A mouse model for mucopolysaccharidosis type III A (Sanfilippo syndrome)

Mucopolysaccharidosis type III A (MPS III A, Sanfilippo syndrome) is a rare, autosomal recessive, lysosomal storage disease characterized by accumulation of heparan sulfate secondary to defective function of the lysosomal enzyme heparan N- sulfatase (sulfamidase). Here we describe a spontaneous mouse mutant that replicates many of the features found in MPS III A in children. Brain sections revealed neurons with distended lysosomes filled with membranous and floccular materials with some having a classical zebra body morphology. Storage materials were also present in lysosomes of cells of many other tissues, and these often stained positively with periodic-acid Schiff reagent. Affected mice usually died at 7-10 months of age exhibiting a distended bladder and hepatosplenomegaly. Heparan sulfate isolated from urine and brain had nonreducing end glucosamine- N -sulfate residues that were digested with recombinant human sulfamidase. Enzyme assays of liver and brain extracts revealed a dramatic reduction in sulfamidase activity. Other lysosomal hydrolases that degrade heparan sulfate or other glycans and glycosaminoglycans were either normal, or were somewhat increased in specific activity. The MPS III A mouse provides an excellent model for evaluating pathogenic mechanisms of disease and for testing treatment strategies, including enzyme or cell replacement and gene therapy.     

Identification of the gene encoding the enzyme deficient in mucopolysaccharidosis IIIC (Sanfilippo disease type C)

Mucopolysaccharidosis IIIC (MPS IIIC), or Sanfilippo C, represents the only MPS disorder in which the responsible gene has not been identified; however, the gene has been localized to the pericentromeric region of chromosome 8. In an ongoing proteomics study of mouse lysosomal membrane proteins, we identified an unknown protein whose human homolog, TMEM76, was encoded by a gene that maps to 8p11.1. A full-length mouse expressed sequence tag was expressed in human MPS IIIC fibroblasts, and its protein product localized to the lysosome and corrected the enzymatic defect. The mouse sequence was used to identify the full-length human homolog (HGSNAT), which encodes a protein with no homology to other proteins of known function but is highly conserved among plants and bacteria. Mutational analyses of two MPS IIIC cell lines identified a splice-junction mutation that accounted for three mutant alleles, and a single base-pair insertion accounted for the fourth.     

Analysis of mammalian fatty acyl-coenzyme A species by mass spectrometry and tandem mass spectrometry

Acyl-CoAs are intermediates of numerous metabolic processes in eukaryotic cells, including beta-oxidation within mitochondria and peroxisomes, and the biosynthesis/remodeling of lipids (e.g. mono-, di-, and triglycerides, phospholipids and sphingolipids). Investigations of lipid metabolism have been advanced by the ability to quantitate acyl-CoA intermediates via liquid chromatography coupled to electrospray ionization-tandem mass spectrometric detection (LC-ESI-MS/MS), which is presently one of the most sensitive and specific analytical methods for both lipids and acyl-CoAs. This review of acyl-CoA analysis by mass spectrometry focuses on mammalian samples and long-chain analytes (i.e. palmitoyl-CoA), particularly reports of streamlined methodology, improved recovery, or expansion of the number of acyl chain-lengths amenable to quantitation.     

Contemporary mass spectrometry for the direct detection of enzyme intermediates

The field of enzymology has long used small-molecule mass spectrometry. However, the direct interrogation of covalent and non-covalent intermediates by large-molecule mass spectrometry of enzymes or large peptide substrates is illuminating an increasingly diverse array of chemistries used in nature. Recent advances now allow improved detection of several modifications formed at sub-stoichiometric levels on the same polypeptide, and elucidation of intermediate dynamics with low millisecond temporal resolution. Highlighting recent applications in both ribosomal and non-ribosomal biosynthesis of natural products, along with acetyl transferases, sulfonucleotide reducatases, and PEP-utilizing enzymes, the utility of small- and large-molecule mass spectrometry to reveal enzyme intermediates and illuminate mechanism is described briefly. From ever more complex mixtures, mass spectrometry continues to evolve into a key technology for a larger number of today's enzymologists.     

