Analysis of amino acids without derivatization in barley extracts by LC-MS-MS

Thiele, Björn; Füllner, K.; Stein, Nadine; Oldiges, Marco; Kuhn, Arnd J.; Hofmann, Diana

doi:10.1007/s00216-008-2167-9

Cited by 101 publications

(70 citation statements)

References 35 publications

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“…Other lower abundance amino acids such as Val and Ile were labeled more slowly. These results are in general agreement with prior data from metabolic labeling of barley plants (Thiele et al, 2008) and revealed that 15% to 30% of many of the major amino acid pools were 15 N labeled in leaves after approximately 10 h of labeling. Alternatively, when the 15 N label was applied 2.5 h after dark (Fig.…”

Section: Labeling Of Amino Acid Pools In Barley Leavessupporting

confidence: 91%

Proteins with High Turnover Rate in Barley Leaves Estimated by Proteome Analysis Combined with in Planta Isotope Labeling

et al. 2014

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Protein turnover is a key component in cellular homeostasis; however, there is little quantitative information on degradation kinetics for individual plant proteins. We have used 15 N labeling of barley (Hordeum vulgare) plants and gas chromatography-mass spectrometry analysis of free amino acids and liquid chromatography-mass spectrometry analysis of proteins to track the enrichment of 15 N into the amino acid pools in barley leaves and then into tryptic peptides derived from newly synthesized proteins. Using information on the rate of growth of barley leaves combined with the rate of degradation of 14 N-labeled proteins, we calculate the turnover rates of 508 different proteins in barley and show that they vary by more than 100-fold. There was approximately a 9-h lag from label application until 15 N incorporation could be reliably quantified in extracted peptides. Using this information and assuming constant translation rates for proteins during the time course, we were able to quantify degradation rates for several proteins that exhibit half-lives on the order of hours. Our workflow, involving a stringent series of mass spectrometry filtering steps, demonstrates that 15 N labeling can be used for large-scale liquid chromatography-mass spectrometry studies of protein turnover in plants. We identify a series of abundant proteins in photosynthesis, photorespiration, and specific subunits of chlorophyll biosynthesis that turn over significantly more rapidly than the average protein involved in these processes. We also highlight a series of proteins that turn over as rapidly as the well-known D1 subunit of photosystem II. While these proteins need further verification for rapid degradation in vivo, they cluster in chlorophyll and thiamine biosynthesis.

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Section: Labeling Of Amino Acid Pools In Barley Leavessupporting

confidence: 91%

Proteins with High Turnover Rate in Barley Leaves Estimated by Proteome Analysis Combined with in Planta Isotope Labeling

et al. 2014

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“…The concentration of glutamic acid in acidified ethanol was 794.3 nmol/ml compared with 202.5 nmol/ ml in acidified water; asparagine concentration varied from 466.1 nmol/ml in acidified ethanol to 110.5 nmol/ml in acidified water and wasn't quantified in the other two extraction solvents. Same results were obtained for valine which wasn't quantified in ACN and acidified water, and whose concentrations decreased from 195.38 nmol/ml in acidified ethanol to 67.22 nmol/ml in ethanol, in accordance with literature data (Pinho, Silva and Macedo, 1994;Thiele et al, 2008).…”

Section: Hplc-dad/-esi-ms Identification and Quantification Of The Frsupporting

confidence: 89%

Effects of Extraction Solvents on the Quantification of Free Amino Acids in Lyophilised Brewer’s Yeast

Stănilă¹,

Diaconeasa²,

Sima³

et al. 2018

BUASVMCN-FST

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The aim of this work was to test some solvents in order to improve the free amino acids extraction from lyophilised brewer's yeast. The brewer' yeast was treated with four types of extraction solvents: Solvent I -acetonitrile 25%/HCl 0.01M (ACN); Solvent II -ethanol 80%; solvent III -HCl 0.05M/deionized water (1/1 volume); Solvent IV -HCl 0.05M/ethanol 80% (1/1 volume). The supernatants were analysed by HPLC-DAD-ESI-MS method. Acetonitrile provided the less quantities and number of amino acids extracted due to its weaker polarity. Solvent II and IV (ethanol, respectively acidified ethanol), which have an increased polarity, extracted 15 amino acids due to the addition of HCl in solvent IV. Solvent III (acidified water) proved to be the best extraction solvent for the amino acids from brewer's yeast providing the separation of 17 compounds:

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“…Cell extraction samples were analyzed with an Agilent 1100 HPLC system (Agilent Technologies, Waldbronn, Germany) coupled with an API4000 mass spectrometer (Applied Biosystems, Concord, Canada) equipped with a TurboIon spray source. Detailed information on separation methods have been reported previously (13,17). The LC-MS data were analyzed as described previously (18).…”

Section: Methodsmentioning

confidence: 99%

Combined Fluxomics and Transcriptomics Analysis of Glucose Catabolism via a Partially Cyclic Pentose Phosphate Pathway in Gluconobacter oxydans 621H

Hanke¹,

Nöh²,

Noack³

et al. 2013

Appl Environ Microbiol

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In this study, the distribution and regulation of periplasmic and cytoplasmic carbon fluxes in Gluconobacter oxydans 621H with glucose were studied by 13 C-based metabolic flux analysis ( 13 C-MFA) in combination with transcriptomics and enzyme assays. For 13 C-MFA, cells were cultivated with specifically 13 C-labeled glucose, and intracellular metabolites were analyzed for their labeling pattern by liquid chromatography-mass spectrometry (LC-MS). In growth phase I, 90% of the glucose was oxidized periplasmically to gluconate and partially further oxidized to 2-ketogluconate. Of the glucose taken up by the cells, 9% was phosphorylated to glucose 6-phosphate, whereas 91% was oxidized by cytoplasmic glucose dehydrogenase to gluconate. Additional gluconate was taken up into the cells by transport. Of the cytoplasmic gluconate, 70% was oxidized to 5-ketogluconate and 30% was phosphorylated to 6-phosphogluconate. In growth phase II, 87% of gluconate was oxidized to 2-ketogluconate in the periplasm and 13% was taken up by the cells and almost completely converted to 6-phosphogluconate. Since G. oxydans lacks phosphofructokinase, glucose 6-phosphate can be metabolized only via the oxidative pentose phosphate pathway (PPP) or the Entner-Doudoroff pathway (EDP).13 C-MFA showed that 6-phosphogluconate is catabolized primarily via the oxidative PPP in both phases I and II (62% and 93%) and demonstrated a cyclic carbon flux through the oxidative PPP. The transcriptome comparison revealed an increased expression of PPP genes in growth phase II, which was supported by enzyme activity measurements and correlated with the increased PPP flux in phase II. Moreover, genes possibly related to a general stress response displayed increased expression in growth phase II.

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Analysis of amino acids without derivatization in barley extracts by LC-MS-MS

Cited by 101 publications

References 35 publications

Proteins with High Turnover Rate in Barley Leaves Estimated by Proteome Analysis Combined with in Planta Isotope Labeling

Proteins with High Turnover Rate in Barley Leaves Estimated by Proteome Analysis Combined with in Planta Isotope Labeling

Effects of Extraction Solvents on the Quantification of Free Amino Acids in Lyophilised Brewer’s Yeast

Combined Fluxomics and Transcriptomics Analysis of Glucose Catabolism via a Partially Cyclic Pentose Phosphate Pathway in Gluconobacter oxydans 621H

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