Biomedical subjects

Wells W Wu

Publications and source records attributed to Wells W Wu.

9 recordsLinked to original sources

PubMed2006

Automated quantification tool for high-throughput proteomics using stable isotope labeling and LC-MSn.

LC-MSn has become a popular option for high-throughput quantitative proteomics, thanks to the availability of stable-isotope labeling reagents. However, the vast quantity of data generated from LC-MSn continues to make the postacquisition quantification analyses challenging, especially in experiments involving multiple samples per experimental condition. To facilitate data analysis, we developed a computer program, QUIL, for automated protein quantification. QUIL accounts for the dynamic nature of spectral background and subtracts this background accordingly during ion chromatogram reconstruction. For elution profile identification, QUIL minimizes the inclusion of coeluted neighbor peaks, yet tolerates imperfect peak shapes. Outlier-resistant methods have been implemented for better protein ratio estimation. The utility of QUIL was validated by quantitative analyses of a standard protein as well as complex protein mixtures, which were labeled with cICAT or 18O and analyzed using LCQ, LTQ, or FT-ICR instruments. For samples that no prior knowledge of relative protein quantities was available, Western blotting was performed for confirmation. For the standard protein, the coefficient of variation (CV) of peptide ratio estimation was 6%. For complex mixtures, the median CV for protein ratio calculations was less than 10%. Computed protein abundance ratios exhibited a relatively high degree of correlation with those obtained from Western blot analyses. Compared with a widely used commercial software tool, QUIL showed improvement in ion chromatogram construction and peak integration and significantly reduced relative errors in abundance ratio assessment.

Animals↗

PubMed2006

High-throughput identification of IMCD proteins using LC-MS/MS.

The inner medullary collecting duct (IMCD) is an important site of vasopressin-regulated water and urea transport. Here we have used protein mass spectrometry to investigate the proteome of the IMCD cell and how it is altered in response to long-term vasopressin administration in rats. IMCDs were isolated from inner medullas of rats, and IMCD proteins were identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We present a WWW-based "IMCD Proteome Database" containing all IMCD proteins identified in this study (n = 704) and prior MS-based identification studies (n = 301). We used the isotope-coded affinity tag (ICAT) technique to identify IMCD proteins that change in abundance in response to vasopressin. Vasopressin analog (dDAVP) or vehicle was infused subcutaneously in Brattleboro rats for 3 days, and IMCDs were isolated for proteomic analysis. dDAVP and control samples were labeled with different cleavable ICAT reagents (mass difference 9 amu) and mixed. This was followed by one-dimensional SDS-PAGE separation, in-gel trypsin digestion, biotin-avidin affinity purification, and LC-MS/MS identification and quantification. Responses to vasopressin for a total of 165 proteins were quantified. Quantification, based on semiquantitative immunoblotting of 16 proteins for which antibodies were available, showed a high degree of correlation with ICAT results. In addition to aquaporin-2 and gamma-epithelial Na channel (gamma-ENaC), five of the immunoblotted proteins were substantially altered in abundance in response to dDAVP, viz., syntaxin-7, Rap1, GAPDH, heat shock protein (HSP)70, and cathepsin D. A 28-protein vasopressin signaling network was constructed using literature-based network analysis software focusing on the newly identified proteins, providing several new hypotheses for future studies.

Animals↗

PubMed2006

Comparative study of three proteomic quantitative methods, DIGE, cICAT, and iTRAQ, using 2D gel- or LC-MALDI TOF/TOF.

A comparative study on the three quantitative methods frequently used in proteomics, 2D DIGE (difference gel electrophoresis), cICAT (cleavable isotope-coded affinity tags) and iTRAQ (isobaric tags for relative and absolute quantification), was carried out. DIGE and cICAT are familiar techniques used in gel- and LC-based quantitative proteomics, respectively. iTRAQ is a new LC-based technique which is gradually gaining in popularity. A systematic comparison among these quantitative methods has not been reported. In this study, we conducted well-designed comparisons using a six-protein mixture, a reconstituted protein mixture (BSA spiked into human plasma devoid of six abundant proteins), and complex HCT-116 cell lysates as the samples. All three techniques yielded quantitative results with reasonable accuracy when the six-protein or the reconstituted protein mixture was used. In DIGE, accurate quantification was sometimes compromised due to comigration or partial comigration of proteins. The iTRAQ method is more susceptible to errors in precursor ion isolation, which could be manifested with increasing sample complexity. The quantification sensitivity of each method was estimated by the number of peptides detected for each protein. In this regard, the global-tagging iTRAQ technique was more sensitive than the cysteine-specific cICAT method, which in turn was as sensitive as, if not more sensitive than, the DIGE technique. Protein profiling on HCT-116 and HCT-116 p53 -/- cell lysates displayed limited overlapping among proteins identified by the three methods, suggesting the complementary nature of these methods.

