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  • 1
    Keywords: SYSTEM ; ACID ; ACIDS ; IONIZATION ; MASS-SPECTROMETRY ; LIQUID-CHROMATOGRAPHY ; PHOSPHOPEPTIDES ; PROTEIN-PHOSPHORYLATION ; phosphoproteins ; PHOSPHORYLATED PEPTIDES ; COLUMNS ; DESFERRIOXAMINE ; Metal ion chelators ; Metal ion mobilization ; Metal-free LC ; Multiply phosphorylated peptides ; Phosphopeptide recovery
    Abstract: It is hypothesized that metal ion-mediated adsorption of phosphorylated peptides on stationary phases of LC-columns is the major cause for their frequently observed poor detection efficiency in LC-MS. To study this phenomenon in more detail, sample solutions spiked with metal ion-mobilizing additives were analyzed by reversed phase muLC-ICP-MS or nanoLC-ESI-MS. Using muLC-ICP-MS, metal ions were analyzed directly as atomic ions. Using electrospray ionization, either metal ion chelates or phosphopeptide standard mixtures injected in subpicomole amounts were analyzed. Deferoxamine, imidazole, ascorbate, citrate, EDTA, and the tetrapeptide pSpSpSpS were tested as sample additives for the interlinked purposes of metal ion-mobilization and improvement of phosphopeptide recovery. Iron probably represents the major metal ion contamination of reversed phase columns. Based on the certified iron level in LC-grade solvents, a daily metal ion load of 〉10 pmol was estimated for typical nanoLC flow rates. In addition, phosphopeptide fractions from IMAC columns were identified as source for metal ion contamination of the LC column, as demonstrated for Ga(3+)-IMAC. The three metal ion-chelating additives, EDTA, citrate and pSpSpSpS, were found to perform best for improving the LC recovery of multiply phosphorylated peptides injected at subpicomole amounts. The benefits of metal ion-mobilizing LC (mimLC) characterized by metal ion complexing sample additives is demonstrated for three different instrumental setups comprising (a) a nanoUPLC-system with direct injection on the analytical column, (b) a nanoLC system with inclusion of a trapping column, and (c) the use of a HPLC-Chip system with integrated trapping and analytical column.
    Type of Publication: Journal article published
    PubMed ID: 20552382
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  • 2
    Abstract: We report the best performance yet for the self-disproportionation of enantiomers (SDE) via achiral chromatography as typically used in laboratories for the isolated yield of the excess enantiomer using N-acetyl beta-amino acid ethyl esters. The results are the most convincing ever demonstration of the capability of the SDE for practical-scale enantiopurification as comparable, or even superior for some systems, to that of recrystallization. For example, from a sample of 94.4 % ee, a yield of 71 % of enantiopure material was isolated in a single chromatographic run. Moreover, the lack of an esoteric structural entity, e.g. strongly polarizing groups, such as, for instance CF3, highlights the fact that the phenomenon is not dependent on the presence of such and thus the process is relevant to any usual-type structure. In contrast to recrystallization, the procedure is predictable, general, and dependable, boding well for its widespread application in routine laboratory settings.
    Type of Publication: Journal article published
    PubMed ID: 26704565
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  • 3
  • 4
    Abstract: Serum carnosinase (CN-1) measurements are at present mainly performed by assessing enzyme activity. This method is time-consuming, not well suited for large series of samples and can be discordant to measurements of CN-1 protein concentrations. To overcome these limitations, we developed sandwich ELISA assays using different anti-CN-1 antibodies, i.e., ATLAS (polyclonal IgG) and RYSK173 (monoclonal IgG1). With the ATLAS-based assay, similar amounts of CN-1 were detected in serum and both EDTA and heparin plasma. The RYSKS173-based assay detected CN-1 in serum in all individuals at significantly lower concentrations compared to the ATLAS-based assay (range: 0.1-1.8 vs. 1-50 mug/ml, RYSK- vs. ATLAS-based, P 〈 0.01). CN-1 detection with the RYSK-based assay was increased in EDTA plasma, albeit at significantly lower concentrations compared to ATLAS. In heparin plasma, CN-1 was also poorly detected with the RYSK-based assay. Addition of DTT to serum increased the detection of CN-1 in the RYSK-based assay almost to the levels found in the ATLAS-based assay. Both ELISA assays were highly reproducible (R: 0.99, P 〈 0.01 and R: 0.93, P 〈 0.01, for the RYSK- and ATLAS-based assays, respectively). Results of the ATLAS-based assay showed a positive correlation with CN-1 activity (R: 0.62, P 〈 0.01), while this was not the case for the RYSK-based assay. However, there was a negative correlation between CN-1 activity and the proportion of CN-1 detected in the RYSK-based assay, i.e., CN-1 detected with the RYSK-based assay/CN-1 detected with the ATLAS-based assay x 100% (Spearman-Rang correlation coefficient: -0.6, P 〈 0.01), suggesting that the RYSK-based assay most likely detects a CN-1 conformation with low CN-1 activity. RYSK173 and ATLAS antibodies reacted similarly in Western blot, irrespective of PNGase treatment. Binding of RYSK173 in serum was not due to differential N-glycosylation as demonstrated by mutant CN-1 cDNA constructs. In conclusion, our study demonstrates a good correlation between enzyme activity and CN-1 protein concentration in ELISA and suggests the presence of different CN-1 conformations in serum. The relevance of these different conformations is still elusive and needs to be addressed in further studies.
