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David C Muddiman

Publications and source records attributed to David C Muddiman.

At least 37 records · Page 2Linked to original sources
PubMed2005

Effect of post-excitation radius on ion abundance, mass measurement accuracy, and isotopic distributions in Fourier transform ion cyclotron resonance mass spectrometry.

We report an evaluation of a modern Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) instrument to determine the general trend of post-excitation radius on total ion abundance, mass measurement accuracy, and isotopic distributions for internally calibrated mass spectra. The optimum post-excitation radius was determined using total ion abundance, mass measurement accuracy (MMA), and isotope ratios. However, despite the utility of internal calibration for achieving ultimate MMA, the internal calibrant ions were insufficient for compensating for sub-optimum ICR cell conditions. The findings presented herein underscore the importance of determining the optimal post-excitation radius in FT-ICR-MS to achieve high ion abundance (low limits of detection), high MMA, and valid isotopic distributions.

Bradykinin↗
PubMed2005

Normalization of relative peptide ratios derived from in-gel digests: applications to protein variant analysis at the peptide level.

The ability to detect protein variants and post-translational modifications by mass spectrometry has become increasingly important. Unfortunately, the ability to detect variants in large intact proteins (>80,000 Da) is limited. Even in the analysis of smaller proteins, algorithms are required to determine the presence of a 2 Da mass shift in an intact 13 kDa protein because the isotopic distribution of the multiply charged ions of the variant overlaps the wild-type distribution. Fortunately, most modern instruments are capable of detecting variants in tryptic peptides derived from intact proteins. If a single common variant protein is known, the presence of a variant tryptic peptide can be easily demonstrated. A more difficult issue is the case where a multiplicity of peptides with multiple amino acid substitutions can be associated with pathology. In these cases a decrease in the relative amount of a variant peptide relative to other internal tryptic fragments would be diagnostic. However, the variability associated with the analysis of in-gel or solution digests of proteins, related to efficiencies in digestion, extraction and ionization, confounds variant analysis at the peptide level. A strategy was developed to normalize for this variability by utilizing multiple isotopically labeled internal standards for multiple peptides derived from the same protein. Erythrocyte spectrin from 36 normal and 25 abnormal osmotic fragility samples was analyzed as a test case. Three isotopically labeled target peptides comprising the alpha/beta-spectrin self-association sites were added to purified digested alpha-spectrin. The utilization of multiple internal standards demonstrates the capability to normalize for sample variability due to ionization efficiency, solvent effects, digestion and extraction efficiency.

Algorithms↗
PubMed2005

Leveling response factors in the electrospray ionization process using a heated capillary interface.

Several investigators have observed a discrepancy in electrospray response of complementary strands from denatured DNA, which has been attributed to the difference in hydrophobicity between the two strands; the more hydrophobic species tend to have higher ion abundances. The implementation of a heated electrospray source has allowed us to "level" the electrospray response for two equimolar complementary strands with different hydrophobicities. As the temperature was increased, the ratio of ion abundances of the less hydrophobic noncoding strand to the more hydrophobic coding strand approached unity. Furthermore, the heated electrospray source was used to denature amplicons containing 7-deaza purines, which can be used to facilitate sequencing by mass spectrometry.

Capillary Action↗
PubMed2005

Primary trabecular meshwork cells incubated in human aqueous humor differ from cells incubated in serum supplements.

