Multiplexed quantitative proteomics using differential metabolic 15N-labeling

There are several advantages of using metabolic labeling in quantitative proteomics. The early pooling of samples compared to post-labeling methods eliminates errors from different sample processing, protein extraction and enzymatic digestion. Metabolic labeling is also highly efficient and relatively inexpensive compared to commercial labeling reagents. However, methods for multiplexed quantitation in the MS-domain (or ‘non-isobaric’ methods), suffer from signal dilution at higher degrees of multiplexing, as the MS/MS signal for peptide identification is lower given the same amount of peptide loaded onto the column or injected into the mass spectrometer. This may partly be overcome by mixing the samples at non-uniform ratios, for instance by increasing the fraction of unlabeled proteins. We have developed an algorithm for arbitrary degrees of nonisobaric multiplexing for relative protein abundance measurements. We have used metabolic labeling with different levels of 15N, but the algorithm is in principle applicable to any isotope or combination of isotopes. Ion trap mass spectrometers are fast and suitable for LC-MS/MS and peptide identification. However, they cannot resolve overlapping isotopic envelopes from different peptides, which makes them less suitable for MS-based quantitation. Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry is less suitable for LC-MS/MS, but provides the resolving power required to resolve overlapping isotopic envelopes. We therefore combined ion trap LC-MS/MS for peptide identification with FTICR LC-MS for quantitation using chromatographic alignment. We applied the method in a heat shock study in a plant model system (A. thaliana) and compared the results with gene expression data from similar experiments in literature.

Role of glutaredoxin 2 and cytosolic thioredoxins in cysteinyl-based redox modification of the 20S proteasome

The yeast 20S proteasome is subject to sulfhydryl redox alterations, such as the oxidation of cysteine residues (Cys-SH) into cysteine sulfenic acid (Cys-SOH), followed by S-glutathionylation (Cys-S-SG). Proteasome S-glutathionylation promotes partial loss of chymotrypsin-like activity and post-acidic cleavage without alteration of the trypsin-like proteasomal activity. Here we show that the 20S proteasome purified from stationary-phase cells was natively S-glutathionylated. Moreover, recombinant glutaredoxin 2 removes glutathione from natively or in vitro S-glutathionylated 20S proteasome, allowing the recovery of chymotrypsin-like activity and post-acidic cleavage. Glutaredoxin 2 deglutathionylase activity was dependent on its entry into the core particle, as demonstrated by stimulating S-glutathionylated proteasome opening. Under these conditions, deglutathionylation of the 20S proteasome and glutaredoxin 2 degradation were increased when compared to non-stimulated samples. Glutaredoxin 2 fragmentation by the 20S proteasome was evaluated by SDS-PAGE and mass spectrometry, and S-glutathionylation was evaluated by either western blot analyses with anti-glutathione IgG or by spectrophotometry with the thiol reactant 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. It was also observed in vivo that glutaredoxin 2 was ubiquitinated in cellular extracts of yeast cells grown in glucose-containing medium. Other cytoplasmic oxido-reductases, namely thioredoxins 1 and 2, were also active in 20S proteasome deglutathionylation by a similar mechanism. These results indicate for the first time that 20S proteasome cysteinyl redox modification is a regulated mechanism coupled to enzymatic deglutathionylase activity.

Absorção e redistribuição de nitrogênio (15N) em Citrus mitis Bl

Com a avaliação da eficiência de uso do nitrogênio, tem-se melhor entendimento dos aspectos nutricionais e respostas à adubação. O presente ensaio teve por objetivo estudar a absorção e redistribuição de nitrogênio (15N) em Citrus mitis Bl.. As fontes de fertilizante utilizadas foram: sulfato de amônio, uréia, nitrato de cálcio e nitrato de potássio. O delineamento experimental utilizado foi inteiramente casualizado, com 4 tratamentos e 3 repetições. Foram realizadas duas amostragens, aos 10 e 20 dias após a aplicação do adubo marcado, a fim de determinar os teores de N nas diferentes partes da planta. Através dos resultados, verificou-se que não houve efeito dos tratamentos sobre o peso de matéria seca e conteúdo de N nas plantas. A eficiência de absorção de N variou com a natureza do fertilizante nitrogenado e com a época de amostragem, ao passo que a redistribuição do N não foi afetada. A eficiência máxima de absorção do N variou de 14% (uréia) e 31% (sulfato de amônio), respectivamente, aos 10 e 20 dias após a aplicação do 15N.

