PubMed Health⌕ Search

Biomedical subjects

Mark P Molloy

Publications and source records attributed to Mark P Molloy.

9 recordsLinked to original sources
PubMed2005

Large-scale evaluation of quantitative reproducibility and proteome coverage using acid cleavable isotope coded affinity tag mass spectrometry for proteomic profiling.

Strategies employing non-gel based methods for quantitative proteomic profiling such as isotope coded affinity tags coupled with mass spectrometry (ICAT-MS) are gaining attention as alternatives to two-dimensional gel electrophoresis (2-DE). We have conducted a large-scale investigation to determine the degree of reproducibility and depth of proteome coverage of a typical ICAT-MS experiment by measuring protein changes in Escherichia coli treated with triclosan, an inhibitor of fatty acid biosynthesis. The entire ICAT-MS experiment was conducted on four independent occasions where more than 24 000 peptides were quantitated using an ion-trap mass spectrometer. Our results demonstrated that quantitatively, the technique provided good reproducibility (median coefficient of variation of ratios was 18.6%), and on average identified more than 450 unique proteins per experiment. However, the method was strongly biased to detect acidic proteins (pI < 7), under-represented small proteins (<10 kDa) and failed to show clear superiority over 2-DE methods in monitoring hydrophobic proteins from cell lysates.

Anti-Infective Agents, Local↗
PubMed2004

Differential expression of the skeletal muscle proteome in mdx mice at different ages.

The mdx mouse is the most commonly used animal model for Duchenne muscular dystrophy (DMD), a disease caused by the absence of dystrophin. Although much has been done to elucidate the structure and function of dystrophin and the dystrophin-associated glycoprotein complex (DGC), little is known about the cascade of molecular events triggered by the absence of dystrophin that lead to muscle degeneration. To study the molecular basis of DMD, we decided to systematically study the skeletal muscle proteome in mdx mice at different ages. By using two-dimensional (2-D) gel electrophoresis, we defined changes in the protein expression pattern between mdx and control muscles. Approximately 46 differentially expressed proteins from the cytosolic fraction of mdx hindlimb muscles at three months of age were detected by 2-D gel analysis, of which 24 were identified by matrix assisted laser desorption/ionization- mass spectrometry. Most of the proteins fell into five groups of functionally related proteins. These functional categories are (i) metabolism and energy production, (ii) serine protease inhibitor family, (iii) growth and differentiation, (iv) calcium homeostasis, and (v) cytoskeletal reorganization and biogenesis. The potential roles of the differentially expressed proteins are discussed in the context of the mdx phenotype. Finally, we analyzed alterations of protein expression in mdx mice at one and six months of age to determine how protein expression changes with disease progression.

Animals↗
PubMed2003

Overcoming technical variation and biological variation in quantitative proteomics.

Quantitative proteomics investigates physiology at the molecular level by measuring relative differences in protein expression between samples under different experimental conditions. A major obstacle to reliably determining quantitative changes in protein expression is to overcome error imposed by technical variation and biological variation. In drug discovery and development the issue of biological variation often rises in concordance with the developmental stage of research, spanning from in vitro assays to clinical trials. In this paper we present case studies to raise awareness to the issues of technical variation and biological variation and the impact this places on applying quantitative proteomics. We defined the degree of technical variation from the process of two-dimensional electrophoresis as 20-30% coefficient of variation. On the other hand, biological variation observed experiment-to-experiment showed a broader degree of variation depending upon the sample type. This was demonstrated with case studies where variation was monitored across experiments with bacteria, established cell lines, primary cultures, and with drug treated human subjects. We discuss technical variation and biological variation as key factors to consider during experimental design, and offer insight into preparing experiments that overcome this challenge to provide statistically significant outcomes for conducting quantitative proteomic research.

Animals↗
PubMed2003

Proteomic analysis of mdx skeletal muscle: Great reduction of adenylate kinase 1 expression and enzymatic activity.