    Type of Publication: Journal article published
    PubMed ID: 22349764
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    Amino acids 11 (1996), S. 99-104 
    ISSN: 1438-2199
    Keywords: Amino acids ; Homocysteine ; Plasma proteins ; Oxidative stress ; Thiols groups ; Atherosclerosis ; Aging
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The oxidative status of plasma proteins after incubation with elevated homocysteine levels has been examined in the presence and absence of transition metal ions. 200µM homocysteine alone does not provoke any loss of plasma thiols groups, but their oxidation significantly enhances as copper concentration increases. No plasma proteins carbonyl groups enhancement has been concurrently found. The physiological relevance of the study is discussed in relationship with the metal-catalyzed oxidation system increment connected with age and nutritional deficiences.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1438-2199
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The session on Membrane Transport was a lively, interactive one with substantial audience participation in the discussion periods. Progress in this area of amino acid science is rapid as the genes encoding the transporter proteins are being cloned and characterized. The contributors to the platform session represented institutions from several countries giving the meeting a truly international flavor. Most of the participants contributed articles to this volume. The platform speakers were Dr. John McGivan (United Kingdom), Dr. Marçal Pastor-Anglada (Spain), Dr. Bruce Stephens (USA), Dr. G. Gazzola and Dr. V. Dall'Asta (Italy), Dr. Carol MacLeod (USA), Miss Maria Rivera-Correa (Puerto Rico), Dr. Ellen Closs (Germany), Dr. Manuel Palacín (Spain), Dr. Ovidio Bussolati (Italy), Dr. Suresh Tate (USA), Dr. S. Nakamura (Japan).
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 1438-2199
    Keywords: Amino acids ; Maple syrup urine disease ; Nuclear magnetic resonance spectroscopy ; Diet management ; Branched chain amino acids ; Branched chain keto acids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary A novel nuclear magnetic resonance method is proposed for the diagnosis and follow-up of patients affected by branched chain ketoaciduria. The method allows quantitation of the branched chain amino acids (BCAA's) such as leucine, isoleucine and valine and of related keto- and hydroxy acids by means of a single spectrum. The method implies short time of analysis, as opposed to the very long time required by the techniques currently in use (amino acid analyzer combined with gaschromatography/mass spectrometry of keto- and hydroxyacids), it is easy and suitable for adjustements of the dietary treatment even on a daily basis. The case of a 15 days old newborn child, presenting muscular hypertonicity was unambiguously diagnosed in few minutes by means of one single NMR spectrum of urine. More interestingly, NMR spectra of serum in the following days were suitable for quantitating amino-, and keto acids as well as other metabolites of relevance in the follow up of the dietary treatment of the disease. After a diet lacking of BCAA's, to eliminate keto acids, a low BCAA diet was introduced, that succeeded in keeping the serum levels of the three amino acids within the normal range, while dropping the related keto acids.
    Type of Medium: Electronic Resource
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  • 8
    ISSN: 1438-2199
    Keywords: Amino acids ; Regulation ; Stress ; Amino acid deprivation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The activities of the transport systems A, B° and XAG- are induced by various forms of stress in renal epithelial cells. Amino acid deprivation induces System A and XAG- in a protein-synthesis dependent process. In the case of System XAG- evidence is presented that induction of transport does not involve an increase in the amount of mRNA for the transporter or of the amount of transport protein. Preliminary evidence for the existence of a novel glycoprotein which is induced in parallel to the induction of these transport systems is presented. It is suggested that the induction of amino acid transport proteins and of some of the so-called stress proteins may be triggered by a common molecular mechanism.
    Type of Medium: Electronic Resource
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  • 9
    ISSN: 1438-2199
    Keywords: Amino acids ; Transport ; System A ; Osmotic regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Mammalian cells accumulate organic osmolytes, either to adapt to permanent osmotic changes or to mediate cell volume increase in cell cycle progression. Amino acids may serve as osmolytes in a great variety of cells. System A, a transport system for neutral amino acids, is induced after hypertonic shock by a mechanism which requires protein synthesis and gene transcription. Indirect evidence supports the view that system A activity increases due to the interaction of pre-existing A carriers with putative activating proteins. The intracellular accumulation of most neutral amino acids after hypertonic shock depends, exclusively, on the increase in system A activity. Long-term activation of system A is dependent on the integrity of cytoskeletal structures, but in a different way depending on whether cells are polarized or not.
    Type of Medium: Electronic Resource
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  • 10
    ISSN: 1438-2199
    Keywords: Cell volume ; Cell cycle ; Glutamine ; Glutamate
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The functional aspects of sodium dependent amino acid transport in mesenchymal cells are the subject of this contribution. In a survey of the cross-talk existing among the various transport mechanisms, particular attention is devoted to the role played by substrates shared by several transport systems, such as L-glutamine. Intracellular levels of glutamine are determined by the activity of System A, the main transducer of ion gradients built on by Na,K-ATPase into neutral amino acid gradients. Changes in the activity of the System are employed to regulate intracellular amino acid pool and, hence, cell volume. System A activity has been found increased in hypertonically shrunken cells and in proliferating cells. Under both these conditions cells have to increase their volume; therefore, System A can be employed as a convenient mechanism to increase cell volume both under hypertonic and isotonic conditions. Although less well characterized, the uptake of anionic amino acids performed by System X− AG may be involved in the maintenance of intracellular amino acid pool under conditions of limited availability of neutral amino acids substrates of System A.
    Type of Medium: Electronic Resource
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