PURPOSE: To determine whether aqueous humor, the in vivo source of nutrients for trabecular meshwork cells, alters cellular and molecular characteristics in primary trabecular monolayer cell cultures when compared with standard culture conditions. METHODS: Human primary trabecular meshwork cell cultures were grown in DMEM supplemented with 50% human aqueous humor (DMEM-AH), heat-denatured DMEM-AH, 10% fetal bovine serum (DMEM-FBS, the standard culture supplement), or heat-denatured DMEM-FBS. Confluent trabecular cells were assayed for cell propagation and morphology for 21 days. Protein expression profiles of trabecular cell lysates were analyzed by two-dimensional polyacrylamide gel electrophoresis. Western blot analysis was used to determine the protein expression of myocilin and TIMP-1 in conditioned media collected from trabecular cells at 5, 10, 15, and 21 days. Myocilin expression was also analyzed by Western immunoblots after addition of dexamethasone (10(-7) M) or ascorbic acid (29 mg/dL). RESULTS: Trabecular cells supplemented with DMEM-AH for 21 days showed decreased cell proliferation when compared with DMEM-FBS (11% vs. 141%). Cellular morphology was also altered: Trabecular cells incubated in DMEM-AH showed larger-, broader-, and flatter-appearing cells than did the more spindle-shaped cells grown in DMEM-FBS. Protein profiles of trabecular cell lysates isolated from cells incubated in DMEM-AH differed from those incubated in DMEM-FBS. In DMEM-AH-conditioned medium, myocilin expression was increased and TIMP-1 expression was decreased at day 21. Induction of myocilin by dexamethasone was observed in conditioned medium isolated from cells treated with DMEM-FBS (442%), but only a 10% increase in myocilin was observed beyond the normal induction in DMEM-AH. Daily administration of ascorbic acid to DMEM-AH failed to increase myocilin expression beyond that obtained with DMEM-AH. CONCLUSIONS: Addition of human aqueous humor rather than FBS to trabecular monolayer cell cultures triggers significant changes in cellular and molecular characteristics. The protein component of aqueous humor is responsible for these changes. Aqueous humor supplementation may maintain cultured trabecular cells in a more physiologic state.

Adult↗
PubMed2005

Reproducibility of retention time using a splitless nanoLC coupled to an ESI-FTICR mass spectrometer.

Replicate injections of a myoglobin tryptic digest, ultrafiltrates of human serum, and ultrafiltrates of human plasma made on a splitless nanoscale liquid chromatography system coupled to a Fourier-transform ion cyclotron resonance mass spectrometer were utilized to assess analytical reproducibility. The mean (across 19 tryptic fragments detected in at least 3 of 24 replicate injections) of the 95% CIM of retention time is +/-6.3 sec and the maximum is +/-11.6 sec; when only those tryptic fragments that were found in 24 of 24 replicates are considered, the maximum 95% CIM of retention time drops to +/-6.7 sec. This represents a deviation of at most seven spectra. Similarly, in the serum (and plasma) filtrates, 95% of the 393 (312) species observed in 3 replicate injections had a 95% CIM of retention time of +/-22.0 (+/-18.5) sec or less. Ion abundance was similarly reproducible, with an average across those tryptic fragments observed in all 24 replicates of the coefficient of variation of ion abundance equal to 37.0%. This reproducibility represents a significant improvement over prior work, which required flow splitting in order to achieve nanoliter-per-minute flow rates. These improvements in retention time reproducibility will also be observed with mass spectrometers employing mass analyzers other than FT-ICR.

Case-Control Studies↗
PubMed2004

Analysis of the low molecular weight fraction of serum by LC-dual ESI-FT-ICR mass spectrometry: precision of retention time, mass, and ion abundance.

This study quantifies the experimental uncertainty for LC retention time, mass measurement precision, and ion abundance obtained from replicate nLC-dual ESI-FT-ICR analyses of the low molecular weight fraction of serum. We used ultrafiltration to enrich the < 10-kDa fraction of components from the high-abundance proteins in a pooled serum sample derived from ovarian cancer patients. The THRASH algorithm for isotope cluster detection was applied to five replicate nLC-dual ESI-FT-ICR chromatograms. A simple two-level grouping algorithm was applied to the more than 7000 isotope clusters found in each replicate and identified 497 molecular species that appeared in at least four of the replicates. In addition, a representative set of 231 isotope clusters, corresponding to 188 unique molecular species, were manually interpreted to verify the automated algorithm and to set its tolerances. For nLC retention time reproducibility, 95% of the 497 species had a 95% confidence interval of the mean of +/- 0.9 min or less without the use of chromatographic alignment procedures. Furthermore, 95% of the 497 species had a mass measurement precision of < or = 3.2 and < or = 6.3 ppm for internally and externally calibrated spectra, respectively. Moreover, 95% of replicate ion abundance measurements, covering an ion abundance range of approximately 3 orders of magnitude, had a coefficient of variation of less than 62% without using any normalization functions. The variability of ion abundance was independent of LC retention time, mass, and ion abundance quartile. These measures of analytical reproducibility establish a statistical rationale for differentiating healthy and disease patient populations for the elucidation of biomarkers in the low molecular fraction of serum.

