Stephanie M. Cologna

Proteomic analysis of 3T3-L1 adipocyte mitochondria during differentiation and enlargement.

The increase in adipose tissue mass arises in part from progressive lipid loading and triglyceride accumulation in adipocytes. Enlarged adipocytes produce the highest levels of pro-inflammatory molecules and reactive oxygen species (ROS). Since mitochondria are the site for major metabolic processes (e.g., TCA cycle) that govern the extent of triglyceride accumulation as well as the primary site of ROS generation, we quantitatively investigated changes in the adipocyte mitochondrial proteome during different stages of differentiation and enlargement. Mitochondrial proteins from 3T3-L1 adipocytes at different stages of lipid accumulation (days 0-18) were digested and labeled using the iTRAQ 8-plex kit. The labeled peptides were fractionated using a liquid phase isoelectric fractionation system (MSWIFT) to increase the depth of proteome coverage and analyzed using LC-MS/MS. A total of 631 proteins in the mitochondrial fraction, including endoplasmic reticulum-associated and golgi-related mitochondrial proteins, were identified and classified into 12 functional categories. A total of 123 proteins demonstrated a statistically significant change in expression in at least one of the time points over the course of the experiment. The identified proteins included enzymes and transporters involved in the TCA cycle, fatty acid oxidation, and ATP synthesis. Our results indicate that cultured adipocytes enter a state of metabolic-overdrive where increased flux through the TCA cycle and increased fatty acid oxidation occur simultaneously. The proteomic data also suggest that accumulation of reduced electron carriers and the resultant oxidative stress may be attractive targets for modulating adipocyte function in metabolic disorders.

Quantitative proteomic analysis of Niemann-Pick disease, type C1 cerebellum identifies protein biomarkers and provides pathological insight.

Niemann-Pick disease, type C1 (NPC1) is a fatal, neurodegenerative disorder for which there is no definitive therapy. In NPC1, a pathological cascade including neuroinflammation, oxidative stress and neuronal apoptosis likely contribute to the clinical phenotype. While the genetic cause of NPC1 is known, we sought to gain a further understanding into the pathophysiology by identifying differentially expressed proteins in Npc1 mutant mouse cerebella. Using two-dimensional gel electrophoresis and mass spectrometry, 77 differentially expressed proteins were identified in Npc1 mutant mice cerebella compared to controls. These include proteins involved in glucose metabolism, detoxification/oxidative stress and Alzheimer disease-related proteins. Furthermore, members of the fatty acid binding protein family, including FABP3, FABP5 and FABP7, were found to have altered expression in the Npc1 mutant cerebellum relative to control. Translating our findings from the murine model to patients, we confirm altered expression of glutathione s-transferase α, superoxide dismutase, and FABP3 in cerebrospinal fluid of NPC1 patients relative to pediatric controls. A subset of NPC1 patients on miglustat, a glycosphingolipid synthesis inhibitor, showed significantly decreased levels of FABP3 compared to patients not on miglustat therapy. This study provides an initial report of dysregulated proteins in NPC1 which will assist with further investigation of NPC1 pathology and facilitate implementation of therapeutic trials.

Human and mouse neuroinflammation markers in Niemann-Pick disease, type C1

Niemann-Pick disease, type C1 (NPC1) is an autosomal recessive lipid storage disorder in which a pathological cascade, including neuroinflammation occurs. While data demonstrating neuroinflammation is prevalent in mouse models, data from NPC1 patients is lacking. The current study focuses on identifying potential markers of neuroinflammation in NPC1 from both the Npc1 mouse model and NPC1 patients. We identified in the mouse model significant changes in expression of genes associated with inflammation and compared these results to the pattern of expression in human cortex and cerebellar tissue. From gene expression array analysis, complement 3 (C3) was increased in mouse and human post-mortem NPC1 brain tissues. We also characterized protein levels of inflammatory markers in cerebrospinal fluid (CSF) from NPC1 patients and controls. We found increased levels of interleukin 3, chemokine (C-X-C motif) ligand 5, interleukin 16 and chemokine ligand 3 (CCL3), and decreased levels of interleukin 4, 10, 13 and 12p40 in CSF from NPC1 patients. CSF markers were evaluated with respect to phenotypic severity. Miglustat treatment in NPC1 patients slightly decreased IL-3, IL-10 and IL-13 CSF levels; however, further studies are needed to establish a strong effect of miglustat on inflammation markers. The identification of inflammatory markers with altered levels in the cerebrospinal fluid of NPC1 patients may provide a means to follow secondary events in NPC1 disease during therapeutic trials.

