Biomedical subjects
Publications and source records attributed to S Naylor.
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Of 311 expressed sequenced tags (ESTs) mapped to single human chromosomes by analysis of a monochromosome somatic cell hybrid panel, 29 were localized to chromosome 3. Analysis of somatic cell hybrid lines containing different regions of chromosome 3 has enabled the regional assignment of these 29 ESTs to 13 of 23 intervals covering chromosome 3. Northern analysis of 25 of the EST clones has provided information on the pattern of expression of potential genes represented by these transcripts in 16 human tissue types. Nine of the clones hybridized solely to a transcript(s) in the testis, 12 hybridized to transcripts in testis and other tissues, and 4 hybridized with transcripts in testis and other tissues but in addition have testis-specific transcript sizes. These ESTs will provide useful markers throughout chromosome 3 for the development of physical and transcription maps. In addition, they provide candidate genes for disease loci mapping to the intervals defined by the chromosome 3 deletion panel.
The eosinophilia-myalgia syndrome (EMS) outbreak that occurred in the USA in 1989 was caused by the intake of L-tryptophan (Trp) produced from one manufacturer, Showa Denko K.K. of Japan. Six compounds present in the Trp were reported to be case-associated contaminants. However, three of these compounds, Peaks C, FF and AAA have remained unidentified. Here, we successfully employ on-line HPLC-electrospray ionization multistage mass spectrometry to structurally characterize Peak C and Peak FF. Peak C was determined by accurate mass-LC-MS to have a protonated molecular ion MH+ = 221.0919 with an empirical formula of C11H13N2O3. By comparing the LC-MS-MS spectra with authentic 5-OHTrp and other structurally similar compounds, as well as considering the chemical reactivity of the indole ring, the structure of Peak C was consistent with 3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo-[2,3-b]-indole-2-carboxy lic acid. Peak FF was also subjected to accurate mass-LC-MS and shown to have MH+ = 338.1524, corresponding to an empirical formula of C19H20N3O3. Comparison of the LC-MS-MS and LC-sCID-MS-MS of spectra derived from Peak FF with a previously characterized contaminant of Trp, namely P31, was consistent with Peak FF being 2-(2-hydroxy indoline)-Trp. Unlike the majority of the contaminants identified in EMS implicated tryptophan, both Peaks C and FF possess an indoline ring. This is significant since a case-associated contaminant found in 5-hydroxy-Trp also contains an indoline ring, and the chemical reactivity of this ring system may possibly play a role in the etiology of EMS.
Fast and efficient analysis of proteins in physiological fluids is of great interest to researchers and clinicians alike. Capillary electrophoresis (CE) has proven to be a potentially valuable tool for the separation of proteins in specimens. However, a generally acknowledged drawback of this technique is the limited sample volumes which can be loaded onto the CE capillary which results in a poor concentration limit of detection. In addition, matrix components in samples may also interfere with separation and detection of analytes. Membrane preconcentration-CE (mPC-CE) has proved to be effective in overcoming these problems. In this report, we describe the systematic evaluation of parameters affecting on-line preconcentration/clean-up and separation of protein mixtures by mPC-CE. Method development was carried out with a standard mixture of proteins (lysozyme, myoglobin, carbonic anhydrase, and human serum albumin). First, using MALDI-TOF-MS, membrane materials with cation-exchange (R-SO3H) or hydrophobic (C2, C8, C18, SDB) characteristics were evaluated for their potential to retain proteins in mPC cartridges. Hydrophobic membranes were found most suitable for this application. Next, all mPC-CE analysis of protein samples were performed in polybrene coated capillaries and parameters affecting sample loading, washing and elution, such as the composition and volume of the elution solvent were investigated. Furthermore, to achieve optimal mPC-CE performance for the separation of protein mixtures parameters affecting postelution focusing and electrophoresis, including the composition of the background electrolyte and a trailing stacking buffer were varied. Optimal conditions for mPC-CE analysis of proteins using a C2 impregnated membrane preconcentration (mPC) cartridge were achieved with a background electrolyte of 5% acetic acid and 2 mM ammonium acetate, 60 nl of 80% acetonitrile in H2O as an elution solvent, and 60 nl of 0.5% ammonium hydroxide as a trailing stacking buffer. The developed method was used successfully to separate proteins in aqueous humor, which contains numerous proteins in a complex matrix of salts.
A quantitative method for detection of amyloid beta peptides using immunoprecipitation-HPLC-mass spectrometry (IP-LC-MS) is described. Comparison of IP-LC-MS with sandwich ELISA revealed comparable results in the analysis of A beta 1-40 and A beta 1-42 derived from fetal guinea pig cell media and cell lysates. The use of IP-LC-MS not only allows a quantitative method for A beta 1-40 and A beta 1-42 peptides present in Alzheimer's disease (AD), but allows detection of other A beta peptide species that may also play a role in the onset of AD in humans.