Algorithms↗
PubMed2004

Analytical performance of a venturi device integrated into an electrospray ionization fourier transform ion cyclotron resonance mass spectrometer for analysis of nucleic acids.

A voltage-assisted venturi device modeled after an industrial air amplifier was used to improve the ion transmission efficiency of a 16.2 kDa oligonucleotide and a 53-mer PCR product in the high-pressure region between an electrospray ionization (ESI) emitter and the sampling orifices of two Fourier transform ion cyclotron resonance mass spectrometers (FT-ICR-MS). The venturi device increased the total ion abundance of the oligonucleotide and the PCR product by more than 6-fold relative to the best achievable signal without the device. Furthermore, the average charge states of the oligonucleotide and PCR product shifted from 12.5- to 14.5- and 10.9- to 12.6-, respectively, with the addition of the venturi device. Specific to FT-ICR mass spectrometry, this increase in the charge state directly translates to an increase in theoretical mass resolving power (>10000 full width half-maximum for the results presented here at 7 T). Adduction was still observed while using the device, suggesting that it is "soft" relative to other high-pressure ion focusing methods.

Cyclotrons↗
PubMed2004

Identification of transthyretin variants by sequential proteomic and genomic analysis.

BACKGROUND: Transthyretin-associated hereditary amyloidosis (ATTR) is an inherited disease in which variants in the primary structure of transthyretin (TTR; prealbumin) lead to the extracellular polymerization of insoluble protein fibrils, causing organ failure and ultimately death when major organs are involved. We have developed an integrated approach to molecular diagnosis with initial analysis of intact plasma TTR by electrospray ionization mass spectrometry (MS) and referral of positive samples for DNA sequence analysis and real-time PCR to confirm the common Gly6Ser polymorphism. METHODS: Samples from 6 patients previously diagnosed with ATTR and from 25 controls with (n = 15) or without (n = 10) polyneuropathy were analyzed in a blinded fashion for the presence of variant TTR. TTR protein was extracted with an immunoaffinity resin from 20 microL of archived plasma samples. The purified TTR was reduced with tris(2-carboxyethyl)phosphine and analyzed by MS. The appearance of two peaks (or a single peak shifted in mass indicative of a homozygous variant), including the wild-type mass of 13,761 Da, was indicative of the presence of a variant, and the individual was referred for DNA sequence analysis. RESULTS: MS analysis of intact reduced TTR correctly identified each of six samples known to contain variant TTR. These results were corroborated by subsequent DNA sequence analysis. Additionally, all Gly6Ser polymorphisms were correctly called based on the +30 mass shift and an equal relative abundance of the +30 polymorphism relative to wild-type TTR. No false-positive results were seen. CONCLUSIONS: This referral method eliminates the necessity of sequencing most samples and allows screening for the familial forms of amyloidosis in a broad patient population in a timely fashion. This method correctly identified all previously known variants and also identified a novel variant, Val94Ala.

Amyloidosis, Familial↗
PubMed2004

Detection of genetic variants of transthyretin by liquid chromatography-dual electrospray ionization fourier-transform ion-cyclotron-resonance mass spectrometry.

BACKGROUND: One of the numerous proteins causing amyloidosis is transthyretin (TTR), a protein usually responsible for the transport of thyroxine and retinol-binding protein. Variants within TTR cause it to aggregate and form insoluble fibers that accumulate in tissue, leading to organ dysfunction. METHODS: TTR was immunoprecipitated from serum by use of a polyclonal antibody and subsequently reduced with tris(2-carboxyethyl)phosphine. The purified TTR was then analyzed by fast-gradient liquid chromatography-dual-electrospray ionization Fourier-transform ion-cyclotron-resonance (FT-ICR) mass spectrometry. DNA sequencing was performed on all samples used in this study. RESULTS: Because of the inherent limitations in achieving high mass measurement accuracy based on the most abundant isotopic mass, we applied a fitting procedure that allowed determination of monoisotopic mass. Wild-type TTR (mean molecular mass, 13,761 Da) and its associated variant forms could be distinguished because of the high molecular mass accuracy afforded by FT-ICR (< or = 3 ppm) except for instances involving isobaric species or when isotopic distributions overlapped significantly. The [M + 11 H+]11+ charge state for all samples was used to determine the mass accuracies for both wild-type and variant forms of the protein. We correctly assigned seven of seven TTR variants. Moreover, using a combination of proteomic and genomic technologies, we discovered and characterized a previously unreported cis double mutation with a mass only 2 Da different from wild-type TTR. Furthermore, DNA sequencing of the TTR gene for all individuals in this study completely agreed with the intact protein measurements. CONCLUSIONS: FT-ICR mass spectrometry has sufficient mass accuracy to identify genetic variants of immunoaffinity-purified TTR. We believe that 91% of known TTR variants could be detected by this technique.

