A new approach to phosphoserine and phosphothreonine analysis in peptides and proteins: chemical modification,enrichment via solid-phase reversible binding,and analysis by mass spectrometry

beta-Elimination of the phosphate group on phosphoserine and phosphothreonine residues and addition of an alkyldithiol is a useful tool for analysis of the phosphorylation states of proteins and peptides. We have explored the influence of several conditions on the efficiency of this PO(4)(3-) elimination reaction upon addition of propanedithiol. In addition to the described influence of different bases, the solvent composition was also found to have a major effect on the yield of the reaction. In particular, an increase in the percentage of DMSO enhances the conversion rate, whereas a higher amount of protic polar solvents, such as water or isopropanol, induces the opposite effect. We have also developed a protocol for enrichment of the modified peptides, which is based on solid-phase covalent capture/release with a dithiopyridino-resin. The procedure for beta-elimination and isolation of phosphorylated peptides by solid-phase capture/release was developed with commercially available alpha-casein. Enriched peptide fragments were characterized by MALDI-TOF mass spectrometric analysis before and after alkylation with iodoacetamide, which allowed rapid confirmation of the purposely introduced thiol moiety. Sensitivity studies, carried out in order to determine the detection limit, demonstrated that samples could be detected even in the low picomolar range by mass spectrometry. The developed solid-phase enrichment procedure based on reversible covalent binding of the modified peptides is more effective and significantly simpler than methods based on the interaction between biotin and avidin, which require additional steps such as tagging the modified peptides and work-up of the samples prior to the affinity capture step.     

Zur Bestimmung von Ammoniak in Lebensmitteln

Zusammenfassung In einer Vakuumapparatur mit modifizierter Destilliervorlage wurden die für ein schonendes Verfahren optimalen Destillationsbedingungen zur quantitativen Freisetzung geringer Ammoniakmengen ermittelt, wobei sich z.B. eine Alkalisierung auf mindestens pH 9 ergab.Die Anwendung dieser Methode auf proteinhaltige Lebensmittel liefert ebenfalls — wenn auch nur in geringem Maße — Ammoniakabspaltungen, die wieder von der Art der Substanzen, der Alkalität der Lösung und der Destillationsdauer abhängig sind. Veränderungen im Verhältnis von Einwaagemenge zu Alkalisierungsmittel liefern daher über Alkalitätsverschiebungen auch jeweils etwas unterschiedliche Ammoniakausbeuten.Diese Vakuumdestillation im Makromaßstab ist bei Ammoniakbestimmungen in Substanzgemischen mit empfindlichen organischen Stickstoffverbindungen der Wasserdampfdestillation bei Normaldruck zwar überlegen, sie kann jedoch nicht als zuverlässige Methode empfohlen werden.

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Determination of ammonia in foodII. Examination of the vacuum distillation

In a vacuum apparatus with modified receiver the optimum conditions for a careful procedure for the quantitative liberation of small amounts of ammonia were tested. An alkalinity of at least pH 9 was necessary.Using this method with foods containing protein, ammonia was released too, though only in small amounts. These are depending not only on the type of substances, but also on the alkalinity of solution and the duration of distillation. Therefore, variations in the relationship between the amount of sample and the solution for alkalisation effect a change of alkalinity and thereby different yields of ammonia.For determinations of ammonia in mixtures of substances with instable nitrogen compounds the macrovacuum-apparatus is superior to the steam distillation applying normal pressure, but cannot be recommended as an reliable method.

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I. Mitteilung: diese Z. 244, 379 (1969).

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Auszug aus der Dissertation W. Sturm: Zur Bestimmung von Ammoniak in Getreidemehlen und Backwaren. Techn. Univ. Braunschweig 1967.     

Effect of nitrogen plasma treatment on the crystallinity and self‐bonding of polyetheretherketone (PEEK) for biomedical applications

Polyetheretherketone (PEEK) is a thermoplastic material with outstanding properties and high potential for biomedical applications, including hermetic encapsulation of active implantable devices. Different biomedical grade PEEK films with initial degree of crystallinity ranging from 8% to 32% (with or without mineral filling) were inspected. PEEK surfaces were treated with nitrogen RF plasma and the effects on materials crystallinity and self‐bonding were evaluated. In particular, the relationship between auto‐adhesive properties and crystalline content of PEEK before and after plasma treatment was examined. PEEK samples showed different bonding strength depending on their degree of crystallinity, with higher self‐bonding performance of mineral‐filled semi‐crystalline films. XRD did not show any modification of the PEEK microstructure as a result of plasma treatment, excluding a significant influence of crystallinity on the self‐bonding mechanisms. Nevertheless, plasma surface treatment successfully improved the self‐bonding strength of all the PEEK films tested, with larger increase in the case of semi‐crystalline unfilled materials. This could be interpreted to the increase in chain mobility that led to interfacial interpenetration of the amorphous phase.     

Experimental investigation of the two-photon widths of the chi(c0) and the chi(c2) mesons

Using 12.7 fb(-1) of data collected with the CLEO detector at CESR, we observed two-photon production of the cc states chi(c0) and chi(c2) in their decay to pi(+)pi(-)pi(+)pi(-). We measured gamma(gammagamma)(chi(c))xB(chi(c)-->pi(+)pi(-)pi(+)pi(-)) to be 75+/-13(stat)+/-8(syst) eV for the chi(c0) and 6.4+/-1.8(stat)+/-0.8(syst) eV for the chi(c2), implying gamma(gammagamma)(chi(c0)) = 3.76+/-0.65(stat)+/-0.41(syst)+/-1.69(br) keV and gamma(gammagamma)(chi(c2)) = 0.53+/-0.15(stat)+/-0.06(syst)+/-0.22(br) keV. Also, cancellation of dominant experimental and theoretical uncertainties permits a precise comparison of gamma(gammagamma)(chi(c0))/gamma(gammagamma)(chi(c2)), evaluated to be 7.4+/-2.4(stat)+/-0.5(syst)+/-0.9(br), with QCD-based predictions.