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Sequence analysis of antigenic major histocompatibility complex (MHC) class I peptides requires minimizing sample loss and enhancing mass spectrometric sensitivity. In order to facilitate such analyses, we have coupled on-line membrane preconcentration-capillary electrophoresis (mPC-CE) with microspray mass spectrometry (mPC-CE-microMS) and tandem mass spectrometry (mPC-CE-microMS/MS). Specifically, cell lysate from approximately 10(9) EG-7 mouse tumor cells was immunoprecipitated and the released MHC class I peptides were subjected to reverse-phase HPLC. An HPLC fraction containing antigenic peptide(s) shown to induce T-cell stimulation was subjected to mPC-CE-microMS. Approximately 10 microL (from 100 microL) of the fraction was pressure-injected and concentrated on a styrenedivinylbenzene (SDB) impregnated membrane. The peptides were eluted from the membrane with approximately 100 nL of 80% methanol, sandwiched between a leading stacking buffer (LSB, also serving as CE separation medium) of approximately 110 nL of 0.1% acetic acid in 10% methanol, and a trailing stacking buffer (TSB) of approximately 110 nL of 0.1% NH4OH. On application of the CE voltage the peptides are subjected to moving boundary transient isotachophoresis and focused. The peptides were separated in a Polybrene-coated capillary with application of -20 kV in reverse polarity mode and subsequently sprayed via an emitter coupled to the CE capillary by a liquid junction containing a platinum wire. An ion at m/z 482.3 was detected and subjected to mPC-CE-microMS/MS and determined to be SIINFEKL, a peptide (OVA) known to be antigenic in the mouse model system. Sensitivity enhancement over conventional mPC-CE-MS and MS/MS was approximately 100-fold.
The significance of proteomic research is coupled with the recent exponential growth of these investigations. Currently, the most popular techniques used for these studies include the coupling of 1- and 2-dimensional electrophoresis with mass spectrometric analysis of the extracted and digested proteins. However, detection limits of gel staining methods have led to a search for complimentary techniques that afford the detection of lower concentrations of biologically relevant proteins. In the present studies, we have evaluated the applicability of on-line capillary electrophoresis - mass spectrometry (CE-MS) for this application. Specifically, we used membrane preconcentration-CE-MS (mPC-CE-MS) to analyze 13 samples of human aqueous humor (AH) from patients with various ocular pathologies (cataract, cataract plus glaucoma, and cataract plus pseudoexfoliation syndrome). This approach enabled rapid analysis of a relatively large volume (1 microL of each specimen, and a protein map for each was created. Measured average molecular weights (Mr) were used to tentatively identify proteins after search of the SWISS-PROT database using TagIdent from ExPaSy. Among those proteins tentatively identified are beta-2 microglobulin (Mr 11731.2), apolipoprotein A1 (Mr 28078.6) and serum albumin (Mr 66400). Proteins with Mr of 4349 (unidentified), 11731.2 (beta-2 microglobulin), 13400-14100 (immunoglobulin fragments), 28078.2 (apolipoprotein A1) and approximately 68000 (serum albumin) were observed in the majority of specimens. Generally no significant differences were noted in the protein composition of aqueous humor samples from different pathologies. However, the absence of an Mr 13345 protein and its oxidized form (Mr 13361) in samples from patients with pseudoexfoliation syndrome was noted. Occasionally the alpha-and beta-chains of hemoglobin, a contaminant in aqueous humor introduced during sampling, were also detected. We conclude from these studies that mPC-CE-MS is an attractive complimentary technique for proteome research, as this approach enables direct mapping and characterization of low concentrations of proteins that are present in complex physiologically derived fluids.
Electrospray ionization mass spectrometry (ESI-MS) was used to measure conformational changes within the DNA-binding domain of the vitamin D receptor (VDR DBD) upon binding zinc (Zn2+). As increasing concentrations of Zn2+ were added to the VDR DBD, a gradual shift in the mass envelope to lower charge states was observed in the multiply charged spectrum. The shift in the charge states was correlated to changes observed in the far-ultraviolet circular dichroic (far-UV CD) spectrum of the protein as it was titrated with Zn2+. Both the multiply charged ESI and far-UV CD spectra of the Zn(2+)-titrated protein show that the binding of the first Zn2+ ion to the protein results in very little conformational change in the protein. The binding of a second Zn2+ ion resulted in a significant alteration in the structure of the protein as indicated by changes in both the multiply charged ESI and far-UV CD spectra. Much smaller changes were seen within the multiply charged ESI or far-UV CD spectra upon increasing the Zn2+ concentration beyond 2 mol/mol of protein. The results presented indicate that ESI-MS in combination with CD is a powerful method to measure gross conformational changes induced by the binding of metals to metalloproteins.
