Biomedical subjects

Brian M Balgley

Publications and source records attributed to Brian M Balgley.

12 recordsLinked to original sources

PubMed2006

Protein extraction from formalin-fixed, paraffin-embedded tissue sections: quality evaluation by mass spectrometry.

A satisfactory protocol of protein extraction has been established based on the heat-induced antigen retrieval (AR) technique widely applied in immunohistochemistry for archival formalin-fixed, paraffin-embedded (FFPE) tissue sections. Based on AR, an initial serial experiment to identify an optimal protocol of heat-induced protein extraction was carried out using FFPE mouse tissues. The optimal protocol for extraction of proteins was then performed on an archival FFPE tissue of human renal carcinoma. FFPE sections were boiled in a retrieval solution of Tris-HCl containing 2% SDS, followed by incubation. Fresh tissue taken from the same case of renal carcinoma was processed for extraction of proteins by a conventional method using radioimmunoprecipitation assay solution, to compare the efficiency of protein extraction from FFPE tissue sections with extraction from fresh tissue. As a control, further sections of the same FFPE sample were processed by the same procedure without heating treatment. Evaluation of the quality of protein extracted from FFPE tissue was done using gel electrophoresis and mass spectrometry, showing most identified proteins extracted from FFPE tissue sections were overlapped with those extracted from fresh tissue.

Animals↗

PubMed2006

Selective enrichment and ultrasensitive identification of trace peptides in proteome analysis using transient capillary isotachophoresis/zone electrophoresis coupled with nano-ESI-MS.

Besides the complexity in protein samples of biological origin, probably the greatest challenge presently facing comprehensive proteome analysis is related to the large variation of protein relative abundances (>6 orders of magnitude), having potential biological significance in mammalian systems. As demonstrated in this work, transient capillary ITP/zone electrophoresis (CITP/CZE) provides selective analyte enrichment through electrokinetic stacking and extremely high resolving power toward protein and peptide mixtures. The result of the CITP process is that major components may be diluted, but trace compounds are concentrated. The on-column transition of CITP to CZE minimizes additional band broadening while providing superior analyte resolution. Online coupling of transient CITP/CZE with nano-ESI-MS allows ultrasensitive detection of trace peptides at levels of subnanomolar concentration or subfemtomole mass in complex peptide mixtures. More importantly, selective enrichment of trace peptides enables the identification and sequence analysis of low-abundance peptides co-migrated with highly abundant species at a concentration ratio of 1:500,000. The combined CITP/CZE-nano-ESI-MS system is demonstrated to be at least one to two orders of magnitude more sensitive than that attained in conventional electrophoretic and chromatographic-based proteome technologies over a wide dynamic concentration range, potentially allowing comprehensive analysis of protein profiles within a small cell population and limited tissue samples using conventional mass spectrometers. Furthermore, the speed of CITP/CZE separation and the lack of column equilibration in CITP/CZE not only improve the throughput of proteome analysis, but also facilitate its seamless integration with other separation technologies in a multidimensional protein identification platform.

Cytochromes c↗

PubMed2006

Capillary separations enabling tissue proteomics-based biomarker discovery.

Development of the capability to enable large-scale proteome studies, analogous to comprehensive gene expression analysis, will clearly have far-reaching impacts on protein biomarker investigations of human diseases such as cancer through interrogation of the archived fresh frozen and formalin-fixed and paraffin-embedded tissue collections. This review therefore focuses on the most recent advances in microdissection techniques and proteome platforms for procuring homogeneous subpopulations of tumor cells or structures and performing comprehensive analysis of protein profiles within tissue specimens, respectively. Developments in capillary separations capable of providing extremely high resolving power and selective analyte enrichment are particularly highlighted for their roles within the broader context of a state-of-the-art integrated tissue proteome effort. The capabilities of CIEF-based multidimensional separations for performing proteome analysis from minute samples create new opportunities in the pursuit of biomarker discovery using enriched and selected cell populations procured from tissue specimens. These proteome technological advances combined with recently developed tissue microdissection techniques provide powerful tools for those seeking to gain a greater understanding at the global level of the cellular machinery associated with human diseases such as cancer.

Biomarkers↗

PubMed2006

Characterization of the human salivary proteome by capillary isoelectric focusing/nanoreversed-phase liquid chromatography coupled with ESI-tandem MS.

Saliva is a readily available body fluid with great diagnostic potential. The foundation for saliva-based diagnostics, however, is the development of a complete catalog of secreted and "leaked" proteins detectable in saliva. By employing a capillary isoelectric focusing-based multidimensional separation platform coupled with electrospray ionization tandem mass spectrometry (MS), a total of 5338 distinct peptides were sequenced, leading to the identification of 1381 distinct proteins. A search of bacterial protein sequences also identified many peptides unique to several organisms and unique to the NCBI nonredundant database. To the best of our knowledge, this proteome study represents the largest catalog of proteins measured from a single saliva sample to date. Data analysis was performed on individual MS/MS spectra using the highly specific peptide identification algorithm, OMSSA. Searches were conducted against a decoyed SwissProt human database to control the false-positive rate at 1%. Furthermore, the well-curated SwissProt sequences represent perhaps the least redundant human protein sequence database (12,484 records versus the 50,009 records found in the International Protein Index human database), therefore minimizing multiple protein inferences from single peptides. This combined bioanalytical and bioinformatic approach has established a solid foundation for building up the human salivary proteome for the realization of the diagnostic potential of saliva.