Analysis of mammalian sphingolipids by liquid chromatography tandem mass spectrometry (LC-MS/MS) and tissue imaging mass spectrometry (TIMS)

Sphingolipids are a highly diverse category of molecules that serve not only as components of biological structures but also as regulators of numerous cell functions. Because so many of the structural features of sphingolipids give rise to their biological activity, there is a need for comprehensive or "sphingolipidomic" methods for identification and quantitation of as many individual subspecies as possible. This review defines sphingolipids as a class, briefly discusses classical methods for their analysis, and focuses primarily on liquid chromatography tandem mass spectrometry (LC-MS/MS) and tissue imaging mass spectrometry (TIMS). Recently, a set of evolving and expanding methods have been developed and rigorously validated for the extraction, identification, separation, and quantitation of sphingolipids by LC-MS/MS. Quantitation of these biomolecules is made possible via the use of an internal standard cocktail. The compounds that can be readily analyzed are free long-chain (sphingoid) bases, sphingoid base 1-phosphates, and more complex species such as ceramides, ceramide 1-phosphates, sphingomyelins, mono- and di-hexosylceramides, sulfatides, and novel compounds such as the 1-deoxy- and 1-(deoxymethyl)-sphingoid bases and their N-acyl-derivatives. These methods can be altered slightly to separate and quantitate isomeric species such as glucosyl/galactosylceramide. Because these techniques require the extraction of sphingolipids from their native environment, any information regarding their localization in histological slices is lost. Therefore, this review also describes methods for TIMS. This technique has been shown to be a powerful tool to determine the localization of individual molecular species of sphingolipids directly from tissue slices.     

Evaluation of a commercial enzyme immunoassay kit (RIDASCREEN) for detection of staphylococcal enterotoxins A, B, C, D, and E in foods.

The RIDASCREEN SET kit (R-Biopharm GmbH, Darmstadt, Germany), a commercial staphylococcal enterotoxin (SE) visual immunoassay kit, was evaluated for its efficacy. The kit utilizes monovalent capture antibodies against SE types A to E (SEA to SEE); therefore, it simultaneously detects and identifies the enterotoxin types. The major advantages of the kit are (i) a high degree of specificity (except for naturally occurring peroxidases, food compositions or ingredients and microbiological products due to growth of nonstaphylococcal microorganisms did not cause false-positive results; additionally, no cross-reactions among reagents of the kits were observed), (ii) excellent sensitivity (minimum detectable limits were 0.20 to 0.30 ng of SEs per ml of extracts of ham, salami, and mushroom and 0.30 to 0.35 ng of SEs per ml of cheese extracts, or 0.50 to 0.75 ng of SEs per g of foods such as noodles, ham, salami, cheese, and turkey), (iii) simplicity (the kit enabled direct assay of SEs in food extracts without the need for lengthy extraction or concentration procedures), (iv) rapidity (it took less than 3 h to complete the analysis of individual enterotoxin types SEA to SEE), and (v) its semiquantitative results (optical density values could be read against a standard curve to estimate the amount of SE in the extract). The RIDASCREEN kit is a convenient, rapid, and reliable tool for the detection and identification of SEs in foods.     

Differentiation and quantification of C1 and C2 C-labeled glucose by tandem mass spectrometry

The fragmentation patterns of various ¹³C-labeled glucose molecules were analyzed by electrospray ionization tandem mass spectrometry. Derivatization of glucose to yield methylglucosamine makes the C-C bond between C1 and C2 a favored cleavage site. This is in contrast to underivatized glucose, which favorably undergoes loss of a fragment containing both C1 and C2. Based on the fragmentation pattern of methylglucoasmine, we developed a method to distinguish and quantify C1 and C2 ¹³C-labeled glucose by derivatization with methylamine followed by multiple reaction monitoring scans in a Q-trap mass spectrometer. Fragment ion ratios in the tandem mass spectra showed an isotope effect with ¹³C or deuterium labeling, so a “correction factor” was introduced to make the quantification more accurate. The current approach can be applied to individually monitor the metabolic origin and fate of C1 and C2 atoms in ¹³C-labeled glucose. This method provides a new means of quantifying glucose isotopomers in metabolic studies.     