Animals↗

PubMed2006

Label-free protein quantification using LC-coupled ion trap or FT mass spectrometry: Reproducibility, linearity, and application with complex proteomes.

A critical step in protein biomarker discovery is the ability to contrast proteomes, a process referred generally as quantitative proteomics. While stable-isotope labeling (e.g., ICAT, 18O- or 15N-labeling, or AQUA) remains the core technology used in mass spectrometry-based proteomic quantification, increasing efforts have been directed to the label-free approach that relies on direct comparison of peptide peak areas between LC-MS runs. This latter approach is attractive to investigators for its simplicity as well as cost effectiveness. In the present study, the reproducibility and linearity of using a label-free approach to highly complex proteomes were evaluated. Various amounts of proteins from different proteomes were subjected to repeated LC-MS analyses using an ion trap or Fourier transform mass spectrometer. Highly reproducible data were obtained between replicated runs, as evidenced by nearly ideal Pearson's correlation coefficients (for ion's peak areas or retention time) and average peak area ratios. In general, more than 50% and nearly 90% of the peptide ion ratios deviated less than 10% and 20%, respectively, from the average in duplicate runs. In addition, the multiplicity ratios of the amounts of proteins used correlated nicely with the observed averaged ratios of peak areas calculated from detected peptides. Furthermore, the removal of abundant proteins from the samples led to an improvement in reproducibility and linearity. A computer program has been written to automate the processing of data sets from experiments with groups of multiple samples for statistical analysis. Algorithms for outlier-resistant mean estimation and for adjusting statistical significance threshold in multiplicity of testing were incorporated to minimize the rate of false positives. The program was applied to quantify changes in proteomes of parental and p53-deficient HCT-116 human cells and found to yield reproducible results. Overall, this study demonstrates an alternative approach that allows global quantification of differentially expressed proteins in complex proteomes. The utility of this method to biomarker discovery is likely to synergize with future improvements in the detecting sensitivity of mass spectrometers.

Amino Acid Sequence↗

PubMed2005

Proteolysis of macrophage inflammatory protein-1alpha isoforms LD78beta and LD78alpha by neutrophil-derived serine proteases.

Macrophage inflammatory protein-1alpha (MIP-1alpha) is a chemokine that leads to leukocyte recruitment and activation at sites of infection. Controlling chemokine activity at sites of infection is important, since excess accumulation of leukocytes may contribute to localized tissue damage. Neutrophil-derived serine proteases modulate the bioactivity of chemokine and cytokine networks through proteolytic cleavage. Because MIP-1alpha is temporally expressed with neutrophils at sites of infection, we examined proteolysis of MIP-1alpha in vitro by the neutrophil-derived serine proteases: cathepsin G, elastase, and proteinase 3. Recombinant human MIP-1alpha isoforms LD78beta and LD78alpha were expressed and purified, and the protease cleavage sites were analyzed by mass spectrometry and peptide sequencing. Chemotactic activities of parent and cleavage molecules were also compared. Both LD78beta and LD78alpha were cleaved by neutrophil lysates at Thr16-Ser17, Phe24-Ile25, Tyr28-Phe29, and Thr31-Ser32. This degradation was inhibited by serine protease inhibitors phenylmethylsulfonyl fluoride and 4-(2-aminoethyl)-benzenesulfonyl fluoride. Incubation of the substrates with individual proteases revealed that cathepsin G preferentially cleaved at Phe24-Ile25 and Tyr28-Phe29, whereas elastase and proteinase 3 cleaved at Thr16-Ser17 and Thr31-Ser32. Proteolysis of LD78beta resulted in loss of chemotactic activity. The role of these proteases in LD78beta and LD78alpha degradation was confirmed by incubation with neutrophil lysates from Papillon-Lefevre syndrome patients, demonstrating that the cell lysates containing inactivated serine proteases could not degrade LD78beta and LD78alpha. These findings suggest that severe periodontal tissue destruction in Papillon-Lefevre syndrome may be related to excess accumulation of LD78beta and LD78alpha and dysregulation of the microbial-induced inflammatory response in the periodontium.

Amino Acid Sequence↗

PubMed2003

Simultaneous determination of trace levels of nine haloacetic acids in biological samples as their pentafluorobenzyl derivatives by gas chromatography/tandem mass spectrometry in electron capture negative ion chemical ionization mode.

