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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

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

We report a now assay of human [delta]-aminolevulinic acid dehydratase (ALAD), an enzyme converting [delta]-aminolevulinic acid (ALA) into porphobilinogen. The assay is developed for use in the clinical diagnosis of [delta]-aminolevulinic acid dehydratase-deficient porphyria, a rare enzymatic deficiency of the heme biosynthetic pathway. The assay involves the incubation of erythrocyte lysate with the natural substrate, ALA, followed by quantitative in situ conversion of porphobilinogen to its butyramide, and liquid-liquid extraction into a mass spectrometer-friendly solvent. Quantitation of the butyrylated porphobilinogen is done by electrospray ionization tandem mass spectrometry, using a deuterium labeled internal standard. The assay stays well within the range wherein ALAD activity is linear with time. The [K.sub.m] of ALAD for ALA was measured as 333 [micro]M, and the [V.sub.max] was 10.3 [micro]M/h. Average enzyme activity among a random sample of 36 anonymous individuals was 277 [micro]mol/L erythrocyte lysate/hour with a standard deviation of 90 [micro]mol/L erythrocyte lysate/hour. The tandem mass spectrometric assay should easily detect the enzyme deficiency, which causes a reduction of activity by 95-99%. The assay shows good reproducibility and low background, requires a simple workup, and uses a commercially available substrate. 10.1021/ac101111m

We report a new assay of human porphobilinogen deaminase (PBGD). Deficiency in this enzyme activity causes acute intermittent porphyria, the most common disorder of heme biosynthesis. The assay involves incubation of blood erythrocyte lysate with porphobilinogen, the natural PBGD substrate. Two subsequent enzymes in the heme biosynthetic pathway, uroporphyrinogen III synthase and uroporphyrinogen decarboxylase, are deactivated by heating so that their activity does not interfere with the PBGD assay. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) is used to monitor the production of uroporphyrinogen I and thus measure the PGBD activity. A simple and efficient workup using liquid--liquid extraction with >90% product recovery was employed to avoid separation by liquid chromatography. The assays show good reproducibility ([+ or -]3.3%) and linear dependence of the uroporphyrinogen I formation on incubation time and protein amount. The [K.sub.m] of PGBD for porphobilinogen was measured as 11.2 [+ or -] 0.5 [micro]M with [V.sub.max] of 0.0041 [+ or -] 0.0002 [micro]M/(min x mg of hemoglobin). The coefficient of variation of PBGD activity among several unaffected individuals (12%) is significantly lower than the decrease due to acute intermittent porphyria (50%).

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.

Diaminohydroxymethyl and triaminomethyl radicals are generated by femtosecond collisional electron transfer to their corresponding cations and characterized by neutralization-reionization mass spectrometry and ab initio/RRKM calculations. The studies have shown that the [pi]-donating N[H.sub.2] groups have increased the stability of the substituted methyl radicals, but the stabilities are not correlated with the radical ionization energies.

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 proline effect in model dipeptides Pro-Gly and Gly-Pro is studied. Kinetic analysis of the Gly-Pro radical system has shown that ring-openings can be triggered by radical sites at both sides of the proline residue in larger peptides and the isomerizations within the proline residue has described the proline effect in electron capture and transfer dissociation.

Surface-enhanced Raman spectroscopy (SERS) was used to detect and characterize polyatomic cations and molecules that were electrosprayed into the gas phase and soft-landed in vacuum on plasma-treated silver substrates. Organic dyes such as crystal violet and Rhodamine B, the nucleobase cytosine, and nucleosides cytidine and 2'-deoxycytidine were immobilized by soft landing on plasma-treated metal surfaces at kinetic energies ranging from near thermal to 200 eV. While enhancing Raman scattering 105-106-fold, the metal surface effectively quenches the fluorescence that does not interfere with the Raman spectra. SERS spectra from submonolayer amounts of soft-landed compounds were sufficiently intense and reproducible to allow identification of Raman active vibrational modes for structure assignment. Soft-landed species appear to be microsolvated on the surface and bound via ion pairing or [pi]-complexation to the Ag atoms and ions in the surface oxide layer. Comparison of spectra from soft-landed and solution samples indicates that the molecules survive soft landing without significant chemical damage even when they strike the surface at hyperthermal collision energies.

We report a new method called metal affinity capture that when coupled with tandem mass spectromery (MAC-MSMS) allows for the selective detection and identification of phosphopeptides in complex mixtures. Phosphopeptides are captured as ternary complexes with [Ga.sup.III] or [Fe.sup.III] and [N.sub.[alpha]],[N.sub.[alpha]]-bis(carboxymethyl)lysine (LysNTA) in solution and electrosprayed as doubly or triply charged positive ions. The gas-phase complexes uniformly dissociate to produce a common (LysNTA + H)+ ion that is used as a specific marker in precursor ion scans. The advantages of MAC-MSMS over the current methods of phosphopeptide detection are as follows. (1) MAC-MSMS uses metal complexes that self-assemble in solution at pH

An experimental and computational study of hydrogen atom adducts to adenine is conducted. The result shows that the relative free energies of adenine radicals in aqueous solution point to the C-8 adducted as the most stable tautomer, which is predicted to be the predominating product at thermal equilibrium in solution at 298 K.

Soft landing of singly charged gas-phase ions on dry metal surfaces that were pretreated in situ by oxygen plasma results in 0.1-2% total yields of recovered intact compounds. Lysine, peptides, crystal violet dye, and a biotin conjugate are found to survive soft landing of hyperthermal ions of up to 50-eV kinetic energy. Soft landing at 40-50-eV ion kinetic energies of a fluorescence-labeled biotin conjugate results in an immobilized fraction that cannot be washed from the surface and is found to contain an intact biotin moiety. The present results represent an ~[10.sup.4] fold improvement in soft-landing efficiency and indicate that plasma-treated metal surfaces can be useful for preparative separation of organic and biological molecules by mass spectrometry. The substantial improvement in soft-landing yields results from a high transmission of electrosprayed ions into the vacuum system, efficient and nondestructive discharge of ions on the metal oxide surface, and facile analyte recovery in the absence of a matrix.

Soft landing on a plasma-treated metal surface of multiply protonated protein ions from the gas phase results in a substantial retention of protein function, as demonstrated for trypsin and streptavidin. The majority of trypsin ions soft-landed at hyperthermal kinetic energies are undamaged and retain 72-98% of enzymatic activity after being washed into solution. A small fraction of trypsin ions that were landed at nominal kinetic energies of 130-200 eV remain tethered to the surface and show ~50% enzymatic activity. The streptavidin tetramer is found to dissociate to monomer units upon multiple charging in electrospray. The majority of soft-landed monomers can be washed into solution where they show affinity to biotin. The layer of streptavidin monomer that is immobilized on the surface can be detected if fluorescence-tagged and retains the ability to reversibly bind biotin. A mechanism is proposed to explain nondestructive protein ion discharge on the surface that considers proton migration from the softlanded cations to the metal oxide layer and metal ion reduction by electron transfer from the bulk metal.