Simultaneous Determination of Multiple Intracellular Primary Metabolites by Ultrahigh Performance Liquid Chromatography Coupled with a Q Exactive HF Mass Spectrometer.

In this study, we established an ultrahigh-performance liquid chromatography-Q Exactive HF MS (UHPLC-HF MS) method for the simultaneous determination of 25 targeted metabolites relating to a broad coverage of central metabolic pathways, such as glycolysis pathway, tricarboxylic acid cycle (TCA), serine biosynthesis pathway (SSP), glutaminolysis pathway, and closely related biosynthetic reactions. A Shodex Asahipak NH2P-50 2D column was used to separate the targeted compounds, and Full MS + PRM detection using an electrospray ionization source in negative mode was employed. The method also integrated a sample purification step by passing through a Waters Sirocco 96 plate to remove protein impurities, ensuring the better resolution and sensitivity of the proposed method. The calibration curves of the method showed good linearity within the range of 1-10 000 µg L-1 with the correlation coefficient no less than 0.99. The method can be used for routine quantification of primary metabolites in a wide variety of cell extract samples. With the help of the method, for the first time, we successfully separate the isomers of 3-phosphoglycerate (3-PG) and 2-phosphoglycerate (2-PG), which lay the groundwork for the accurate quantification of metabolites of the tumor cells, the study of PGAM1 inhibitors, and the development of neotype anticancer drugs.

The identification of six novel proteins with fibronectin or collagen type I binding activity from Streptococcus suis serotype 2.

Streptococcus suis, a major swine pathogen, is an emerging zoonotic agent that causes meningitis and septic shock. Bacterial cell wall and secreted proteins are often involved in interactions with extracellular matrix proteins (ECMs), which play important roles in the initial steps of pathogenesis. In this study, 2D SDS-PAGE, western blotting-based binding affinity measurements, and microtiter plate binding assays were used to identify cell wall and secreted proteins from S. suis that interact with fibronectin and collagen type I. We identified six proteins from S. suis, including three proteins (translation elongation factor G, oligopeptide-binding protein OppA precursor, and phosphoglycerate mutase) that show both fibronectin and collagen type I binding activity. To the best of our knowledge, these three newly identified proteins had no previously reported fibronectin or collagen type I binding activity. Overall, the aim in this study was to identify proteins with ECM binding activity from S. suis and it represents the first report of six new proteins from S. suis that interact with fibronectin or collagen type I.

Analysis of proteins in human gingival crevicular fluid by mass spectrometry.

BACKGROUND AND OBJECTIVE: Gingival crevicular fluid is a bodily fluid transuded from periodontal tissues into the gingival crevice and periodontal pocket, and contains many species of components. Proteins in gingival crevicular fluid have been studied as markers for periodontal diseases. Mass spectrometric analysis is used for the analyses of proteins, lipids, saccharides and metals, and expected as an approach for disease diagnosis. For better analysis of the protein components in gingival crevicular fluid, we investigated proteins in gingival crevicular fluid samples from the healthy gingival crevice and periodontal pocket using mass spectrometry. MATERIAL AND METHODS: Gingival crevicular fluid samples were collected from subjects who gave their informed consent and were periodontally healthy or had diseased pockets. These samples were electrophoretically separated, and each fraction on the gels was analysed by nano liquid chromatography coupled with tandem mass spectrometry. Antimicrobial peptides detected in gingival crevicular fluid were confirmed by western blotting. RESULTS: One hundred and four proteins were detected in gingival crevicular fluid samples from both healthy sites and sites of periodontitis; 64 proteins were contained only in gingival crevicular fluid from healthy sites and 63 proteins were observed only in gingival crevicular fluid from periodontitis sites. These proteins were blood-, cytoskeleton-, immunity-, inflammation- and lipid-related proteins and enzymes. Some proteins, including ceruloplasmin, glycogen phosphorylase, glutathione S-transferase, phosphoglycerate mutase, psoriasin, S100A11 and resistin, were identified for the first time in gingival crevicular fluid. Antimicrobial peptides, such as lactoferrin, α1-antitrypsin, lipocalin, S100A7, S100A8, S100A9 and cathelicidin, were observed by mass spectrometry and western blotting. CONCLUSION: Multiple protein components in gingival crevicular fluid were analysed at the same time using mass spectrometry, and this approach may be useful for the diagnosis of periodontal diseases.