Amyloidosis, Familial↗
PubMed2004

Androgens negatively regulate forkhead transcription factor FKHR (FOXO1) through a proteolytic mechanism in prostate cancer cells.

The ability of androgens to inhibit apoptosis in both normal and malignant prostatic cells has been well documented. However, the underlying mechanisms are understood poorly. Here we demonstrated that forkhead transcription factor FKHR (FOXO1)-induced death of LNCaP cells was blocked by a synthetic androgen R1881. Androgen treatment also resulted in a reduction in transcriptional activity of FKHR in these cells. Moreover, treatment of LNCaP cells with R1881 led to a decrease in the intact FKHR protein (70 kDa) and an increase in a faster migrating protein band (60 kDa). Androgen-enhanced appearance of the 60-kDa protein was diminished specifically by lysosomal acidic cysteine protease inhibitors. Mass spectrometry analyses of the purified FLAG-tagged 70- and 60-kDa proteins demonstrated that the 60-kDa species is a FKHR protein product that lacks about 120 amino acid residues of the C-terminal end. Mutagenesis of the basic amino acid Arg(537) in the protease cleavage region, as suggested by mass spectrometry, abrogated both the androgen-induced accumulation of the 60-kDa product and decrease in cell death induced by FKHR, suggesting that the residue Arg(537) is a potential protease cleavage site. Finally, ectopic expression of the first 537 amino acids of FKHR produced an inhibitory effect on transcriptional activity of the intact protein. Together, these results suggest that androgens induce increased activity of an acidic cysteine protease, which in turn cleaves FKHR. This provides a mechanism by which androgens protect prostate cancer cells from the killing effect of FKHR.

Amino Acid Sequence↗
PubMed2004

Determination of the relative energies of activation for the dissociation of aromatic versus aliphatic phosphopeptides by ESI-FTICR-MS and IRMPD.

Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (ESI-FTICR-MS) coupled with infrared multiphoton dissociation (IRMPD) is potentially a powerful method for rapid phosphopeptide mapping of complex proteolytic digests. The dissociation of deprotonated phosphopeptides by IRMPD is energetically favorable over unmodified deprotonated peptides because of a lower energy of activation and a higher internal energy under identical irradiation conditions. The energies of activation for dissociation are determined for model peptides phosphorylated on an aliphatic side chain (serine) and an aromatic side chain (tyrosine). The determination of phosphorylation location provides important biochemical information identifying the kinase involved in specific phosphorylation mechanisms. The data presented in this manuscript also support the theory that for phosphopeptides, the phosphate moiety's P-O stretch is in direct resonance with the infrared laser (10.6 microm), thus increasing the relative absorptivity of the modified species. A greater extinction coefficient affords more extensive photon absorption and subsequently a greater internal energy at the rapid exchange limit.

Peptide Mapping↗
PubMed2004

A method for calculating 16O/18O peptide ion ratios for the relative quantification of proteomes.