The coupling of the widely used separation technique of conventional sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) with the mass accuracy measurement capability of mass spectrometry (MS) provides a very powerful analytical technique. However, at present, there is no simple, definitive method for coupling the two methods. Typically, separated proteins are extracted from the gel, either as the native protein or as a peptide mixture after in-gel proteolytic digestion, and then analyzed by mass spectrometry. However, the various extraction techniques described previously have been labor intensive and require a large number of steps. The mass spectrometry analysis of very low concentrations of in vivo derived proteins requires minimum sample handling and on-line concentration. Therefore, we have developed an efficient microelectroelution technique that is applied in a single step manner and contains an on-line concentration device. Initial results from this system have shown a high efficiency of analyte elution from the gel and a simple, robust technique for the coupling of SDS-containing gels with MALDI-TOF-MS analysis and a capability of analyzing proteins at the subpicomole level.
In the present study we describe conditions that permit the characterization of noncovalent protein-substrate complexes in aqueous solution by microspray electrospray ionization-mass spectrometry (ESI-MS), using a heated transfer capillary at low temperature (45 degrees C). Specifically, we examined the binding of calmodulin to two polypeptides; the calmodulin-binding domain of calmodulin-dependent protein kinase II (CamK-II) and melittin. Calmodulin, a well known calcium-binding protein, binds to a number of small amphipathic peptides in a calcium-dependent manner. Our results directly show that both peptides form equimolar complexes with calmodulin only in the presence of calcium. The stoichiometry necessary for the formation of each complex was 1:1:4 for calmodulin:peptide (melittin or CamK-II):Ca2+, respectively. Furthermore, it is demonstrated that the detection of the complex in ESI-MS is source temperature dependent.
On-line HPLC/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) in conjunction with NMR has been successfully employed to identify and structurally characterize seven contaminants found in three different commercial preparations of melatonin. Six of these contaminants were identified as analogues of impurities found in contaminated L-tryptophan (an over-the-counter dietary supplement) associated with the eosinophilia-myalgia syndrome (EMS) epidemic that occurred in the United States during 1989. In particular, our studies identified two compounds with MH+ = 249 to be hydroxymelatonin isomers. Four other compounds with MH+ = 477 were identified as melatonin-formaldehyde condensation products. These compounds are structural analogues of L-tryptophan contaminants, namely, 'peak C' and 'peak E' that were previously implicated as etiological agents causing EMS. It has been reported that melatonin consumption has resulted in eosinophilia in some humans taking high doses of this supplement. Although there has not been a major outbreak of EMS-like symptoms from consumption of melatonin, this study clearly suggests that tighter control and regulation of nutritional supplements sold and used as drugs is necessary.
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The vitamin D receptor (VDR) binds to specific DNA sequences termed vitamin D response elements (VDREs) thereby enhancing or repressing transcription. We have used electrospray ionization mass spectrometry to examine the interaction between the DNA-binding domain of the vitamin D receptor (VDR DBD) with a double-stranded DNA (dsDNA) sequence containing the VDRE from the mouse osteopontin gene. The VDR DBD was shown to bind to the appropriate DNA sequence only when bound to 2 moles of zinc (Zn2+) or cadmium (Cd2+) per mole of protein. Additional binding of Zn2+ or Cd2+ by the protein caused the protein to dissociate from the dsDNA. These results show that the VDR DBD/DNA metal-dependent association occurs when the receptor is occupied by 2 moles of Zn2+ per mole of protein and that further binding of Zn2+ to the protein causes dissociation of the complex.
We have identified three new members of the olfactory receptor (OR) gene family within a large segment of DNA that is duplicated with high similarity near many human telomeres. This segment is present at 3q, 15q, and 19p in each of 45 unrelated humans sampled from various populations. Additional copies are present polymorphically at 11 other subtelomeric locations. The frequency with which the block is present at some locations varies among populations. While humans carry seven to 11 copies of the OR-containing block, it is located in chimpanzee and gorilla predominantly at a single site, which is not orthologous to any of the locations in the human genome. The observation that sequences flanking the OR-containing segment are duplicated on larger and different sets of chromosomes than the OR block itself demonstrates that the segment is part of a much larger, complex patchwork of subtelomeric duplications. The population analyses and structural results suggest the types of processes that have shaped these regions during evolution. From its sequence, one of the OR genes in this duplicated block appears to be potentially functional. Our findings raise the possibility that functional diversity in the OR family is generated in part through duplications and inter-chromosomal rearrangements of the DNA near human telomeres.