Proteomic analysis of epicardial and subcutaneous adipose tissue reveals differences in proteins involved in oxidative stress.

Epicardial adipose tissue (EAT) is an endocrine organ adjacent to coronary arteries and myocardium without anatomy barriers. Locally produced adipokines may reflect or affect to cardiovascular physiology and pathology. Our aim was to study the protein expression profiles of EAT and subcutaneous adipose tissue (SAT) to identify local candidate molecules characterizing EAT in patients with cardiovascular disease. EAT and SAT samples were collected from 55 patients undergoing heart surgery. Proteins from these tissues were separated by two-dimensional (2D) gel electrophoresis, and differences between them were identified by MALDI-TOF/TOF spectra. Differences in protein levels were further investigated by real-time RT-PCR and Western blots, and production of reactive oxygen species (ROS) in EAT and SAT was evaluated by nitroblue tetrazolium chloride assays. ROS production was higher in EAT than SAT. We have found mRNA differences for catalase, glutathione S-transferase P, and protein disulfide isomerase, and 2D Western blots additionally showed post-translational differences for phosphoglycerate mutase 1; all four are related to oxidative stress pathways. EAT suffers greater oxidative stress than SAT in patients with cardiovascular diseases and exhibits associated proteomic differences that suggest the possibility of its association with myocardial stress in these patients.

Peony root extract upregulates transthyretin and phosphoglycerate mutase in mouse cobalt focus seizure.

Cobalt focus is a seizure focus model in which cerebral neurons exhibit long-lasting severe spike discharges, followed by neuronal death. However, the neuronal death is prevented when peony root extract (PR) is administered prior to cobalt application. We tested the hypothesis that PR modulates the expression of neuroprotective proteins in the cerebrum of mouse cobalt focus by proteomic analysis using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry to screen for differentially expressed proteins. Analyses revealed that transthyretin, a carrier protein for thyroid hormones and retinoids, and the brain form of phosphoglycerate mutase, a glycolytic enzyme, were upregulated in the cobalt-treated mouse cerebrum and further increased by PR administration in association with upregulation of neurogranin/RC3, a target of the transcriptional activation by thyroid hormones and retinoids. These findings suggest that PR-induced protection of mouse cerebral neurons involves neurotrophic events caused by thyroid hormones and/or retinoids and enhanced glycolysis.

Proteomic analysis of brain proteins in the gracile axonal dystrophy (gad) mouse, a syndrome that emanates from dysfunctional ubiquitin carboxyl-terminal hydrolase L-1, reveals oxidation of key proteins.

Ubiquitin carboxyl-terminal hydrolase L-1 (UCH L-1) is a crucial enzyme for proteasomal protein degradation that generates free monomeric ubiquitin. Our previous proteomic study identified UCH L-1 as one specific target of protein oxidation in Alzheimer's disease (AD) brain, establishing a link between the effect of oxidative stress on protein and the proteasomal dysfunction in AD. However, it is unclear how protein oxidation affects function, owing to the different responses of proteins to oxidation. Analysis of systems in which the oxidized protein displays lowered or null activity might be an excellent model for investigating the effect of the protein of interest in cellular metabolism and evaluating how the cell responds to the stress caused by oxidation of a specific protein. The gracile axonal dystrophy (gad) mouse is an autosomal recessive spontaneous mutant with a deletion on chromosome 5 within the gene encoding UCH L-1. The mouse displays axonal degeneration of the gracile tract. The aim of this proteomic study on gad mouse brain, with dysfunctional UCH L-1, was to determine differences in brain protein oxidation levels between control and gad samples. The results showed increased protein oxidation in thioredoxin peroxidase (peroxiredoxin), phosphoglycerate mutase, Rab GDP dissociation inhibitor alpha/ATP synthase and neurofilament-L in the gad mouse brain. These findings are discussed with reference to the effect of specific protein oxidation on potential mechanisms of neurodegeneration that pertain to the gad mouse.