Transporte do 15N e produtividade do tomateiro enxertado irrigado com água carbonatada

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influência do etil-trinexapac no acúmulo, na distribuição de nitrogênio (15N) e na massa de grãos de arroz de terras altas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efeito do déficit de água no aproveitamento do nitrogênio - 15n na cultura do trigo

Três fontes de nitrogênio - 15n foram utilizadas: sulfato de amônio, nitrato de amônio e uréia. Elas foram aplicadas no plantio de dois modos: a lanço e a seguir incorporadas ao solo, ou no sulco de plantio. Determinou-se a eficiência de utilização do nitrogênio - 15n aplicado no plantio e no perfilhamento, sendo respectivamente, em media 14 e 13% do nitrogênio aplicado e utilizados pelo trigo. Obtiveram-se valores de 16 e 12% de eficiência do nitrogênio - 15n aplicado no sulco de plantio ou a lanço e a seguir incorporado ao solo, respectivamente.

Utilização do nitrogênio (15N) residual de coberturas de solo e da uréia pela cultura do milho

Geralmente, grande parte do N de fertilizantes minerais e de plantas de cobertura de solo não é aproveitada pelo milho no cultivo imediato à aplicação, o qual pode ser absorvido pelas culturas cultivadas subseqüentemente. O objetivo deste trabalho foi avaliar o aproveitamento pelo milho do N residual da uréia, da crotalária (Crotalaria juncea) e do milheto (Pennisetum americanum) marcados com 15N, aplicados ao milho cultivado em sistema plantio direto, no ano agrícola anterior, num Latossolo Vermelho distroférrico no Cerrado. O estudo foi desenvolvido na fazenda experimental da Faculdade de Engenharia de Ilha Solteira-UNESP, Selvíria (MS), em áreas distintas. O delineamento experimental foi de blocos ao acaso com 15 tratamentos e quatro repetições, aplicados ao milho em 2001/02 e 2002/03. Os tratamentos foram dispostos em esquema fatorial 3 x 5, compreendendo a combinação de três coberturas de solo: crotalária juncea, milheto e vegetação espontânea (pousio), e cinco doses de N-uréia: 0, 30, 80, 130 e 180 kg ha-1. Após a colheita do milho, as duas áreas permaneceram em pousio nas entressafras e, em seguida, cultivadas novamente com milho, safras 2002/03 (experimento 1) e 2003/04 (experimento 2), utilizando adubação similar em todas as parcelas, para distinguir o efeito do N residual. O aproveitamento médio do N residual da parte aérea do milheto e da crotalária pelo milho foi inferior a 3,5 e 3 %, respectivamente, da quantidade inicial. A quantidade de N residual da uréia absorvida pelo milho aumentou de forma quadrática, no experimento 1, e linear, no experimento 2, em relação à dose de N aplicada, sendo o aproveitamento desta inferior a 3 %. As coberturas de solo não influenciaram o aproveitamento pelo milho do N residual da uréia, e vice-versa.

Efeito da oferta dietética de proteína sobre o ganho muscular, balanço nitrogenado e cinética da 15N-glicina de atletas em treinamento de musculação

