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This article pays tribute to Frantisek "Frank" Turecek, a scientist and mountaineer. The author shares their personal experiences with Frank, including their first meeting in Prague and later joining his research group at the University of Washington. They discuss Frank's work in studying short-lived radicals and his passion for climbing mountains. The article also highlights Frank's contributions to the field of mass spectrometry and his dedication to mentoring graduate students. It provides a personal perspective on Frank's adventurous spirit and his achievements in both science and mountaineering. [Extracted from the article]

We report a new assay of N-acetylgalactosamine-4-sulfatase (aryl sulfatase B) activity in dried blood spots (DBS) for the early detection of mucopolysaccharidosis VI (Maroteaux--Lamy syndrome) in newborn screening. The assay uses a synthetic substrate consisting of N-acetylgalactosamine-4-sulfate moiety glycosidically linked to a hydrophobic residue and furnished with a tert-butyloxy-carbamido group as a marker for specific mass spectrometric fragmentation. Incubation with aryl sulfatase B present in DBS converts the substrate to a desulfated product which is detected by electrospray tandem mass spectrometry and quantified using a homologous internal standard. Assay and workup procedures were optimized to be compatible with the work flow in newborn screening laboratories. Analysis of DBS from human newborns showed clear distinction of aryl sulfatasc B activity from 89 healthy individuals where it ranged between 1.4 and 16.9 [micro]mol/(h L of blood), with an average activity of 7.4 [micro]mol/(h L of blood), and an MPS-VI patient that had an activity of 0.12 [micro]mol/(h L of blood). Results are also reported for the aryl sulfatase B assay in DBS from groups of normal felines and felines affected with MPS-VI. 10.1021/ac102090v

[Cav.sub.v]1 channels initiate excitation-contraction coupling in skeletal and cardiac muscle. During the fight-or-flight response, epinephrine released by the adrenal medulla and norepinephrine released from sympathetic nerves increase muscle contractility by activation of the [beta]-adrenergic receptor/cAMP-dependent protein kinase pathway and up-regulation of [ca.sub.v]1 channels in skeletal and cardiac muscle. Although the physiological mechanism of this pathway is well defined, the molecular mechanism and the sites of protein phosphorylation required for [Ca.sub.v]1 channel regulation are unknown. To identify the regulatory sites of phosphorylation under physiologically relevant conditions, [Cav.sub.v]1.1 channels were purified from skeletal muscle and sites of phosphorylation on the [alpha]1 subunit were identified by mass spectrometry. Two phosphorylation sites were identified in the proximal C-terminal domain, serine 1575 (S1575) and threonine 1579 (T1579), which are conserved in cardiac [Ca.sub.v]1.2 channels (S1700 and T1704, respectively). In vitro phosphorylation revealed that [Ca.sub.v]1.1-S1575 is a substrate for both cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase II, whereas [ca.sub.v]1.1-T1579 is a substrate for casein kinase 2. Treatment of rabbits with isoproterenol to activate [beta]-adrenergic receptors increased phosphorylation of S1575 in skeletal muscle [ca.sub.v]1.1 channels in vivo, and treatment with propranolol to inhibit [beta]-adrenergic receptors reduced phosphorylation. As S1575 and T1579 in [ca.sub.v]1.1 channels and their homologs in [ca.sub.v]1.2 channels are located at a key regulatory interface between the distal and proximal C-terminal domains, it is likely that phosphorylation of these sites in skeletal and cardiac muscle is directly involved in calcium channel regulation in response to the sympathetic nervous system in the fight-or-flight response. adrenalin | calcium channels | cyclic AMP | mass spectometry | protein kinase A doi/ 10.1073/pnas.1012384107

Combined liquid chromatography--electrospray ionization-tandem mass spectrometry (LC--ESI-MS/MS) is a powerful method for the analysis of oxygenated metabolites of polyunsaturated fatty acids including eicosanoids. Here we describe the synthesis of a new derivatization reagent N-(4-aminomethyiphenyl)pyridinium (AMPP) that can be coupled to eicosanoids via an amide linkage in quantitative yield. Conversion of the carboxylic acid of eicosanoids to a cationic AMPP amide improves sensitivity of detection by 10- to 20-fold compared to negative mode electrospray ionization detection of underivatized analytes. This charge reversal derivatization allows detection of cations rather than anions in the electrospray ionization mass spectrometer, which enhances sensitivity. Another factor is that AMPP amides undergo considerable collision-induced dissociation in the analyte portion rather than exclusively in the cationic tag portion, which allows isobaric derivatives to be distinguished by tandem mass spectrometry, and this further enhances sensitivity and specificity. This simple derivatization method allows prostaglandins, thromboxane [B.sub.2], leukotriene [B.sub.4], hydroxyeicosatetraenoic acid isomers, and arachidonic acid to be quantified in complex biological samples with limits of quantification in the 200-900 fg range. One can anticipate that the AMPP derivatization method can be extended to other carboxylic acid analytes for enhanced sensitivity detection. 10.1021/ac100720p