Cross-species identification of novel Candida albicans immunogenic proteins by combination of two-dimensional polyacrylamide gel electrophoresis and mass spectrometry.

We have previously reported the usefulness of two-dimensional gel electrophoresis followed by Western blotting with sera from patients with systemic candidiasis in the detection of the major Candida albicans antigens (Pitarch et al., Electrophoresis 1999, 20, 1001-1010). The identification of these antigens would be useful for the characterization of good markers for the disease, and for the development of efficient diagnostic strategies. In this work we have used nanoelectrospray tandem mass spectrometry to obtain amino acid sequence information from the immunogenic proteins previously detected. We report here the cross-species identification of these antigens by matching of tandem mass spectrometry data to Saccharomyces cerevisiae proteins. Using this approach, we unambiguously identified the four C. albicans immunogenic proteins analyzed, namely aconitase, pyruvate kinase, phosphoglycerate mutase and methionine synthase. Furthermore, we report for the first time that aconitase, methionine synthase and phosphoglycerate mutase have antigenic properties in C. albicans.

Distribution of phosphoglycerate mutase isozymes in rat, rabbit and human tissues.

The distribution of phosphoglycerate mutase isozymes (types MM, MB and BB) in rat, rabbit and human tissues has been studied by electrophoresis on cellulose acetate and by highly-resolutive ion exchange chromatography. In the three species, muscle is the tissue with higher phosphoglycerate mutase activity. Heart is the only tissue with the three phosphoglycerate mutase isozymes in substantial amounts. Skeletal muscle contains mostly type MM isozyme and the other tissues possess almost exclusively type BB isozyme. Even in the presence of inhibitors, adenylate kinase can interfere with the staining reactions when large samples are analyzed and a long period of incubation is required.

Analysis of the relationship between the decrease in pH and accumulation of 3-phosphoglyceric acid in developing forespores of Bacillus species.

Analysis of the pH decrease and 3-phosphoglyceric acid (3PGA) accumulation in the forespore compartment of sporulating cells of Bacillus subtilis showed that the pH decrease of 1 to 1.2 units at approximately 4 h of sporulation preceded 3PGA accumulation, as observed previously in B. megaterium. These data, as well as analysis of the forespore pH decrease in asporogenous mutants of B. subtilis, indicated that sigma G-dependent forespore transcription, but not sigma K-dependent mother cell transcription, is required for the forespore pH decrease. Further analysis of these asporogenous mutants showed an excellent correlation between the forespore pH decrease and the forespore's accumulation of 3PGA. These latter results are consistent with our previous suggestion that the decrease in forespore pH results in greatly decreased activity of phosphoglycerate mutase in the forespore, which in turn leads to 3PGA accumulation. In further support of this suggestion, we found that (i) elevating the pH of developing forespores of B. megaterium resulted in rapid utilization of the forespore's 3PGA depot and (ii) increasing forespore levels of PGM approximately 10-fold in B. subtilis resulted in a large decrease in the spore's depot of 3PGA. The B. subtilis strain with a high phosphoglycerate mutase level sporulated, and the spores germinated and went through outgrowth normally, indicating that forespore accumulation of a large 3PGA depot is not essential for these processes.

[Phosphoglyceric acid mutase].

Human phosphoglyceric acid mutase comprises M-, B- and MB-type isozymes composed of the combination of the muscle-specific (M) and nonmuscle-specific (B) subunits. Human DNAs encoding M and B subunits were respectively reconstructed at their 5' regions without changing the amino acid sequences, and expressed directly in Escherichia coli under controls of the trp promoter. M- and B-type isozymes were over-produced in the bacterial cytoplasm as soluble, active forms, which have been purified and characterized. MB-type was synthesized in vitro by recombining M- and B-type. All the three recombinant isozymes thus obtained were the same in properties tested as the naturally-occurring ones. Polyclonal IgGs specific to the M-type, B-type and MB-type isozymes were prepared from rabbits immunized with the respective isozymes mainly by treating with the columns bound with the M- or B-type isozyme. A method for the immunoassay of the MB-type isozyme which exists specifically in cardiac muscle, is now under development.