The effect of increased protein intake on the muscle mass gain, nitrogen balance and N-15-glycine kinetics was studied in six young, healthy subjects practitioners of strength training (> 2 years), without use of anabolic steroids and in agreement with the ethical principles of the research. All athletes received adequate diet (0.88g protein/kg/day) during 2 weeks prior the study (D1), and thereafter with diet providing 1.5g of protein/kg/day and 30kcal/g of protein (D2 diet) for the subsequent 2 weeks. Later on, they all received diet with 2.5g of protein/kg/day (D3 diet) and 30 kcal/g protein for the last two weeks. Body composition, food intake, blood biochemistry, nitrogen balance (NB) and 15N-glycine kinetics were determined at the beginning, after D1 (M0) and in the last days of the D2 (M1) and D3 (M2). The results showed at the end of the study (4 weeks) significant increase in muscle mass (1.63 +/- 0.9kg), without difference between D2 and D3. The NB followed the protein/energy consumption (M0 = -7.8g/day; M1 = 5.6g/day and D3 = 16.6g/day), the protein synthesis followed the NB, with M0 < (M1= M2) (M1 = 49.8 +/- 12.2g N/day and M2 = 52.5 +/- 14.0g N/day). Protein catabolism rate was similarly kept among diets. Thus, the results of the NB and N-15-glycine kinetics indicate that the recommended protein intake for these athletes is higher than the one for sedentary adults (0.88g/kg) and lower than 2.5g/kg, around 1.5g of protein/kg/day, with adjustment of the energy consumption to 30 kcal/g of protein.

Traceability of animal byproducts in quail (Coturnix coturnix japonica) tissues using carbon (13C/12C) and nitrogen (15N/14N) stable isotopes

Consistent information on meat products consumed by the public is essential. The technique of stable isotopes is a powerful tool to recover consumers' confidence, as it allows the detection of animal byproduct residues in poultry meat, particularly in quail meat. This study aimed at checking the presence of poultry byproduct mixtures in quail diets by applying the technique of carbon (13C/12C) and nitrogen (15N/14N) stable isotopes in quail breast muscle, keel, and tibia. Sixty four one-day-old male quails were obtained from a commercial farm. Birds were housed in an experimental house from one to 42 days of age, and were randomly distributed into 8 experimental treatments, and fed diets containing poultry offal meal (POM), bovine meat and bone meal (MBM) or poultry feather meal (PFM), or their mixtures. Four birds per treatment were slaughtered at 42 days of age, and breast (Pectoralis major), keel, and tibia were collected for analyses. The inclusion of animal byproducts in quail diets was detected by 13C e 15N analyses in the tissues of the birds; however, it was not possible to specify which byproducts were used. It was concluded that quail meat can be certified by the technique of stable isotopes.

The 20S proteasome alpha(5) subunit of Arabidopsis thaliana carries an RNase activity and interacts in planta with the Lettuce mosaic potyvirus HcPro protein

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Traceability of animal meals in Japanese quail eggs using the technique of 13C e 15N* stable isotopes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Balanço do nitrogênio da uréia (15N) no sistema solo-planta na implantação da semeadura direta na cultura do milho

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Aproveitamento do nitrogênio (15N) da crotalária e do milheto pelo milho sob plantio direto em Latossolo Vermelho de Cerrado

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Protein-energy status and 15N-glycine kinetic study of child a cirrhotic patients fed low- to high-protein energy diets

In five male cirrhotic patients (Child A) and in four age- and sex-matched healthy control subjects, whole-body protein turnover was measured using a single oral dose of N-15-glycine as a tracer and urinary ammonia as end product. Subjects were studied in the fasting and feeding state, with different levels of protein and energy intake. The patients were underweight and presented lower plasma transthyretin and retinol-binding protein levels. When compared with controls, the kinetic studies showed patients to be hypometabolic in the fasting (Do) state and with the control diet [D-1 = (0.85 g of protein/154 kJ). kg(-1). day(-1)]. However, when corrected by body weight, the kinetic differences between groups disappeared, whereas the N-retention in the feeding state showed better results for the patients due mainly to their efficient breakdown decrease. When fed high-level protein or energy diets [D-2 = (0.9 g protein/195 kJ) and D-3 = (1.56 g protein/158 kJ). kg(-1). day(-1)], the patients showed D-0 = D-1 = D-2 < D-3 for N-flux and (D-0 = D-1) < D-3 (D-2 is intermediary) for protein synthesis. Thus, the present data suggest that the remaining mass of the undernourished mild cirrhotic patients has fairly good protein synthesis activity and also that protein, rather than energy intake, would be the limiting factor for increasing their whole-body protein synthesis.