the new window, search and select the specialized personal database file. Mark the box include spectra for compound if present.Click the Append button. In the new dialog box, select Yes to check the new compounds added. Select No to keep searching for more compounds of interest.Second, new compounds can be added manually if they are not available in the instrument's general library. For this open, the specialized personal database. Once opened, select PCDL, and then the allow editing option.Select the edit compounds option. Click the add new button. In the upper section of the window, complete the information of the new compound.Fill in the formula, name, IUPAC name, CAS number, Chemspider ID, and other identifiers. Using information available in the free online libraries, Chemspider, PubChem, And Phenol Explorer to fill in the information of the new compound of interest. Once finished, click the button save as new to save the new compound information in the specialized personal database.Repeat the process with all the compounds of interest to complete the specialized personal database. Use the instrument's qualitative manager software to identify phenolic compounds and phenolic metabolites. Open the sample file.In the Chromatogram panel, select define Chromatograms and extract the total ion chromatogram TIC, extracted ion-chromatogram of MS EIC and EIC of MS/MS. Select the integrate chromatogram option. In the find compounds panel, select find by formula options.In the new window, select formula source and then the database slash library option. Find the personal database previously created and click on open. Select the formula matching option and set a mass match tolerance of five parts per million, ppm.Select the negative ions option and select only the minus H dialog box. In results option, mark the extract EIC, extract cleaned spectrum, extract raw spectrum, and include structured dialog boxes. Select the results filters option.And then mark warn if score is, then set the score match at 80%Then mark do not match if the score is and set the score at 75%Then click on the find compounds by formula to identify compounds of interest in the sample. The step by step process for the identification of phenolic metabolites through the semi-targeted UPLC MS/MS analysis of plasma samples is shown in the next figure. First, the total ion-chromatogram was obtained using a self-created PCDL database that contained 645 phenolic compounds and their metabolites, obtained from free online databases.Next, the extracted ion-chromatogram and fragmentation pattern, or the ions with less than five ppm mass, match tolerance were obtained. Finally, the isotopic distribution of the detected peaks was compared to that of the theoretical compounds. From this analysis, a total of 25 phenolic compounds and metabolites were identified in the plasma samples.To evaluate the effectiveness of the design method in the identification of the absorb phenolic compounds or their metabolites, five samples of the study participants obtained prior and after the 30 day intervention were analyzed. The relative abundance of each compound was calculated by dividing the area under the curve after treatment by AUC before treatment. Table four shows the list of 12 phenol compounds that showed an increase in plasma after the 30 day consumption of the brosimum alicastrum flour containing foods.Phenylacetic acid was only metabolite found consistently in higher concentration after the treatment. Glycitin, a glycosylated isoflavone, and three hydroxyphenylvaleric acid increased in three of the five samples, but decreased in the other two. Gomisin M2, a lignan, was detected in three of the five samples only after the nutritional intervention.The other phenolic compounds and metabolites were found only in one sample and only after the treatment. The method developed present paper, shows that in most parts it was well suited for the objective for which it was created. The sample per treatment was simple and effective.UPLC separation and semi-targeted MS/MS identification of phenolic metabolites were achieved.

semi-targeted-ultra-high-performance-chromatography-coupled-to-mass

Research

JoVE Journal

Biochemistry

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 ...

Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing plant metabolites because it provides molecular weights with high sensitivity and specificity. Leaf spray MS is an ambient ionization technique where plant tissue is used for direct chemical analysis via electrospray, eliminating chromatography from the process. This approach to sampling metabolites allows for a wide range of chemical classes to be detected simultaneously from intact plant tissues, minimizing the amount of sample preparation needed. When used with a high-resolution, accurate mass MS, leaf spray MS facilitates the rapid detection of metabolites of interest. It is also possible to collect tandem mass fragmentation data with this technique to facilitate a compound identification. The combination of accurate mass measurements and fragmentation is beneficial in confirming compound identities. The leaf spray MS technique requires only minor modifications to a nanospray ionization source and is a useful tool to further expand the capabilities of a mass spectrometer. Here, fresh leaf tissue from Sceletium tortuosum (Aizoaceae), a traditional medicinal plant from South Africa, is analyzed; numerous mesembrine alkaloids are detected with leaf spray MS.

Leaf spray mass spectrometry is a direct chemical analysis technique that minimizes the sample preparation and eliminates chromatography, allowing for the rapid detection of small molecules from plant tissues.

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing ...

A Plasma Sample Preparation for Mass Spectrometry using an Automated Workstation

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves numerous incubation and liquid transfer steps in order to achieve denaturation, reduction, alkylation, and cleavage. Adapting this workflow onto an automated workstation can increase efficiency and reduce coefficients of variance, thereby providing more reliable data for statistical comparisons between sample types. We previously described an automated proteomic sample preparation workflow1. Here, we report the development of a more efficient and better controlled workflow with the following advantages: 1) The number of liquid transfer steps is reduced from nine to six by combining reagents; 2) Pipetting time is reduced by selective tip pipetting using a 96-position pipetting head with multiple channels; 3) Potential throughput is increased by the availability of up to 45 deck positions; 4) Complete enclosure of the system provides improved temperature and environmental control and reduces the potential for contamination of samples or reagents; and 5) The addition of stable isotope labeled peptides, as well as &#946;-galactosidase protein, to each sample makes monitoring and quality control possible throughout the entire process. These hardware and process improvements provide good reproducibility and improve intra-assay and inter-assay precision (CV of less than 20%) for LC-MS based protein and peptide quantification. The entire workflow for digesting 96 samples in a 96-well plate can be completed in approximately 5 hours.

Here, we present an automated plasma protein digestion method for mass spectrometry-based quantitative proteomic analysis. In this protocol, the liquid transferring and incubation steps for protein denaturation, reduction, alkylation, and trypsin digestion reactions are streamlined and automated. It takes approximately five hours to prepare a 96-well plate with desired precision.

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves ...

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 ...