A method is described for the identification and relative quantification of proteomes using accurate mass tags (AMT) generated by nLC-dual ESI-FT-ICR-MS on a 7T instrument in conjunction with stable isotope labeling using 16O/18O ratios. AMTs were used for putative peptide identification, followed by confirmation of peptide identity by tandem mass spectrometry. For a combined set of 58 tryptic peptides from bovine serum albumin (BSA) and human transferrin, a mean mass measurement accuracy of 1.9 ppm +/-0.94 ppm (CIM99%) was obtained. This subset of tryptic peptides was used to measure 16O/18O ratios of 0.36 +/- 0.09 (CIM99%) for BSA (micro = 0.33) and 1.48 +/- 0.47 (CIM99%) for transferrin (micro = 1.0) using a method for calculating 16O/18O ratios from overlapping isotopic multiplets arising from mixtures of 16O, 18O1, and 18O2 labeled C-termini. The model amino acid averagine was used to calculate a representative molecular formula for estimating and subtracting the contributions of naturally occurring isotopes solely as a function of peptide molecular weight. The method was tested against simulated composite 16O/18O spectra where peptide molecular weight, 16O/18O ratio, 18O1/18O2 ratios, and number of sulfur atoms were varied. Relative errors of 20% or less were incurred when the 16O/18O ratios were less than three, even for peptides where the number of sulfur atoms was over- or under-estimated. These data demonstrate that for biomarker discovery, it is advantageous to label the proteome representing the disease state with 18O; and the method is not sensitive to variations in 18O1/18O2 ratio. This approach allows a comprehensive differentiation of expression levels and tentative identification via AMTs, followed by targeted analysis of over- and under-expressed peptides using tandem mass spectrometry, for applications such as the discovery of disease biomarkers.

Amino Acid Sequence↗
PubMed2004

Identification and characterization of novel, naturally processed measles virus class II HLA-DRB1 peptides.

Previously, we identified a naturally processed and presented measles virus (MV) 19-amino-acid peptide, ASDVETAEGGEIHELLRLQ (MV-P), derived from the phosphoprotein and eluted from the human leukocyte antigen (HLA) class II molecule by using mass spectrometry. We report here the identification of a 14-amino-acid peptide, SAGKVSSTLASELG, derived from the MV nucleoprotein (MV-N) bound to HLA-DRB1*0301. Peripheral blood mononuclear cells (PBMC) from 281 previously vaccinated measles-mumps-rubella II (MMR-II) subjects (HLA discordant) were studied for peptide recognition by T cells. Significant gamma interferon (IFN-gamma) responses to MV-P and MV-N peptides were observed in 55.9 and 15.3% of subjects, respectively. MV-P- and MV-N-specific interleukin-4 (IL-4) responses were detected in 19.2 and 23.1%, respectively, of PBMC samples. Peptide-specific cytokine responses and HLA-DRB1 allele associations revealed that, for the MV-P peptide, the allele with the strongest association with both IFN-gamma (P = 0.02) and IL-4 (P = 0.03) secretion was DRB1*0301. For MV-N, the allele with the strongest association with IFN-gamma secretion was DRB1*1501 (P = 0.04), and the alleles with the strongest associations with IL-4 secretion were DRB1*1103 and DRB1*1303 (P = 0.01). These results indicate that HLA class II MV proteins can be processed, presented, and identified, and the ability to generate cell-mediated immune responses can be demonstrated. This information is promising for new vaccine design strategies with peptide-based vaccines.

Alleles↗
PubMed2003

Coordination-driven self-assembly of supramolecular cages: heteroatom-containing and complementary trigonal prisms.

The self-assembly of three nanoscopic prisms of approximate size 1 x 4 nm is reported. Tetrahedral carbon, silicon, and phosphorus were used as structure-defining elements in these coordination-based cages. A carbon-based assembly completes a pair of nanoscopic complementary 3-D structures. The formation of the structures is supported by multinuclear NMR, ESI FT-ICR mass spectrometry, and elemental analysis data.

Anthracenes↗
PubMed2003

Naturally processed measles virus peptide eluted from class II HLA-DRB1*03 recognized by T lymphocytes from human blood.

This is the first report of the direct identification of a HLA-DRB1*03 measles-derived peptide from measles virus infected EBV-transformed B cells. We purified HLA-DR3-peptide complexes from EBV-B cells infected with measles virus (Edmonston strain) and sequenced the HLA-DR3-peptides by mass spectrometry. A class II peptide, derived from a measles phosphoprotein, ASDVETAEGGEIHELLRLQ (P1, residues 179-197), exhibited the capacity to stimulate peripheral blood mononuclear cells to proliferate. Our data provides direct evidence that the antigenic peptide of measles virus was processed by antigen-presenting cells, presented in the context of HLA class II molecules, and was recognized by peripheral blood T cells from healthy individuals previously immunized with measles vaccine. The approach described herein provides a useful methodology for the future identification of HLA-presented pathogen-derived epitopes using mass spectrometry. The study of cell-mediated immune responses to the measles-derived peptide in immune persons should provide significant insight into the design and development of new vaccines.