S-Methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO) is a known metabolite of the aversion therapy drug disulfiram (DSF). MeDTC-SO is also a potent inhibitor of human mitochondrial aldehyde dehydrogenase (hmALDH) with an IC50 of 1.5 microM. Inhibition of the enzyme by MeDTC-SO resulted in the addition of approximately 100 Da to the molecular mass of the intact protein, as determined by on-line HPLC-electrospray ionization MS (LC-MS). Dialysis of the inhibited protein did not reverse the inhibition, and the molecular mass of 54,533 Da (+/- 0.01%) remained unchanged, indicating that a covalent modification of the protein had occurred. Proteolytic digestion of hmALDH under basic conditions using trypsin at pH 7.8 revealed that the adduct was base labile. However, treating the adducted protein with endopeptidase-Glu-C at pH 3.7 produced a peptide adduct at MH+ = 4924, tentatively attributable to a carbamoylated peptide. This peptide contains three adjacent cysteines, one of which has been implicated as a key amino acid in the highly conserved active site region of ALDH. A pepsin digestion of hmALDH carried out at pH 3.7 and subsequent LC-MS analysis revealed an ion at MH2(2+) = 501.5, corresponding to the carbamoylated peptide FNQGQC1C2C3. This peptide contains the same adjacent active site cysteines. This latter peptide was subjected to LC-MS/MS, which enabled us to determine that the site of carbamoylation was at Cys2. The MS/MS product ion data also confirmed the presence of a carbamoyl group as the adduct species.
In 1992, the Health Care Financing Administration (HCFA) implemented a major change in the methodology of the quality of care oversight activities conducted by Medicare Peer Review Organizations. The Health Care Quality Improvement Program (HCQIP) represented a shift in oversight activity direction from identifying and dealing with individual clinical errors to helping providers improve mainstream care. The change in the oversight activities of Peer Review Organizations has been so substantial that the organizations are now commonly referred to as Quality Improvement Organizations (QIOs). Since its introduction, the HCQIP has developed multiple cooperative projects between QIOs and participating hospitals to examine specific processes of care and to ultimately improve the quality of care provided to Medicare patients. This report describes one project in North Carolina focusing on inpatient treatment of patients with a principal diagnosis of unstable angina, one of the most frequent causes of hospital admissions for Medicare patients. Based on the guidelines for treating unstable angina issued by the Agency for Health Care Policy and Research, 5 measures of good medical care for these patients were selected as quality of care indicators. A total of 16 hospitals in North Carolina each provided medical records of approximately 50 Medicare patients discharged with a principal diagnosis of unstable angina. Our findings indicated that guidelines-recommended standard of care were met in only a minority of patients. These indicators of care--including ordering an electrocardiogram within the first hour of admission and admitting high-risk patients to the intensive care unit--all occurred in <50% of the patients. Moreover, use of drugs that improve outcomes in patients with unstable angina was lower than expected. Only 17% of eligible patients with unstable angina were discharged on a lipid-lowering medication. Although there was variation in compliance with the guidelines between types of hospitals, all hospitals had an opportunity to improve in at least one quality of care indicator. The data demonstrate that significant variances exist between published guidelines and actual practices. Given the high rates of readmission for patients with coronary disease, there is opportunity to improve compliance with recommended guidelines of good care. The new oversight activity direction taken by Medicare should ultimately improve care for more patients than could ever be achieved through individual case review.
The use of capillary electrophoresis (CE) for clinically relevant assays is attractive since it often presents many advantages over contemporary methods. The small-diameter tubing that holds the separation medium has led to the development of multicapillary instruments, and simultaneous sample analysis. Furthermore, CE is compatible with a wide range of detectors, including UV-Vis, fluorescence, laser-induced fluorescence, electrochemistry, mass spectrometry, radiometric, and more recently nuclear magnetic resonance, and laser-induced circular dichroism systems. Selection of an appropriate detector can yield highly specific analyte detection with good mass sensitivity. Another attractive feature of CE is the low consumption of sample and reagents. However, it is paradoxical that this advantage also leads to severe limitation, namely poor concentration sensitivity. Often high analyte concentrations are required in order to have injection of sufficient material for detection. In this regard, a series of devices that are broadly termed 'analyte concentrators' have been developed for analyte preconcentration on-line with the CE capillary. These devices have been used primarily for non-specific analyte preconcentration using packing material of the C18 type. Alternatively, the use of very specific antibody-containing cartridges and enzyme-immobilized microreactors have been demonstrated. In the current report, we review the likely impact of the technology of capillary electrophoresis and the role of the CE analyte concentrator-microreactor on the analysis of biomolecules, present on complex matrices, in a clinical laboratory. Specific examples of the direct analysis of physiologically-derived fluids and microdialysates are presented, and a personal view of the future of CE in the clinical environment is given.