The synthesis of substrates and two assays for the detection of N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase (uncovering enzyme).

A method for the synthesis and purification of large quantities of four radiolabeled substrates for quantitation of uncovering enzyme is described. Four substrates, [3H]GlcNAc-alpha-P-Man alpha Me, [3H]GlcNAc-alpha-P-uteroferrin, [3H]GlcNAc alpha-P-Man alpha 1-2Man-O-Me, and [3H]GlcNAc alpha-P-Man9GlcNAc, were enzymatically synthesized using GlcNAc-phosphotransferase from Acanthamoeba castellanii and uridine diphosphate N-acetyl-[3H]glucosamine and, as acceptor, methyl-alpha-D-mannopyranoside (Man alpha Me), uteroferrin, Man alpha 1-2Man-O-methyl, or Man9GlcNAc. The isolation of the [3H]GlcNAc-P-modified product of each reaction is detailed. Two assays for the detection of uncovering enzyme activity using [3H]GlcNAc-alpha-P-uteroferrin and [3H]GlcNAc-alpha-P-Man alpha Me are outlined. The ability to easily synthesize four relevant substrates for uncovering enzyme offers flexibility in assaying uncovering enzyme.     

Neonatal onset of organic acidemia (propionic) diagnosed by tandem mass spectrometry

Propionic acidemia is an autosomal recessive disorder as a result of a deficient activity of propionyl-CoA carboxylase. Propionyl CoA is metabolized by propionyl-CoA carboxylase to methylmalonyl CoA. Propionic acidemia is a major cause of ketotic hyperglycinemia. This disorder is characterized by episodic vomiting, dehydratation, feeding intolerante, lethargy, hypotonia, metabolic acidosis, ketosis and hyperammonemia. The patient presented herein was a full-term female newborn with encephalopathy in the first days of life. She presented hypoglycemia, metabolic acidosis with increased anion gap, ketosis, hyperammonemia, anemia, leukopenia and thrombocytopenia. The brain ultrasonography was normal. The tandem mass expectrometry done by Pediatrix was abnormal, with the acylcarnitine results consistent with an organic acidemia. The parents are consanguineus and have a history of abortus, miscarriage and neonatal death, characteristics suggestive of the presence of genetic defects.     

Determination of tylosins A, B, C and D in bee larvae by liquid chromatography coupled to ion trap-tandem mass spectrometry

A LC-MS/MS method has been developed to simultaneously quantify tylosins A, B, C and D in bee larvae, compounds currently used to treat one of the most lethal diseases affecting honey bees around the world, American Foulbrood (AFB). The influence of different aqueous media, temperature and light exposure on the stability of these four compounds was studied. The analytes were extracted from bee larvae with methanol and chromatographic separation was achieved on a Luna C(18) (150 × 4.6 mm i.d.) using a ternary gradient composed of a diluted formic acid, methanol and acetonitrile mobile phase. To facilitate sampling, bee larvae were initially dried at 60°C for 4h and afterwards, they were diluted to avoid problems of pressure. MSD-Ion Trap detection was employed with electrospray ionization (ESI). The calibration curves were linear over a wide range of concentrations and the method was validated as sensitive, precise and accurate within the limits of quantification (LOQ, 1.4-4.0 ng/g). The validated method was successfully employed to study bee larvae in field tests of bee hives treated with two formulations containing tylosin. In both cases it was evident that the minimal inhibitory concentration (MIC) had been reached.     