Adolescent↗
PubMed2003

Dual electrospray ionization source for confident generation of accurate mass tags using liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry.

Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) has rapidly established a prominent role in proteomics because of its unparalleled resolving power, sensitivity and ability to achieve high mass measurement accuracy (MMA) simultaneously. However, space-charge effects must be quantitatively, routinely, and confidently corrected because they are known to profoundly influence MMA. We argue that the most effective way to account for space-charge effects is to introduce an internal mass calibrant (IMC) using a dual electrospray ionization (ESI) source where the IMC is added from a separate ESI emitter. The major disadvantage of our initial dual ESI source to achieve high MMA, and arguably the only one, was the time required to switch between the analyte emitter and IMC emitter (i.e., >300 ms). While this "switching time" was acceptable for direct infusion experiments, it did not lend itself to high-throughput applications or when conducting on-line liquid separations. In this report, we completely redesigned the dual ESI source and demonstrate several key attributes. First, the new design allows for facile alignment of ESI emitters, undetectable vibration, and the ability to extend to multiple emitters. Second, the switching time was reduced to <50 ms, which allowed the analyte and IMC to be accumulated "simultaneously" in the external ion reservoir and injected as a single ion packet into the ion cyclotron resonance cell, eliminating the need for a separate accumulation and ion injection event for the IMC. Third, by using a high concentration of the IMC, the residence time on this emitter could be reduced to approximately 80 ms, allowing for more time spent accumulating analyte ions of significantly lower concentration. Fourth, multiplexed on-line separations can be carried out providing increased throughput. Specifically, the new dual ESI source has demonstrated its ability to produce a stable ion current over a 45-min time period at 7 T resulting in mass accuracies of 1.08 ppm +/- 0.11 ppm (mean +/- confidence interval of the mean at 95% confidence; N = 160). In addition, the analysis of a tryptic digest of apomyoglobin by nanoLC-dual ESI-FT-ICR afforded an average MMA of -1.09 versus -74.5 ppm for externally calibrated data. Furthermore, we demonstrate that the amplitude of a peptide being electrosprayed at 25 nM can be linearly increased, ultimately allowing for dynamic analyte/IMC abundance modulation. Finally, we demonstrate that this source can reliably be used for multiplexing measurements from two (eventually more) flow streams.

Amino Acid Sequence↗
PubMed2003

Implications of hydrophobicity and free energy of solvation for characterization of nucleic acids by electrospray ionization mass spectrometry.

Electrospray ionization (ESI) is a dynamic process that, when coupled with mass spectrometry (MS), serves as an invaluable tool for analysis of biomolecules. Our group, as well as others, has observed that there is a bias in signal intensity for one strand of a PCR amplicon over the complementary strand in an ESI mass spectrum. In this report, we have investigated the contributions of hydrophobicity and free energy of solvation to relative signal intensities in ESI-MS spectra of nucleic acids. We developed approaches for predicting which strand of the PCR amplicon will be the most intense: one based on a rate equation for calculating ion flux using values from the literature for hydrophobicity and free energy of solvation and the other based on the percentage of the relatively hydrophilic guanines present in the strand. A trend in signal intensity for deoxyribonucleotide triphosphates, oligonucleotides, and PCR amplicons was observed that was consistent with our model. On the basis of the observation that increased hydrophobicity correlates with greater signal intensity, we selectively enhanced the signal intensity of a 20-mer with the addition of an alkyl chain to the 5' terminus, which subsequently improved the limit of detection to 1 nM, an improvement by 1 order of magnitude. This was extended to a 53-bp PCR amplicon by modifying one primer with the hydrophobic moiety, which resulted in a 16% increase in signal intensity. We capitalized on this result to determine allele frequencies from pooled DNA for single-nucleotide polymorphisms down to 1%.

Deoxyribonucleotides↗