Chromatography, Liquid↗

PubMed2005

Proteome analysis of microdissected tumor tissue using a capillary isoelectric focusing-based multidimensional separation platform coupled with ESI-tandem MS.

This study demonstrates the ability to perform sensitive proteome analysis on the limited protein quantities available through tissue microdissection. Capillary isoelectric focusing combined with nano-reversed-phase liquid chromatography in an automated and integrated platform not only provides systematic resolution of complex peptide mixtures based on their differences in isoelectric point and hydrophobicity but also eliminates peptide loss and analyte dilution. In comparison with strong cation exchange chromatography, the significant advantages of electrokinetic focusing-based separations include high resolving power, high concentration and narrow analyte bands, and effective usage of electrospray ionization-tandem MS toward peptide identifications. Through the use of capillary isoelectric focusing-based multidimensional peptide separations, a total of 6866 fully tryptic peptides were detected, leading to the identification of 1820 distinct proteins. Each distinct protein was identified by at least one distinct peptide sequence. These high mass accuracy and high-confidence identifications were generated from three proteome runs of a single glioblastoma multiforme tissue sample, each run consuming only 10 microg of total protein, an amount corresponding to 20,000 selectively isolated cells. Instead of performing multiple runs of multidimensional separations, the overall peak capacity can be greatly enhanced for mining deeper into tissue proteomics by increasing the number of CIEF fractions without an accompanying increase in sample consumption.

Brain Neoplasms↗

PubMed2005

Effects of chromatography conditions on intact protein separations for top-down proteomics.

For top-down proteomics, nano-reversed phase liquid chromatography (RPLC) plays a major role in both single and multidimensional protein separations in an effort to increase the overall peak capacity for the resolution of complex protein mixtures prior to mass spectrometry analysis. Effects of various chromatography conditions, including alkyl chain length in the stationary phase, capillary column temperature, and ion-pairing agent, on the resolution of intact proteins are studied using nano-RPLC-electrospray ionization-mass spectrometry. Optimal chromatography conditions include the use of C18 column heated at 60 degrees C and the addition of trifluoroacetic acid instead of heptafluorobutyric acid as the ion-paring agent in the mobile phase. Under optimized chromatography conditions, there are no significant differences in the separation performance of yeast cell lysates present in the native versus denatured states. Denatured yeast proteins resolved and eluted from nano-RPLC can be subjected to proteolytic digestion in an on- or off-line approach to provide improved protein sequence coverage toward protein identification in a combined top-down/bottom-up proteome platform.

Chromatography, High Pressure Liquid↗

PubMed2005

Electrospray interfacing of polymer microfluidics to MALDI-MS.

The off-line coupling of polymer microfluidics to MALDI-MS is presented using electrospray deposition. Using polycarbonate microfluidic chips with integrated hydrophobic membrane electrospray tips, peptides and proteins are deposited onto a stainless steel target followed by MALDI-MS analysis. Microchip electrospray deposition is found to yield excellent spatial control and homogeneity of deposited peptide spots, and significantly improved MALDI-MS spectral reproducibility compared to traditional target preparation methods. A detection limit of 3.5 fmol is demonstrated for angiotensin. Furthermore, multiple electrospray tips on a single chip provide the ability to simultaneously elute parallel sample streams onto a MALDI target for high-throughput multiplexed analysis. Using a three-element electrospray tip array with 150 microm spacing, the simultaneous deposition of bradykinin, fibrinopeptide, and angiotensin is achieved with no cross talk between deposited samples. In addition, in-line proteolytic digestion of intact proteins is successfully achieved during the electrospray process by binding trypsin within the electrospray membrane, eliminating the need for on-probe digestion prior to MALDI-MS. The technology offers promise for a range of microfluidic platforms designed for high-throughput multiplexed proteomic analyses in which simultaneous on-chip separations require an effective interface to MS.

Microfluidic Analytical Techniques↗

PubMed2005

Tissue proteomics using capillary isoelectric focusing-based multidimensional separations.

The capabilities of capillary isoelectric focusing-based multidimensional separations for performing proteome analysis from minute samples create new opportunities in the pursuit of biomarker discovery using enriched and selected cell populations procured from tissue specimens. In this article, recent advances in online integration of capillary isoelectric focusing with nano-reversed phase liquid chromatography for achieving high-resolution peptide and protein separations prior to mass spectrometry analysis are reviewed, along with its potential application to tissue proteomics. These proteome technological advances combined with recently developed tissue microdissection techniques, provide powerful tools for those seeking to gain a greater understanding at the global level of the cellular machinery associated with human diseases such as cancer.