New iridium(I) complexes with labile ligands: reactivity and structural characterization by atmospheric pressure mass and tandem mass spectrometry

Reaction of diphosphine complexes [IrCl{(C₆F₅)₂P(CH₂)₂P(C₆F₅)₂}]₂ (I) and [IrCl(dppe)]₂ (II) with coordinating solvents (acetonitrile, acetone, DMSO) leads to several square-planar complexes of the type [IrCl(diphosphine)(solvent)] which are stable only in solution ([IrCl{(C₆F₅)₂P(CH₂)₂P(C₆F₅)₂}(NCCH₃)] (III) and [IrCl{(C₆F₅)₂P(CH₂)₂P(C₆F₅)₂}(acetone)], IV) and/or can be detected only under APCI-MS/MS conditions ([IrCl(dppe)(solvent)]). When III is allowed to react with CO for at least 30 min, the unusual five coordinated trans-dicarbonyl complex [IrCl{(C₆F₅)₂P(CH₂)₂P(C₆F₅)₂}(CO)₂] (Vb) is formed, as characterized by ¹H and ³¹P NMR, FT-IR, TGA and APCI-MS/MS.A new and stable square-planar complex [Ir(OCH₃)(cod)(PClPh₂)] (IX) was also synthesized. Its APCI-MS/MS spectrum is simple and unique as it shows exclusively the loss of a neutral C₃H₂ species. Along with the APCI-MS and APCI-MS/MS analyses, whenever it was possible all complexes were also characterized by ¹H and ³¹P NMR spectroscopy.     

Validation of salivary cortisol and testosterone assays in chimpanzees by liquid chromatography‐tandem mass spectrometry

Owing to its high temporal sensitivity, saliva has distinct advantages for measuring steroids, compared with other noninvasive samples such as urine and feces. Here, we report the validity of assaying salivary cortisol (C) and testosterone (T) using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) in captive male chimpanzees, Pan troglodytes. For both the C and T concentrations, we found positive relationships between saliva and plasma. The concentrations of C and T in saliva showed clear patterns of diurnal fluctuation, whereas those in urine and feces did not. These results suggest that the salivary steroid concentrations can be regarded as good indicators of circulating steroid levels. We also developed and validated an efficient method for collecting saliva samples from cotton rope. Although rope includes inherent steroid‐like compounds and may affect the accuracy of steroid measurements, our rope‐washing procedures effectively removed intrinsic steroidal materials. There was a significant association between the C and T concentrations measured from saliva collected from rope licked by the chimpanzees and those measured from saliva collected directly from the mouth. Salivary T values estimated by LC/MS‐MS were similar to those measured by radioimmunoassay. The results indicate the usefulness of saliva as a noninvasive steroid measure and that steroids in the saliva of chimpanzees can be accurately measured by LC‐MS/MS. Am. J. Primatol. 71:696–706, 2009. © 2009 Wiley‐Liss, Inc.     

Quantitative bioanalysis of peptides by liquid chromatography coupled to (tandem) mass spectrometry

With the growing interest for peptides and proteins in different kinds of fields, e.g. pharmacy, clinical diagnostics or food industry, the quantification of these compounds is becoming more and more important. Quantitative analysis of these analytes in biological matrices, however, remains a challenging task, due to the complexity of both the matrix and the analytical characteristics of these large bio-molecules. Liquid chromatography coupled to (tandem) mass spectrometry (LC-MS or LC-MS/MS) is the preferred analytical technique for peptide analysis as it allows very selective and sensitive measurements. This article summarizes the numerous published LC-MS applications for the quantification of peptides in biological matrices and discusses all different issues herewith concerned. This includes chromatographic aspects as the selection and effects of mobile and stationary phase, flow rate and temperature, as well as mass spectrometric characteristics such as ionization and detection modes, collision-induced dissociation of peptides and factors influencing the mass spectrometric response. For both techniques the main properties of all described methods have been listed, creating a comprehensive overview with the peptide analytes divided into different classes. Likewise, all other issues concerned with quantitative bioanalysis have been evaluated in detail, including extensive consideration of several different applied sample pre-treatment techniques and reflection of subjects as the choice for an internal standard and assay validation. Furthermore, several issues which are of particular interest for the quantitative bioanalysis of peptide compounds like peptide adsorption and degradation have been regarded.     