Combining Isoelectric Point-Based Fractionation, Liquid Chromatography and Mass Spectrometry to Improve Peptide Detection and Protein Identification

The off-line coupling of an isoelectric trapping device termed membrane separated wells for isoelectric focusing and trapping (MSWIFT) to mass spectrometry-based proteomic studies is described. The MSWIFT is a high capacity, high-throughput, mass spectrometry-compatible isoelectric trapping device that provides isoelectric point (pI)-based separations of complex mixtures of peptides. In MSWIFT, separation and analyte trapping are achieved by migrating the peptide ions through membranes having fixed pH values until the peptide pI is bracketed by the pH values of adjacent membranes. The pH values of the membranes can be tuned, thus affording a high degree of experimental flexibility. Specific advantages of using MSWIFT for sample prefractionation include: (1) small sample volumes (∼200 μL), (2) customized membranes over a large pH range, (3) flexibility in the number of desired fractions, (4) membrane compatibility with a variety of solvents systems, and (5) resulting fractions do not require sample cleanup before MS analysis. Here, we demonstrate the utility of MSWIFT for mass spectrometry-based detection of peptides in improving dynamic range and the reduction of ion suppression effects for high-throughput separations of tryptic peptides.

A novel approach to collision-induced dissociation (CID) for ion mobility-mass spectrometry experiments

Collision induced dissociation (CID) combined with matrix assisted laser desorption ionization-ion mobility-mass spectrometry (MALDI-IM-MS) is described. In this approach, peptide ions are separated on the basis of mobility in a 15 cm drift cell. Following mobility separation, the ions exit the drift cell and enter a 5 cm vacuum interface with a high field region (up to 1000 V/cm) to undergo collisional activation. Ion transmission and ion kinetic energies in the interface are theoretically evaluated accounting for the pressure gradient, interface dimensions, and electric fields. Using this CID technique, we have successfully fragmented and sequenced a number of model peptide ions as well as peptide ions obtained by a tryptic digest. This instrument configuration allows for the simultaneous determination of peptide mass, peptide-ion sequence, and collision-cross section of MALDI-generated ions, providing information critical to the identification of unknown components in complex proteomic samples.

Isoelectric Point Separations of Peptides and Proteins

The separation of ampholytic components according to isoelectric point has played an important role in isolating, reducing complexity and improving peptide and protein detection. This brief review outlines the basics of isoelectric focusing, including a summary of the historical achievements and considerations in experimental design. Derivative methodologies of isoelectric focusing are also discussed including common detection methods used. Applications in a variety of fields using isoelectric point based separations are provided as well as an outlook on the field for future studies.

Characterization of hydroxypropyl-beta-cyclodextrins used in the treatment of Niemann-Pick Disease type C1

2-Hydroxypropyl-beta-cyclodextrin (HPβCD) has gained recent attention as a potential therapeutic intervention in the treatment of the rare autosomal-recessive, neurodegenerative lysosomal storage disorder Niemann-Pick Disease Type C1 (NPC1). Notably, HPβCD formulations are not comprised of a single molecular species, but instead are complex mixtures of species with differing degrees of hydroxypropylation of the cyclodextrin ring. The degree of substitution is a critical aspect of the complex mixture as it influences binding to other molecules and thus could potentially modulate biological effects. VTS-270 (Kleptose HPB) and Trappsol® Cyclo™ are HPβCD products under investigation as novel treatments for NPC1. The purpose of the present work is to compare these two different products; analyses were based on ion distribution and abundance profiles using mass spectrometry methodology as a means for assessing key molecular distinctions between products. The method incorporated electrospray ionization and analysis with a linear low-field ion mobility quadrupole time-of-flight instrument. We observed that the number of hydroxypropyl groups (the degrees of substitution) are substantially different between the two products and greater in Trappsol Cyclo than in VTS-270. The principal ions of both samples are ammonium adducts. Isotope clusters for each of the major ions show doubly charged homodimers of the ammonium adducts. In addition, both products show doubly charged homodimers from adduction of both a proton and ammonium. Doubly charged heterodimers are also present, but are more intense in Trappsol Cyclo than in VTS-270. Based on the analytical differences observed between VTS-270 and Trappsol Cyclo with respect to the degree of substitution, the composition and fingerprint of the complex mixture, and the impurity profiles, these products cannot be considered to be the same; the potential biological and clinical implications of these differences are not presently known.