To investigate the pathophysiological events of Duchenne muscular dystrophy, we analyzed alterations of protein expression in hindlimb muscles of three month old mdx mice using two-dimensional gel electrophoresis and mass spectrometry. About 40 differentially expressed proteins from the cytosolic fraction and 20 from the microsomal fraction of mdx hindlimb muscles were identified. Among these altered proteins, adenylate kinase 1 (AK 1) was particular interesting because its decrease in abundance was so dramatic (> four-fold). Enzymatic assays demonstrated that AK 1 activity was also decreased in mdx mice. Furthermore, the expression and enzymatic activity of AK 1 were consistently reduced in mdx mice at one and six months of age, suggesting a direct relationship between the lack of dystrophin and alteration of AK 1. Along with AK 1, creatine kinase (CK) provides a major pathway for regulation of nucleotide ratios and energy metabolism in muscles. To gain a better understanding of mechanisms of energy metabolism, we also investigated CK activities in these mdx mice at different ages. Our results suggested that decreased AK 1 expression and activity might result in redistribution of energy flow through the alternative CK system, thus a compensatory potential might limit cellular energy failure in mdx skeletal muscle.

Adenylyl Cyclases↗
PubMed2003

Purification and characterization of a serine protease and chitinases from Paecilomyces lilacinus and detection of chitinase activity on 2D gels.

The filamentous fungus Paecilomyces lilacinus is currently developed as a biocontrol agent against plant parasitic nematodes. Nematode eggs and cuticles are the infection sites for biocontrol agents that penetrate by the production of lytic enzymes. P. lilacinus was cultured in liquid media and proteases and chitinases were induced by the introduction of egg yolk and chitin, respectively. A serine protease was purified from a culture medium using Sepharose-bacitracin affinity column. The protease occurred in three forms, two of which were C-terminally truncated. Chitinase activity was also observed in the culture supernatant, and after separation by isoelectric focusing six proteins were detected that showed activity. Chitinase activity was further confirmed on non-denaturing one-dimensional (1D) and two-dimensional (2D) gels using a sandwich assay with glycol chitin as a substrate. Two of the proteins had similarities with endochitinases as shown by their N-terminal amino acid sequences.

Amino Acid Sequence↗
PubMed2002

Profiling the alkaline membrane proteome of Caulobacter crescentus with two-dimensional electrophoresis and mass spectrometry.

Attempts at protein profiling in the alkaline pH region using isoelectric focusing have often proved difficult, greatly limiting the scope of proteome analysis. We investigated several parameters using custom pH 8-11 immobilized pH gradients to separate a Caulobacter crescentus membrane preparation. These included sample application, quenching endoosomotic flow and gel matrix composition. Among these factors, the sample application position was the predominant parameter to affect two-dimensional gel quality. Separated proteins were silver stained and profiled using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The use of a prototype MALDI-Q-Tof mass spectrometer assisted identification of several proteins by providing highly informative peptide fragmentation data from the sample digests. Thirty-two unique alkaline proteins were identified in this study, which complements our previously described C. crescentus membrane proteome. Our experiments point towards new options for proteomic researchers aiming to both extend the scope of analysis, and simplify methods of identifying proteins with high confidence.

2-Propanol↗
PubMed2002

Proteomics: technologies and applications.

Enabling technologies for proteomics (studies examining the protein complement of the genome) have been in development for over 20 years. However, more recently, the field has become formalized by combining the techniques for large-scale protein separation (two-dimensional electrophoresis) with very precise, high fidelity approaches to the analysis and characterisation of the separated proteins (mass spectrometry). These approaches bring to reality the powerful scope of proteomics, enabling the interpretation of cell physiology through profiling the molecular biodescriptors of gene expression, the proteins. Applications of proteomics are wide ranging and can be found within many disciplines, although one of the more developed fields that is examined here covers the arena of toxicological and pharmacological profiling. In this paper, the current technologies that make proteomics possible will be looked at. Some areas where proteomics has delivered valuable biological insight will also be considered, and there will be a look towards future areas of development.

Electrophoresis, Gel, Two-Dimensional↗