Haloacetic acids (HAAs) are environmentally and medically important chemicals. No analytical method is currently available to analyze EPA-regulated HAAs in biological samples at environmentally relevant low concentrations. Clinical studies of this class of chemicals are also limited by the lack of analytical techniques of high sensitivity and precision. We now report a new analytical method using gas chromatography/ion trap mass spectrometry for quantifying nine HAAs present inplasma, urine, and water at picogram per milliliter levels. The derivatization reactions of HAAs with pentafluorobenzyl bromide were optimized and detection with an electron capture negative ion chemical ionization mode was employed to enhance the sensitivity. Selected ion monitoring and selected reaction monitoring methods were utilized for quantitation. The detection limits of HAAs in plasma, urine, and water were 25-1000 pg/mL. Accuracies varied from 86.6 to 118.1% (intraday) and 81.7 to 119.6% (interday). Precisions (CV) varied from 0.9 to 19.9% (intraday) and 0.8 to 19.8% (interday), and linearities (r2) varied from 0.9732 to 0.9998 (intraday) and 0.9422 to 0.9987 (interday), respectively. Methyl tertbutyl ether and diethyl ether provided the highest extraction recoveries for the HAAs (74.9-107.2%). The method was applied successfully to a kinetic investigation of low levels of HAAs in humans consuming chlorinated drinking water.

Acetates↗

PubMed2003

A strategy for metabolite identification using triple-quadrupole mass spectrometry with enhanced resolution and accurate mass capability.

Using a single platform of a triple-quadrupole mass spectrometer equipped with enhanced resolution and accurate mass capabilities, a strategy for metabolite identification of a drug in a biological matrix has been demonstrated. The strategy is based on first screening for metabolites via neutral loss and precursor ion scan schemes, devised as the result of the product ion spectrum of a matrix-free standard of the drug. The accurate masses of the precursor ions identified via the two scan schemes plus the precursor ions of structurally likely metabolites are then determined by enhanced resolution, accurate mass (AM) selected ion monitoring (SIM). The identities of the metabolites are further established by determining the accurate masses of the product ions via enhanced resolution AM selected reaction monitoring (SRM). The feasibility of the strategy was demonstrated using a liver microsome incubation sample of nefazodone, an antidepressant drug. The neutral loss and precursor ion screening runs were able to identify most of the metabolites of nefazodone. The subsequent SIM and SRM experiments gave mass accuracy of better than +/-0.003 u for the masses of the precursor and product ions of nefazodone and all the metabolites. The ability to perform metabolite screening by using the scan features followed by accurate mass determinations on the same instrument is an attractive feature of using a triple-quadrupole mass spectrometer with enhanced resolution and accurate mass capability.

Animals↗

PubMed2003

The relationship between disinfection by-product formation and structural characteristics of humic substances in chloramination.

The influence of structural characteristics of humic substances on disinfection by-product (DBP) formation was investigated for seven humic substances isolated from aquatic and terrestrial sources. The structural characterizations included 13C nuclear magnetic resonance (13C NMR) spectroscopy and ultraviolet (UV) spectroscopy. The aqueous humic substances were chloraminated at pH 7.0 and 8.5, with and without the presence of the bromide ion, and analyzed for total organic halogen (TOX), trihalomethanes (THMs), and haloacetic acids (HAAs). Aromatic contents determined by 13C NMR and differential UV absorbance at 254 nm statistically correlated with TOX formation for the humic substances investigated at p < 0.08. In contrast, a lack of correlation was observed for THM and HAA formation and these parameters. This paper also compiles relevant literature and discusses the contrasting reaction response of DBP precursor material to chlorination and chloramination.

Chloramines↗

PubMed2002

Correlations between differential absorbance and the formation of individual DBPs.

This study examined correlations between the differential absorbance at 272nm (deltaA272) and the formation of disinfection by-products (DBPs) in chlorinated water from the Tolt River, a water source for Seattle, WA. The DBPs investigated included chloroform (CHCl3), dichlorobromomethane (CHCl2Br), mono-, di- and trichloroacetic acids (MCAA, DCAA, and TCAA, respectively), chloral hydrate (CH), dichloroacetonitrile (DCAN) and 1,1,1-trichloropropanone (TCP). Whereas the kinetics of DBP formation are complex and are non-linear, the same DBP data represented as a function of deltaA272 are quite simple. Absorbance decreases when the water is chlorinated, i.e., deltaA272 is always negative. The DBP vs. -deltaA272 correlations can almost always be quantified by linear equations, at least above some threshold value of -deltaA272, with R2 values > 0.95. The only DBP that did not follow this trend was CH, for which an exponential relationship better described the data. TCP and DCAN were unstable at pH 7 and 8, but at pH 6 linear correlations between their concentrations and -deltaA272 were as strong as those for the more stable DBPs. The threshold -deltaA272 value is approximately the same for many of the DBPs studied, supporting the hypothesis that individual DBPs are released following the formation of a common intermediate, or at least a small group of similar intermediates. The DBP vs. -deltaA272 correlations may have practical value since they provide an alternative approach for monitoring the formation of individual DBP species on-line, but the generality of the relationships needs to be further examined.

Absorption↗