Proteomic analysis of Saccharomyces cerevisiae grown on glucose or glycerol

<p>The goal of this research was to use two-dimensional electrophoresis to examine changes in abundance of enzymes of the glycolytic pathway in the yeast Saccharomyces cerevisiae grown on carbon sources that support either fermentation to ethanol or oxidative metabolism. Large-scale profiling of protein abundances (expression proteomics) often detects changes in protein abundance between physiological states. Such changes in enzyme abundance are often interpreted as evidence of metabolic change although most textbooks emphasise control of enzyme activities not enzyme amount. Two-dimensional difference gel electrophoresis (2DDIGE) was therefore used to examine differences in protein abundance between S. cerevisiae strain BY4741 grown on either glucose (fermentation) or glycerol. Growth on 2% glucose, but not on glycerol, was accompanied by extensive production of ethanol. Doubling times for growth were 2 h 5 min in glucose and 9 h 41 min in glycerol. Conditions for extraction and two-dimensional electrophoresis of proteins were established. One hundred and seventy nine proteins were identified by MALDI mass spectrometry of tryptic digests of protein spots excised from Coomassie stained gels. All of the enzymes for conversion of glucose to ethanol, except for the second enzyme of glycolysis phosphoglucose isomerase, were identified using twodimensional electrophoresis of 100 μg of protein from cells grown on 2% glucose. Identification of proteins excised from the DIGE gels was more challenging, partly because of the lower amount of protein. Eight of the proteins that showed statistically significant differences in abundance (≥ 2-fold, p ≤ 0.01) between glucose and glycerol were identified by mass spectrometry of proteins excised from the 2DDIGE gels, and a further 18 varying proteins were matched to proteins identified from the Coomassie stained gels. Of these total 26 identified or matched proteins, subunits of five of the enzymes for conversion of glucose to ethanol were more abundant from the fermentative cells grown on glucose. The more abundant glycolytic enzymes were phosphofructokinase 2, fructose-1,6-bisphosphate aldolase, triosephosphate isomerase and enolase, plus pyruvate decarboxylase that was required for conversion of the glycolytic product pyruvate to acetaldehyde. The alcohol dehydrogenases Adh1 and Adh4 that convert acetaldehyde to ethanol were detected but did not vary significantly between growth on glucose or glycerol. The results confirmed that in this case changes in abundance of some enzymes were consistent with the altered metabolic output. Future studies should examine whether changes in the abundance and activity of these enzymes are responsible for the differences in metabolism.</p>

Proteomic analysis of Saccharomyces cerevisiae grown on glucose or glycerol

<p>The goal of this research was to use two-dimensional electrophoresis to examine changes in abundance of enzymes of the glycolytic pathway in the yeast Saccharomyces cerevisiae grown on carbon sources that support either fermentation to ethanol or oxidative metabolism. Large-scale profiling of protein abundances (expression proteomics) often detects changes in protein abundance between physiological states. Such changes in enzyme abundance are often interpreted as evidence of metabolic change although most textbooks emphasise control of enzyme activities not enzyme amount. Two-dimensional difference gel electrophoresis (2DDIGE) was therefore used to examine differences in protein abundance between S. cerevisiae strain BY4741 grown on either glucose (fermentation) or glycerol. Growth on 2% glucose, but not on glycerol, was accompanied by extensive production of ethanol. Doubling times for growth were 2 h 5 min in glucose and 9 h 41 min in glycerol. Conditions for extraction and two-dimensional electrophoresis of proteins were established. One hundred and seventy nine proteins were identified by MALDI mass spectrometry of tryptic digests of protein spots excised from Coomassie stained gels. All of the enzymes for conversion of glucose to ethanol, except for the second enzyme of glycolysis phosphoglucose isomerase, were identified using twodimensional electrophoresis of 100 μg of protein from cells grown on 2% glucose. Identification of proteins excised from the DIGE gels was more challenging, partly because of the lower amount of protein. Eight of the proteins that showed statistically significant differences in abundance (≥ 2-fold, p ≤ 0.01) between glucose and glycerol were identified by mass spectrometry of proteins excised from the 2DDIGE gels, and a further 18 varying proteins were matched to proteins identified from the Coomassie stained gels. Of these total 26 identified or matched proteins, subunits of five of the enzymes for conversion of glucose to ethanol were more abundant from the fermentative cells grown on glucose. The more abundant glycolytic enzymes were phosphofructokinase 2, fructose-1,6-bisphosphate aldolase, triosephosphate isomerase and enolase, plus pyruvate decarboxylase that was required for conversion of the glycolytic product pyruvate to acetaldehyde. The alcohol dehydrogenases Adh1 and Adh4 that convert acetaldehyde to ethanol were detected but did not vary significantly between growth on glucose or glycerol. The results confirmed that in this case changes in abundance of some enzymes were consistent with the altered metabolic output. Future studies should examine whether changes in the abundance and activity of these enzymes are responsible for the differences in metabolism.</p>

The effect of exogenous factors on the polyenzymatic activity of RuBisCO and ATP synthase of chloroplasts from pea leaves