Pyruvate produces inotropic responses in the adult reperfused heart. Pyruvate oxidation and anaplerotic entry into the tricarboxylic acid (TCA) cycle via carboxylation are linked to the stimulation of contractile function. The goals of this study were to determine if these metabolic pathways operate and are maintained in the developing myocardium after reperfusion. Immature male swine (age: 10-18 days) were subjected to cardiopulmonary bypass (CPB). Intracoronary infusion of [2-[sup.13]C]pyruvate (to achieve an estimated final concentration of 8 mM) was given for 35 min, starting either during weaning (group I) and after its discontinuation (group II) or without (control) CPB. Hemodynamic data were collected. [sup.13]C NMR spectroscopy was used to determine the fraction of pyruvate entering the TCA cycle via pyruvate carboxylation (PC) to total TCA cycle entry (PC plus decarboxlyation via pyruvate dehydrogenase). Liquid chromatography-mass spectrometry was used to determine total glutamate enrichment. Pyruvate infusion starting during the weaning of mechanical circulatory support improved maximum dP/dt (P < 0.05) but waiting to start the infusion until after the discontinuation of CPB did not. Glutamate fractional enrichment was confirmed by liquid chromatography-mass spectroscopy as adequate (>5%) to provide signal to noise in the NMR experiment in all groups. The ratio of pyruvate carboxylase to total pyruvate entry into the TCA cycle did not differ between groups (group I: 20 [+ or -] 4%, group II: 23 [+ or -] 7%, and control: 27 [+ or -] 7%). These data show that robust PC operates in the neonatal pig heart and is maintained during reperfusion under conditions that emulate CPB and reperfusion in human infants. cardiac metabolism; pyruvate carboxylation; pyruvate decarboxlyation; citric acid cycle; tricarboxylic acid cycle

We report new assays of enzymes uroporphyrinogen decarboxylase (UROD) and coproporphyrinogen III oxidase (CPO) in the heme biosynthetic pathway. The assays were developed for use in clinical diagnostics of inherited disorders porphyria cutanea tarda and hereditary coproporphyria, respectvely. Electrospray ionization tandem mass spectrometry is used to monitor the decarboxylafion of pentaporphyrinogen I or uroporphyrinogen III catalyzed by UROD and to determine the enzyme activity in human erythrocytes by measuring the production of coproporphyrinogen I or III. The [K.sub.m] value for pentaporphyrinogen I was measured as 0.17 [+ or -] 0.03 [micro]M. A mass spectrometric assay was also developed for the two-step decarboxylative oxidation of coproporphyrinogen III to protoporphyrinogen IX catalyzed by CPO in mitochondria from human lymphocytes ([K.sub.m] = 0.066 [+ or -] 0.009 [micro]M). The assays show good reproducibility, use simple workup by liquid-liquid extraction of enzymatic products, and employ commercially available substrates and internal standards.

We report substantial in situ enrichment of phosphopeptides in peptide mixtures using zirconium oxide coated plates for detection by MALDI-TOF mass spectrometry. The novel feature of this approach rests on the specific preparation of zirconium oxide coatings using reactive landing on stainless steel support of gas-phase positive ions produced by electrospray of zirconium(IV)-n-propoxide solutions in 1-propanol. Reactive landing was found to produce durable functionalized surfaces for selective phosphopeptide capture and desorption-ionization by MALDI. Enrichment factors on the order of 20-90 were achieved for several monophosphorylated peptides relative to abundant nonphosphorylated peptides in tryptic digests. We demonstrate the ability of the zirconium oxide functionalized MALDI surfaces to facilitate detection of enriched phosphopeptides in mid-femtomole amounts of m-casein digests per MALDI spot.

The formation of transient, hypervalent ammonium radicals via the fast collisional reduction of benzylalkylammonium ions in the gas phase was examined. Likewise, the dissociations occurring in the ammonium center, aromatic ring or substituents of the intermediate radicals were investigated. It was found that the dissociations, which depend on the substituents at the amine nitrogen atom and in the aromatic ring, occur via C-N and N-H bond cleavages and result in stable molecules and the corresponding radicals.