Biomedical subjects

Dale Patterson

Publications and source records attributed to Dale Patterson.

4 recordsLinked to original sources

PubMed2005

High throughput proteome screening for biomarker detection.

Mass spectrometry-based quantitative proteomics has become an important component of biological and clinical research. Current methods, while highly developed and powerful, are falling short of their goal of routinely analyzing whole proteomes mainly because the wealth of proteomic information accumulated from prior studies is not used for the planning or interpretation of present experiments. The consequence of this situation is that in every proteomic experiment the proteome is rediscovered. In this report we describe an approach for quantitative proteomics that builds on the extensive prior knowledge of proteomes and a platform for the implementation of the method. The method is based on the selection and chemical synthesis of isotopically labeled reference peptides that uniquely identify a particular protein and the addition of a panel of such peptides to the sample mixture consisting of tryptic peptides from the proteome in question. The platform consists of a peptide separation module for the generation of ordered peptide arrays from the combined peptide sample on the sample plate of a MALDI mass spectrometer, a high throughput MALDI-TOF/TOF mass spectrometer, and a suite of software tools for the selective analysis of the targeted peptides and the interpretation of the results. Applying the method to the analysis of the human blood serum proteome we demonstrate the feasibility of using mass spectrometry-based proteomics as a high throughput screening technology for the detection and quantification of targeted proteins in a complex system.

Automation↗

PubMed2004

Depth of proteome issues: a yeast isotope-coded affinity tag reagent study.

As a test case for optimizing how to perform proteomics experiments, we chose a yeast model system in which the UPF1 gene, a protein involved in nonsense-mediated mRNA decay, was knocked out by homologous recombination. The results from five complete isotope-coded affinity tag (ICAT) experiments were combined, two using matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS) and three using electrospray MS/MS. We sought to assess the reproducibility of peptide identification and to develop an informatics structure that characterizes the identification process as well as possible, especially with regard to tenuous identifications. The cleavable form of the ICAT reagent system was used for quantification. Most proteins did not change significantly in expression as a consequence of the upf1 knockout. As expected, the Upf1 protein itself was down-regulated, and there were reproducible increases in expression of proteins involved in arginine biosynthesis. Initially, it seemed that about 10% of the proteins had changed in expression level, but after more thorough examination of the data it turned out that most of these apparent changes could be explained by artifacts of quantification caused by overlapping heavy/light pairs. About 700 proteins altogether were identified with high confidence and quantified. Many peptides with chemical modifications were identified, as well as peptides with noncanonical tryptic termini. Nearly all of these modified peptides corresponded to the most abundant yeast proteins, and some would otherwise have been attributed to "single hit" proteins at low confidence. To improve our confidence in the identifications, in MALDI experiments, the parent masses for the peptides were calibrated against nearby components. In addition, five novel parameters reflecting different aspects of identification were collected for each spectrum in addition to the Mascot score that was originally used. The interrelationship between these scoring parameters and confidence in protein identification is discussed.

Amino Acid Sequence↗

PubMed2003

Nanocapillary liquid chromatography interfaced to tandem matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry: mapping the nuclear proteome of human fibroblasts.

Miniaturized liquid chromatography nanoseparation in combination with minigel fractionation of human primary cell nuclei is presented. We obtained high-sensitivity and high-throughput identification of expressed proteins by subcellular fractionation and nanocapillary liquid chromatography interfaced to both electrospray ionization (ESI)- and matrix-assisted laser desorption/ionisation (MALDI) tandem mass spectrometry. The reversed-phase nanocapillary eluents were applied directly onto the MALDI target plate as discrete crystal spots using in-line matrix infusion. When working with primary cells, only a limited amount of sample is available. To maximize the number of identified proteins from a restricted amount of sample, miniaturized sample preparation protocols and nanoflow separation is a necessity, especially when working with low-abundant proteins. From the same isolated nuclear sample, complementary separation of intact proteins by two-dimensional (2-D) gel electrophoresis was made. In total 594 gene products from the nuclear preparations were identified out of which 261 were unique. Several proteins involved in transcriptional events were identified such as TATA-binding protein, EBNA-co-activator, and interleukin enhancer binding proteins, indicating that sufficient proteomic depth is obtained to study transcriptional controlling events. Our results suggest that by sample prefractionation and downscaled nanoflow separation along with a combined mass spectrometry strategy, it is possible to identify a large number of nuclear proteins from human primary cells. These findings are of particular importance due to the disease link of these targets cells.

Cell Nucleus↗

PubMedDate not supplied

Proteome annotations and identifications of the human pulmonary fibroblast.

We hereby report on a three year project initiative undertaken by our research team encompassing large-scale protein expression profiling and annotations of human primary lung fibroblast cells. An overview is given of proteomic studies of the fibroblast target cell involved in several diseases such as asthma, idiopatic pulmonary disease, and COPD. It has been the objective within our research team to map and identify the protein expressions occurring in both activated-, as well as resting cell states. The JGGL database www.2DDB.org has been built around these data, allowing advanced hypothesis building using the interactive query bioinformatic tools developed. Gene ontology has been applied to these annotations, classifying and correlating protein expressions to function. The localization as well as the biological processes involved for the annotations are being presented including an annotation-, and sequence-identification strategy, resulting in close to 2000 protein identities. Both gel based, high resolution 2D-gels, and liquid-phase separation (three-dimensional HPLC), as well as the combination of gel- and LC-based approaches (1D-gels and nano-capillary LC, reversed-phase) were utilized. Protein sequencing and structure identities were acquired by a combination of MALDI-, and electrospray-mass spectrometry techniques. Phenotypical and morphological characterizations were also made for this human disease target cell in both stimulated- and resting-cell states. The use of functional assays that demonstrate the key regulating role of growth factors and cytokine stimuli such as PDGF, TGF-beta, and EGF and the effect of ECM molecules such as Biglycan, are also presented and discussed.

Amino Acid Sequence↗