Aim. To isolate and purify protein complexes – ATP synthase and RuBisCO – from pea leaf chloroplasts and study the effect of a microbiological fertilizer “Extracon” and sulfonamide inhibitors acetazolamide and ethoxyzolamide on the enzymatic activity of these proteins.Materials and methods. Chloroplasts were isolated from the leaves of two-week-old pea sprouts, protein complexes of purified thylakoid membranes were solubilized with digitonin (10 mg of digitonin per 1 mg of protein), the protein concentration was determined according to Lowry. Native electrophoresis with displacement of the charge of the soluble protein fraction from the chloroplast stroma, as well as membrane proteins, was carried out in the modified system of Anderson et al., Kolisnichenko et al. A modified Lemmley system was applied to the protein electrophoresis in the polyacrylamide gel in the presence of sodium dodecyl sulfate. The methods of Alain and Hintsik, as well as Gomorrah were used to determine the ATPase activity in the polyacrylamide gel. Visualization of the carbonic anhydrase activity in the polyacrylamide gel was performed by the method of Edwards and Petton. Results and discussion. Using physicochemical methods of potentiometry, spectrophotometry the ATPase, carbonic anhydrase and esterase activities of the enzymes were studied. The results obtained indicate that specific carbonic anhydrase inhibitors (acetazolamide and ethoxyzolamide) also block the esterase and ATPase activity of the enzyme complexes. “Extracon” (a multifunctional microbiological preparation) almost 1.5 times increases the activity of the enzymes, showing a complex activating effect of the fertilizer on both light and dark reactions of photosynthesis.Conclusions. The method of identification and isolation of RuBisCO and ATP synthase on the basis of two-dimensional electrophoresis and electrophoretic elution has been proposed. It allows determining the presence of certain enzyme activity of complexes at first in SDS plates (express analysis) and further to study the effect of various factors of endogenous and exogenous origin on the enzymatic properties of electrophoretically pure enzymes. The use of two-dimensional electrophoresis as a tool for assessing the impact of various factors of endogenous and exogenous origin on the plant cell and the plant as a whole through constant monitoring of the work and activity of enzyme systems of the plant cell is promising.

Adaptation of the two-dimensional electrophoresis method for canned meat

Studies of the qualitative indicators of canned meat in accordance with regulatory documents are carried out on average samples of specimens, but when studying by proteomic methods, such sampling does not allow high-quality separation of protein components due to the high fat content in the product. When two-dimensional electrophoresis was carried out on an average sample, fragments of the main muscle and connective tissue proteins of beef were found in small quantities, but the electrophoretogram was not very informative. A significantly better separation was achieved after removing the fat fraction from the product. When studying broth from canned meat, the largest amount of intensely coloured high-molecular-weight protein fractions with a mass of more than 50 kDa was revealed. The electrophoretogram of the meat pieces showed a wide range of proteins across the entire molecular weight range of the polyacrylamide gel, including major muscle proteins. The study of broth together with meat pieces but after fat removal is optimal for the primary screening of the protein component of canned meat.

Plasma apolipopotein C-2 elevation is associated with Takayasu arteritis

Takayasu arteritis (TAK) is an autoimmune systemic arteritis of unknown etiology. Although a number of investigators have attempted to determine biomarkers for diagnosing TAK, there exist no specific serological markers of this intractable disease. We undertook the exploration of novel serological markers which could be useful for an accurate diagnosis of TAK using an unbiased proteomics approach. The purified plasma samples from untreated patients with TAK and healthy individuals were separated by two-dimensional electrophoresis. The differentially expressed protein spots were detected by gel comparison and identified using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MS). Next, we validated plasma concentrations of identified proteins by enzyme-linked immunosorbent assay (ELISA). Two-dimensional electrophoresis and numerical analysis revealed 19 spots and 3 spot clusters whose sum of the sample averages was ≥ 0.01, and the average concentrations were ≥ 1.5 times in the patient group compared with the control group. Among them, 10 spots and spot clusters that met the condition of the average spot concentration being 2.5 times more than that in the control group were selected. After processing these spots using MS and conducting MS/MS ion search, we identified 10 proteins: apolipoprotein C-2 (ApoC-2), actin, apolipoprotein A-1, complement C3, kininogen-1, vitronectin, α2-macroglobulin, 14–3–3 protein ζ/δ, complement C4, and inter-α-trypsin inhibitor heavy chain H4 isoform 1 precursor. Finally, ELISA demonstrated that plasma ApoC-2 level was significantly elevated in patients with TAK compared with that in healthy individuals. Thus, ApoC-2 would be a promising candidate biomarker for TAK diagnosis.

Bacterial proteomics and its application for pathogenesis studies

: Bacteria build their structures by implementing several macromolecules such as proteins, polysaccharides, phospholipids, and nucleic acids, which leads to preserve their lives and play an essential role in their pathogenesis. There are two genomic and proteomic methods to study various macromolecules of bacteria, which are complementary methods and provide comprehensive information. Proteomic approaches are used to identify proteins and their cell applications. Furthermore, to study bacterial proteins, macromolecules are involved in the bacteria's structures and functions. These protein-based methods provide comprehensive information about the cells, such as the external structures, internal compositions, post-translational modifications, and mechanisms of particular actions such as biofilm formation, antibiotic resistance, and adaptation to the environment, which are helpful in promoting bacterial pathogenesis. These methods use various devices such as MALDI-TOF MS, LC-MS, and two-dimensional electrophoresis, which are valuable tools for studying different structural and functional proteins of the bacteria and their mechanisms of pathogenesis that causes rapid, easy, and accurate diagnosis of the infections.

Identification of mare colostrum proteins

Colostrum is an essential feed of foals. It is a source of nutrients and functional proteins significant for foals’ growth and development. In the presented research using two-dimensional electrophoresis coupled via spectrometry mass MALDI-TOF in the mares’ colostrum (whey proteins fraction) were identified 24 proteins representing 15 different gene products. The identified proteins were involved in supporting foals’ immature immune systems and in the transport of various compounds. Further research of mares’ colostrum will allow determining more gene products. An in-depth analysis of mares’ milk will provide information about biochemical processes occurring in the mammary gland of the mare during the lactation period.