Accurate mass measurement and tandem mass spectrometry of intact globin chains identify the low proportion variant hemoglobin Lepore-Boston-Washington from the blood of a heterozygote.

Within a mixture of proteins, minor polymorphic components are difficult to identify using a conventional proteomic approach. Their identification generally requires multi-dimensional separation steps, before or after proteolytic cleavage, followed by sequence analysis of the proteolytic products. In this study, we investigated the potential of tandem mass spectrometry for protein characterization by identifying the delta-beta hybrid human hemoglobin variant Lepore-Boston-Washington using electrospray ionization tandem mass spectrometry.

A proteomic approach to the identification of cytochrome P450 isoforms in male and female rat liver by nanoscale liquid chromatography-electrospray ionization-tandem mass spectrometry.

Nanoscale reversed-phase liquid chromatography (LC) combined with electrospray ionization-tandem mass spectrometry (ESI-MS/MS) has been used as a method for the direct identification of multiple cytochrome P450 (P450) isoforms found in male and female rat liver. In this targeted proteomic approach, rat liver microsomes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by in-gel tryptic digestion of the proteins present in the 48- to 62-kDa bands. The resultant peptides were extracted and analyzed by LC-ESI-MS/MS.

Observation of an intact noncovalent homotrimer of detergent-solubilized rat microsomal glutathione transferase-1 by electrospray mass spectrometry.

Microsomal glutathione transferase-1 (MGST1) is a membrane-bound enzyme involved in the detoxification of xenobiotics and the protection of cells against oxidative stress. The proposed active form of the enzyme is a noncovalently associated homotrimer that binds one substrate glutathione molecule/trimer. In this study, this complex has been directly observed by electrospray mass spectrometry analysis of active rat liver MGST1 reconstituted in a minimum amount of detergent.

Neurosteroids in rat brain: extraction, isolation, and analysis by nanoscale liquid chromatography-electrospray mass spectrometry.

A method designed for the analysis of sulfated neurosteroids and unconjugated ketonic neurosteroids in rat brain using nanoscale liquid chromatography-electrospray (nano-LC-ES) mass spectrometry is described. Neurosteroids in rat brain tissue were extracted, purified, and separated into two groups, neutral unconjugated steroids and steroid sulfates, by employing solid-phase partition, cation- and anion-exchange chromatography. The steroid sulfate fraction was analyzed by nano-LC-ES mass spectrometry.

Characterisation of alpha-1 giardin: an immunodominant Giardia lamblia annexin with glycosaminoglycan-binding activity.

Alpha-1 giardin is an immunodominant protein in the intestinal protozoan parasite Giardia lamblia. The Triage((R)) parasite panel, used to detect copro-antigens in stool from giardiasis patients, reacts with an epitope between amino acids 160 and 200 in alpha-1 giardin. This region of the protein is also highly immunogenic during human infections.

Distinct but parallel evolutionary patterns between alcohol and aldehyde dehydrogenases: addition of fish/human betaine aldehyde dehydrogenase divergence.

Alcohol dehydrogenases (ADHs) of the MDR type (medium-chain dehydrogenases/reductases) have diverged into two evolutionary groups in eukaryotes: a set of 'constant' enzymes (class III) typical of basal enzymes, and a set of 'variable' enzymes (remaining classes) suggesting 'evolving' forms. The variable set has larger overall variability, different segment variability, and variability also in functional segments. Using a major aldehyde dehydrogenase (ALDH) from cod liver and fish ALDHs deduced from the draft genome sequence of Fugu rubripes (Japanese puffer fish), we found that ALDHs form more complex patterns than the ADHs.

Tandem mass spectrometry in the study of fatty acids, bile acids, and steroids.

Over the last 50 years, the mass spectrometry of lipids has evolved to become one of the most mature techniques in biomolecule analysis. Many volatile and non-polar lipids are directly amenable to analysis by gas-chromatography-mass spectrometry (GC-MS), a technique that combines the unsurpassed separation properties of gas-chromatography with the sensitivity and selectivity of electron ionization mass spectrometry. Less volatile and/or thermally labile lipids can be analyzed by GC-MS, following appropriate sample derivatization.

Identification of immunoreactive proteins during acute human giardiasis.

The protozoan Giardia lamblia is a major cause of parasite-induced diarrhea in humans. Humoral immunity has been shown to be important for clearance of the infection, but only a few antigens have been identified. In this study, we focused on the immunoreactivity of nonvariant antigens.

Accurate mass measurement by electrospray ionization quadrupole mass spectrometry: detection of variants differing by <6 Da from normal in human hemoglobin heterozygotes.

Mass spectrometry has a basic limitation when human hemoglobin variants are analyzed, because it cannot resolve two globin chains that differ in mass by <6 Da. Several common beta-chain variants differ by 1 Da from normal and, hence, when present in heterozygotes, are not resolved from the normal beta-chain. Normal and variant chains appear together in the spectrum as a single entity, whose mass is the abundance weighted mean of the two chains.

Artifactually low glycated haemoglobin in a patient with severe hypertriglyceridaemia.

This report describes a case of artifactually low glycated haemoglobin (Hb) in a patient with type II diabetes and severe hypertriglyceridaemia. The effect of hypertriglyceridaemia on glycated Hb determination using the Abbott Vision method was investigated in a series of patients with diabetes. The interference of triglycerides in glycated Hb assays was also investigated by two other methods, the Beckman Synchron CX4 delta immunoturbidimetric method, and the Primus affinity chromatography high performance liquid chromatography assay.

Derivatisation for the characterisation of neutral oxosteroids by electrospray and matrix-assisted laser desorption/ionisation tandem mass spectrometry: the Girard P derivative.