Rapid and quantitative analysis of unconjugated C(27) bile acids in plasma and blood samples by tandem mass spectrometry

A subgroup of peroxisomal disorders, peroxisome biogenesis defects (PBD), can be differentiated by elevated levels of C(27) bile acids in plasma and bile. Patients with peroxisomal disorders, who lack the ability to chain-shorten the C(27) bile acid intermediates into C(24) bile acids, show elevated levels of C(27) bile acids, notably 3 alpha,7 alpha-dihydroxy-5 beta-cholest-26-oic acid and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid. C(27) bile acids are normally estimated against other bile acid standards, by time-consuming gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry methods, in plasma (minimum of 50 microl). In this article we describe the quantitation of unconjugated di- and trihydroxy C(27) bile acids in 5-microl plasma samples and 3-mm blood spots, using deuterium-labeled internal standards. The synthesis of (2)H(3)-labeled di- and trihydroxycoprostanic acids is described. The sample preparation and analysis by electrospray tandem mass spectrometry (ES-MS/MS) takes less than 1 h and features dimethylaminoethyl ester derivatives. The levels of the di- and trihydroxy bile acids are significantly higher in PBD patients than in age-matched control subjects for both plasma and blood spots collected at birth (some stored for up to 18 years). Excellent correlation is observed between the C(26:0)/C(22:0) very long chain fatty acid (VLCFA) ratio and the levels of trihydroxy C(27) bile acids in plasma from PBD patients.The ES-MS/MS method can be used to rapidly screen for PBD patients in plasma samples with elevated C(26:0)/C(22:0) VLCFA ratios and in archived collections of neonatal blood spots. - Johnson, D. W., H. J. ten Brink, R. C. Schuit, and C. Jakobs. Rapid and quantitative analysis of unconjugated C(27) bile acids in plasma and blood samples by tandem mass spectrometry. J. Lipid Res. 2001. 42: 9;-16.     

Measurement of carnitine biosynthesis enzyme activities by tandem mass spectrometry: differences between the mouse and the rat

Although the mouse frequently is used to study metabolism and deficiencies therein, little is known about carnitine biosynthesis in this animal. To this point, only laborious procedures have been described to measure the activity of carnitine biosynthesis enzymes using subcellular fractions as the enzyme source. We developed two simple tandem mass spectrometry-based methods to determine the activity of three carnitine biosynthesis enzymes (6-N-trimethyllysine dioxygenase, 4-trimethylaminobutyraldehyde dehydrogenase, and 4-trimethylaminobutyric acid dioxygenase) in total homogenates that can be prepared from frozen tissue. The new assays were used to characterize these enzymes in mouse liver homogenate. Because carnitine biosynthesis has been studied extensively in the rat, we compared the mouse tissue distribution of carnitine biosynthesis enzyme activities and levels of the biosynthesis metabolites with those in the rat to determine which tissues contribute to carnitine biosynthesis in these species. Surprisingly, large differences in enzyme activities were found between the rat and the mouse, whereas carnitine biosynthesis metabolite levels were very similar in both species, possibly due to the different kinetic properties of the first enzyme of carnitine biosynthesis. Also, muscle carnitine levels were found to vary considerably between these two species, suggesting that there is a metabolic dissimilarity between the mouse and the rat.     

A rapid screening procedure for cholesterol and dehydrocholesterol by electrospray ionization tandem mass spectrometry

The mono-(dimethylaminoethyl) succinyl (MDMAES) ester is a new derivative for rapid, mild, and sensitive electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis of cholesterol and dehydrocholesterol. It is an order of magnitude more sensitive than the previous most practical alternative, the N-methylpyridyl ether derivative. The MDMAES derivative was used to develop a rapid screening procedure for the biochemical diagnosis of Smith-Lemli-Opitz syndrome (SLOS) by measuring the dehydrocholesterol/cholesterol ratio in plasma (5 microl) and plasma spotted onto filter paper. Details of the synthesis of [25,26,26,26,27,27,27-(2)H7]-7-dehydrocholesterol, used as a standard for quantitation, are included. The measurement of total sterols as MDMAES esters, after base hydrolysis of plasma, afforded a dehydrocholesterol/cholesterol ratio of 0.05-2.95 for SLOS patient samples (n = 5) compared with 0.001-0.003 for normal adult controls (n = 20). Direct hexane extraction of plasma without base hydrolysis enabled the measurement of free sterols with a total sample analysis time of <1 h. The free dehydrocholesterol/cholesterol ratio was 0.10-4.47 for SLOS patient samples (n = 5) and 0.003-0.011 for normal adult controls (n = 20).     