Electrophoresis, Capillary↗

PubMed2003

Capillary isoelectric focusing-based multidimensional concentration/separation platform for proteome analysis.

An integrated proteome concentration/separation approach involving on-line combination of capillary isoelectric focusing (CIEF) with capillary reversed-phase liquid chromatography (CRPLC) is developed for providing significant analyte concentration and extremely high resolving power toward protein and peptide mixtures. Upon completion of analyte focusing, the self-sharpening effect greatly restricts analyte diffusion and contributes to analyte stacking in narrowly focused bands with a concentration factor of approximately 240. In addition to analyte focusing, CIEF as the first separation dimension resolves proteins/peptides on the basis of their differences in pI and offers greater resolving power than that achieved in strong cation exchange chromatography. The grouping of two highly resolving and completely orthogonal separation techniques of CIEF and CRPLC, together with analyte focusing and concentration, significantly enhances the dynamic range and sensitivity of conventional mass spectrometry toward the identification of low-abundance proteins. The CIEF-based multidimensional separation/concentration platform enables the identification of a greater number of yeast soluble proteins than methods presented in the literature, yet requires a protein loading of only 9.6 microg. This protein loading is 2-3 orders of magnitude lower than those employed by the reported non-gel-based proteome techniques. The distribution of a codon adaptation index value for identified yeast proteins approximates to that predicted for the entire yeast proteome and supports the capability of CIEF-based proteome separation technology for achieving comprehensive proteome analysis. By reducing the inner diameter of chromatography columns from 180 microm to 100 microm, the required protein loading is further decreased from 9.6 microg to 960 ng, illustrating the potential usage of this proteome technology for the analysis of protein profiles within small cell populations or limited tissue samples.

Chromatography, Liquid↗

PubMed2002

Utilizing renewable resources to create functional polymers: chitosan-based associative thickener.

There is a growing interest in utilizing renewable resources and exploiting biological reactions for environmentally friendly products and processes. We report the use of the enzyme tyrosinase to graft the natural phenol, catechin, onto the biopolymer chitosan. Chemical evidence for grafting was obtained by UV/visible spectrophotometry and electrospray mass spectrometry. Rheological measurements demonstrate that the catechin-modified chitosan behaves as an associative thickener. Specifically, the viscosity increases dramatically with concentration of this modified chitosan. Furthermore, when the catechin-modified chitosan is dissolved at low concentrations (0.6% w/w), steady shear measurements show shear thinning behavior, while oscillatory measurements show weak gel behavior. These results demonstrate the potential for utilizing renewable resources and biochemical processing to functionalize biopolymersto offertechnically useful properties. To suggest the relative environmental impacts of chitosan derivatives with existing water-soluble polymers, we used the framework of a life cycle assessment.

Biocompatible Materials↗

PubMedDate not supplied

Integrated capillary isoelectric focusing/nano-reversed phase liquid chromatography coupled with ESI-MS for characterization of intact yeast proteins.

An integrated protein concentration/separation platform, combining capillary isoelectric focusing (CIEF) with nano-reversed phase liquid chromatography (nano-RPLC), is developed to provide significant protein concentration and high resolving power for the analysis of complex protein mixtures. Upon completion of protein focusing, the proteins are sequentially and hydrodynamically loaded into individual trap columns using a group of microinjection and microselection valves. Repeated pro-tein loadings and injections into trap columns are carried out automatically until the entire CIEF cap-illary content is sampled and fractionated. Each CIEF fraction "parked" in separate trap columns is further resolved using nano-RPLC, and the eluants are analyzed using electrospray ionization-mass spectrometry.

Automation↗

PubMedDate not supplied

Large scale analysis of MASCOT results using a Mass Accuracy-based THreshold (MATH) effectively improves data interpretation.

In this report, we take a heuristic approach to studying the effects of mass tolerance settings and database size on the sensitivity and specificity of MASCOT. We also examine the efficacy of the MASCOT Identity Threshold as a discriminator when applied to QqTOF data with an average mass accuracy of 10 ppm or better. As predicted, arbitrarily large mass tolerance settings negatively affect MASCOT's specificity, and to a lesser degree, sensitivity. Increased mass tolerances also render the generation of a significance threshold less effective. To study these effects, we used Bayes' Law to calculate MASCOT's predictive values. With a relatively small search database (Human IPI), MASCOT had a mean positive predictive value of 0.993 when combined with MASCOT's Identity Threshold. However, the corresponding average negative predictive value, or the probability that an ion was not present given no score or a score below threshold, was reduced as mass tolerances were tightened, and had an average value of 0.717. This value was improved upon by extrapolating an empirical threshold using a reversed database search and a new algorithm to rapidly identify false positive identifications. Using the empirical threshold reduced false negative identifications on the average 17% while limiting the false positive rate to below 5%; even larger reductions were obtained using mass tolerances approaching two times the actual error of the experimental data. A simple application of this strategy to the analysis of a microdissected glioblastoma multiforme sample analyzed by IEF/LC-MS/MS is reported, as is a description of the tools required to implement a large scale analysis using this alternative approach.

Algorithms↗