The identification, quantification and localisation of steroids in biological fluids and tissues are subjects of considerable importance. Not only do steroids have classical hormonal properties via binding to nuclear receptors, they can also elicit cellular responses via interactions with other proteins. For mass spectrometric analysis, neutral steroids are not readily ionised by either electrospray (ES) or matrix-assisted laser desorption/ionisation (MALDI).

Co-localization of the mammalian hemochromatosis gene product (HFE) and a newly identified transferrin receptor (TfR2) in intestinal tissue and cells.

Mutations in the HFE gene and a newly identified second transferrin receptor gene, TfR2, cause hemochromatosis. The cognate proteins, HFE and TfR2, are therefore of key importance in human iron homeostasis. HFE is expressed in small intestinal crypt cells where transferrin-iron entry may determine subsequent iron absorption by mature enterocytes, but the physiological function of TfR2 is unknown.

Reduction of S-nitrosoglutathione by human alcohol dehydrogenase 3 is an irreversible reaction as analysed by electrospray mass spectrometry.

Human alcohol dehydrogenase 3/glutathione-dependent formaldehyde dehydrogenase was shown to rapidly and irreversibly catalyse the reductive breakdown of S-nitrosoglutathione. The steady-state kinetics of S-nitrosoglutathione reduction was studied for the wild-type and two mutated forms of human alcohol dehydrogenase 3, mutations that have previously been shown to affect the oxidative efficiency for the substrate S-hydroxymethylglutathione. Wild-type enzyme readily reduces S-nitrosoglutathione with a kcat/Km approximately twice the kcat/Km for S-hydroxymethylglutathione oxidation, resulting in the highest catalytic efficiency yet identified for a human alcohol dehydrogenase.

On-column electrochemical reactions accompanying the electrospray process.

It is well-established that electrochemical reactions can occur within the capillary of an electrospray (ES) devise coupled to a mass spectrometer. In fact, such reactions must occur to maintain charge balance during the ES process. However, electrochemical reactions occurring distal to the capillary as a result of the high potential applied to the capillary have not been thoroughly investigated.

Capillary liquid chromatography/electrospray mass spectrometry for analysis of steroid sulfates in biological samples.

A new procedure for capillary liquid chromatography-electrospray (CLC-ES) mass spectrometry is described. Using this procedure, coupling of a CLC column to a low-flow-rate ES interface is made simple. A 5-cm precolumn and a 35-cm analytical column, both fused-silica capillaries with an i.

The mammalian alcohol dehydrogenases interact in several metabolic pathways.

Mammalian alcohol dehydrogenases (ADHs), including ADH1-ADH5/6, interact extensively in the oxidation and reduction of alcohols and aldehydes. ADH1 and ADH2 are involved in several metabolic pathways besides the oxidation of ethanol and have also been shown to be involved in drug transformations. The ADH2 enzymes show further complexity among the species, e.

Identification of a potent antibacterial factor isolated from bronchoalveolar lavage fluid: guanidine, N-[3-[(aminoiminomethyl)amino]propyl]-N-dodecyl-, a potential source of error in the analysis of antibacterial agents.

The widespread use of antibiotics in modern society has encouraged the search for new antibacterial compounds. In this laboratory investigations are being made to identify and characterise novel antibacterial peptides. With this in mind, the antibacterial properties of human bronchoalveolar lavage (BAL) fluid from sarcoidosis patients is being investigated.

Hydrogen/deuterium exchange and aggregation of a polyvaline and a polyleucine alpha-helix investigated by matrix-assisted laser desorption ionization mass spectrometry.

The membrane-associated pulmonary surfactant protein C (SP-C), containing a polyvaline alpha-helix, and a synthetic SP-C analogue with a polyleucine helix (SP-C(Leu)) were studied by hydrogen/deuterium exchange matrix-assisted laser desorption ionization (MALDI) mass spectrometry. SP-C, but not SP-C(Leu), formed abundant amyloid fibrils under experimental conditions. In CD(3)OD/D(2)O, 91:9 (v/v), containing 2 mM ammonium acetate, SP-C(Leu) and SP-C exchanged 40% of their exchangeable hydrogens within 1 min.

Multiple milia due to radiotherapy.

We report the appearance of multiple milia in a 61 year old woman appearing 7 years after irradiation for breast cancer. The lesions were confined to the irradiated field.

Alkaline hydrolysis of oxaliplatin--isolation and identification of the oxalato monodentate intermediate.

The alkaline degradation of the chemotherapeutic agent oxaliplatin has been studied using liquid chromatography. The oxalato ligand is lost in two consecutive steps. First, the oxalato ring is opened, forming an oxalato monodentate intermediate, as identified by electrospray ionization mass spectrometry.

Electrospray tandem mass spectrometry of intact beta-chain hemoglobin variants.

In this report, we present data to illustrate how human hemoglobin (Hb) variants can be identified by electrospray tandem mass spectrometry (MS/MS) of the intact Hb chains following the one-step dilution of whole blood. MS/MS spectra were recorded on a series of intact beta-chain human Hb variants. The resultant spectra were interpreted, and using the information gleaned from the fragmentation patterns of known variants, two unknown beta-chain variants were characterized solely by this mass spectrometric method.

Frameshift mutation in the V2 domain of human keratin 1 results in striate palmoplantar keratoderma.

The striate form of palmoplantar keratoderma is a rare autosomal dominant disorder affecting palm and sole skin. Genetic heterogeneity of striate palmoplantar keratoderma has been demonstrated with pathogenic mutations in the desmosomal proteins desmoplakin and desmoglein 1. We have studied a four-generation family of British descent with striate palmoplantar keratoderma.