Immunocytochemical localization of glycolytic and fermentative enzymes in Zymomonas mobilis.

Gold-labeled antibodies were used to examine the subcellular locations of 11 glycolytic and fermentative enzymes in Zymomonas mobilis. Glucose-fructose oxidoreductase was clearly localized in the periplasmic region. Phosphogluconate lactonase and alcohol dehydrogenase I were concentrated in the cytoplasm near the plasma membrane. The eight remaining enzymes were more evenly distributed within the cytoplasmic matrix. Selected enzyme pairs were labeled on opposite sides of the same thin section to examine the frequency of colocalization. Results from these experiments provide evidence that glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and alcohol dehydrogenase I form an enzyme complex.

Nuclear location of phosphoglycerate mutase BB isozyme in rat tissues.

We have previously reported (Ureña et al. Eur. J. Cell Biol. 1990) that in skeletal muscle, type MM phosphoglycerate mutase isozyme is present in the nucleus as well as in the cytosol. To determine whether type BB phosphoglycerate mutase isozyme is also present in nucleus, the subcellular location of this isozyme was studied in different rat tissues by cell fractionation and immunogold techniques. With the aid of high affinity-purified anti-phosphoglycerate mutase BB isozyme antibodies, the isozyme was located in the nucleus of neuronal, astroglial and liver cells but not in the nucleus of oligodendroglial and endothelial cells. Biochemical studies on purified nuclear fractions also demonstrated the presence of phosphoglycerate mutase activity in the nucleus. Both immunocytochemical and biochemical techniques showed that nuclear phosphoglycerate mutase-specific activity depended on the type of cell.

Prenatal diagnosis by isoenzymic differentiation of Treacher Collins' syndrome induced by retinoids in rats.

A series of branchial arch malformations was induced in 618 embryos from 72 pregnant rats by a single intraperitoneal injection of 10 mg/kg etretinate at 8.5 days of gestation. The litters developed several malformations, including microtia, low set and dorsally placed outer ears, defective middle ear ossicles, short cochleas, defectively differentiated Meckel's cartilages, micrognathia, rudimentary malar bones, lateral facial clefts, fistulas and skin tags, all of which were similar to Treacher Collins' syndrome in man. The defects were accompanied by a pathological differentiation pattern of various isoenzymes in maxillary and mandibular processes. These isoenzymes could be detected in amniotic fluid from the 9th to the 20th days of pregnancy and showed a pathological differentiation pattern here as well. We conclude that a teratogenically induced syndrome affecting the first and second branchial arches is accompanied by a pathological differentiation pattern that can be traced by determinations of isoenzymes in the branchial arches as well as in amniotic fluid.

Distribution and developmental transition of phosphoglycerate mutase and creatine phosphokinase isozymes in rat muscles of different fiber-type composition.

Phosphoglycerate mutase and creatine phosphokinase have in mammals three isozymes (types MM, MB and BB) with similar tissue distribution and developmental transition in muscle cells. To assess whether the phenotype and the developmental switch of these isozymes differ in the diverse types of muscle fibers, the enzymatic activities and the isozyme patterns, analyzed by cellulose acetate electrophoresis, have been determined in rat soleus, extensor digitorum longus and gastrocnemius muscles during postnatal development. Both phosphoglycerate mutase and creatine phosphokinase activity increased in the three muscles, the increase in extensor digitorum longus and gastrocnemius being higher than in soleus. For the two enzymes the increase in activity was due to the progressive increment of the muscle-specific forms. It is concluded that whereas phosphoglycerate mutase and creatine phosphokinase type-B subunits are present at similar levels in both type I and type II muscle fibers, phosphoglycerate mutase and creatine phosphokinase type-M subunits exhibit much higher levels in type II fibers.

Characterization of phosphoglycerate mutase isoenzymes from free dissected facial processes.