A dataset of human cornea proteins identified by Peptide mass fingerprinting and tandem mass spectrometry

Diseases of the cornea are extremely common and cause severe visual impairment worldwide. To explore the basic molecular mechanisms involved in corneal health and disease, the present study characterizes the proteome of the normal human cornea. All proteins were extracted from the central 7-mm region of 12 normal human donor corneas containing all layers: epithelium, Bowman's layer, stroma, Descemet's membrane, and endothelium. Proteins were fractionated and identified using two different procedures: (i) two-dimensional gel electrophoresis and protein identification by MALDI-MS and (ii) strong cation exchange or one-dimensional SDS gel electrophoresis followed by LC-MS/MS. All together, 141 distinct proteins were identified of which 99 had not previously been identified in any mammalian corneas by direct protein identification methods. The characterized proteins are involved in many processes including antiangiogenesis, antimicrobial defense, protection from and transport of heme and iron, tissue protection against UV radiation and oxidative stress, cell metabolism, and maintenance of intracellular and extracellular structures and stability. This proteome study of the healthy human cornea provides a basis for further analysis of corneal diseases and the design of bioengineered corneas.     

Design and synthesis of substrate and internal standard conjugates for profiling enzyme activity in the Sanfilippo syndrome by affinity chromatography/electrospray ionization mass spectrometry.

We describe the design and synthesis of substrate and internal standard conjugates for application in profiling enzyme activity of the enzymes alpha-D-2-deoxy-2-N-sulfonamido-glucosamine sulfamidase, alpha-D-2-deoxy-2-N-acetyl-glucosamine hydrolase, acetyl-coenzymeA:alpha-D-2-deoxy-2-amino-glucosamine transferase, and alpha-D-2-deoxy-2-N-acetyl-glucosamine-6-sulfate sulfatase. Deficiency of any one of these enzymes results in a single clinical phenotype known as Sanfilippo syndrome. Such substrates have been proven effective in the confirmation of enzyme deficiency by a combination of affinity chromatography (AC) and electrospray ionization mass spectrometry (ESIMS), which forms the foundation for a new analytical technology (ACESIMS) of general interest and application to clinical and biomedical research.     

Simultaneous determination of decitabine and vorinostat (Suberoylanalide hydroxamic acid, SAHA) by liquid chromatography tandem mass spectrometry for clinical studies

A reverse-phase high-performance liquid chromatography method with electrospray ionization and detection by tandem mass spectrometry is described for the simultaneous quantitative determination of decitabine (5-aza-2'-deoxycytidine) and vorinostat (Suberoylanalide hydroxamic acid, SAHA) in human plasma. The method involves a simple acetonitrile precipitation step and centrifugation followed by injection of the supernatant onto a C18 150mmx2.1mm I.D., 3microm HPLC column at 36 degrees C. Separation of decitabine, SAHA and their respective internal standards was achieved with a gradient elution and detection was via the mass spectrometer operated in selected reaction monitoring mode. The method was within the defined validation parameters for linearity, repeatability, reproducibility and stability. The limit of detection was determined as 1.0 and 0.125ngml(-1) and lower limits of quantitation were 10 and 1ngml(-1) for decitabine and SAHA, respectively. Effects of sample preparation on stability were also evaluated in human plasma. For clinical sample handling tetrahydrouridine, an inhibitor of cytidine deaminase was found to help prevent decitabine degradation. The method is currently being used in clinical pharmacokinetic studies for the evaluation of decitabine and SAHA combination therapies.