To characterize the three phosphoglycerate mutase (PGM) isoenzymes present in rat facial processes (types MM, BB, and MB), their sensitivity to reagents of the sulfhydryl groups and to heat treatment has been studied. Type BB PGM was not affected by the -SH group reagents; type MB PGM was inhibited about 50%, and type MM PGM was fully inhibited. Type MB PGM showed a greater heat lability than type MM PGM. There was a developmental change from type BB PGM from the 9th embryonic day to isoenzymes MB and MM on the 15th embryonic day. Isoenzyme development was first seen in mandibular processes, followed by maxillary, lateral nasal, and medial nasal processes.

Isolation of a cDNA encoding the muscle-specific subunit of human phosphoglycerate mutase.

We have isolated a full-length cDNA specifying the muscle-specific subunit of human phosphoglycerate mutase (PGAM-M). The cDNA encodes a deduced protein 253 amino acids in length and contains an unusually short (37 nucleotides) 3'-untranslated region. The deduced human PGAM-M protein is clearly related to yeast PGAM and to human diphosphoglycerate mutase. Genomic Southern analysis using the PGAM-M cDNA as a probe implies the presence of a large PGAM gene family in the human genome, while Northern analysis demonstrates tissue-specific transcription of this isoenzyme gene.

Biochemical and immunological arguments for homology between red cell and liver phosphoglyceromutase isozymes.

Phosphoglyceromutase (2,3-bisphospho-D-glycerate:2- phospho-D-glycerate phosphotransferase, EC 2.7.5.3) from the red cell is compared with the muscle and the liver forms of the enzyme obtained from rabbits. Partially purified samples were used for this study. The electrophoretic mobility of red cell phosphoglyceromutase is completely different from that of muscle and similar to that of liver. The muscle isozyme can hybridize with liver or red blood cell phosphoglyceromutase. N-Ethylmaleimide, an SH group reagent, completely inhibits muscle phosphoglyceromutase activity and slightly inhibits the activity of the enzyme from liver and from red cells. Both liver and red cell isozymes are completely inactivated by heating at 60 degrees C for 15 min, whereas a 25% decrease in inactivity is noted for the muscle enzyme. Finally, chicken antibody directed against human red cell phosphoglyceromutase reacts with the enzyme isolated from rabbit red cells and liver, but not with that obtained from muscle.

Macro-structural organization of phosphoglycerate mutase.

The three-dimensional structure of phosphoglycerate mutase has been analyzed using a contoured distance matrix and by visual inspection using three-dimensional computer graphics. Three folding lobes have been identified and their internal structure tentatively characterized. The active site is located at a lobe interface with a channel providing possible access from above and below. The arrangement of active site residues on two lobes suggests that the active site might be conformationally flexible. The remaining interface not associated with the active site channel appears to be predominately hydrophobic and thus may contribute to inter-lobe stability.

The reconstitution of denatured phosphoglycerate mutase.

The reconstitution of the tetrameric enzyme yeast phosphoglycerate mutase after denaturation in guanidine hydrochloride has been studied. Denaturation is almost completely reversible at enzyme concentrations greater than 10 micrograms/ml. Cross-linking by glutaraldehyde has been used to monitor the reassociation of the subunits; the kinetics of this process has been analyzed in terms of a model involving an equilibrium between monomer and dimer followed by a bimolecular association of two dimers to give a tetramer. Reactivation is found to parallel the appearance of tetramer. Structural changes during reconstitution have been measured by circular dichroism and fluorescence. Both methods reveal complex kinetics indicating the rapid formation of structured monomers (half-time less than 10 s), followed by slow subunit association. For comparison, preliminary reconstitution experiments were performed on the dimeric phosphoglycerate mutase from rabbit muscle.

Age-related changes of glyceraldehyde-3-phosphate-dehydrogenase (E.C. 1.2.1.12), 3-phosphoglyceratekinase (E.C. 2.7.2.3), phosphoglyceratemutase (E.C. 2.7.5.3), and enolase (E.C. 4.2.1.11) in bovine lenses.

The specific activities of the four investigated enzymes of the carbohydrate metabolism decrease with ageing. In the old tissue, phosphoglyceratemutase and enolase sustain changes in their molecular weight, and their substrate affinity is modified.