Defective Cytochrome P450-Catalysed Drug Metabolism in Niemann-Pick Type C Disease

Niemann-Pick type C (NPC) disease is a neurodegenerative lysosomal storage disease caused by mutations in either the NPC1 or NPC2 gene. NPC is characterised by storage of multiple lipids in the late endosomal/lysosomal compartment, resulting in cellular and organ system dysfunction. The underlying molecular mechanisms that lead to the range of clinical presentations in NPC are not fully understood. While evaluating potential small molecule therapies in Npc1-/- mice, we observed a consistent pattern of toxicity associated with drugs metabolised by the cytochrome P450 system, suggesting a potential drug metabolism defect in NPC1 disease. Investigation of the P450 system in the context of NPC1 dysfunction revealed significant changes in the gene expression of many P450 associated genes across the full lifespan of Npc1-/- mice, decreased activity of cytochrome P450 reductase, and a global decrease of multiple cytochrome P450 catalysed dealkylation reactions. In vivo drug metabolism studies using a prototypic P450 metabolised drug, midazolam, confirmed dysfunction in drug clearance in the Npc1-/- mouse. Expression of the Phase II enzyme uridinediphosphate-glucuronosyltransferase (UGT) was also significantly reduced in Npc1-/- mice. Interestingly, reduced activity within the P450 system was also observed in heterozygous Npc1+/- mice. The reduced activity of P450 enzymes may be the result of bile acid deficiency/imbalance in Npc1-/- mice, as bile acid treatment significantly rescued P450 enzyme activity in Npc1-/- mice and has the potential to be an adjunctive therapy for NPC disease patients. The dysfunction in the cytochrome P450 system were recapitulated in the NPC1 feline model. Additionally, we present the first evidence that there are alterations in the P450 system in NPC1 patients.

Efficient Electrophoretic Method to Remove Neutral Additives from Protein Solutions Followed by Mass Spectrometry Analysis

A mass spectrometry (MS)-compatible, isoelectric point-based separation method for removal of neutral additives from protein solutions is described. The separation is performed by electrophoretic migration and trapping using a device referred to as membrane separated wells for isoelectric focusing and trapping (MSWIFT). Electrophoretic separation in the MSWIFT device is fast; the entire process can be carried out in a matter of minutes, and it does not require further sample cleanup prior to MS analysis. Proof-of-concept experiments in which neutral additives (e.g., Triton X-100, Tween 20, poly(ethylene glycol)) are removed from protein solutions using the MSWIFT device followed by MS analysis are described. Coupling the MSWIFT separation with ion mobility MS provides additional separation via the gas phase and assists in achieving higher quality ESI mass spectra when small amounts of additives remain in solution.

An Efficient Approach to Evaluate Reporter Ion Behavior from MALDI-MS/MS Data for Quantification Studies Using Isobaric Tags

Protein quantification, identification, and abundance determination are important aspects of proteome characterization and are crucial in understanding biological mechanisms and human diseases. Different strategies are available to quantify proteins using mass spectrometric detection, and most are performed at the peptide level and include both targeted and untargeted methodologies. Discovery-based or untargeted approaches oftentimes use covalent tagging strategies (i.e., iTRAQ, TMT), where reporter ion signals collected in the tandem MS experiment are used for quantification. Herein we investigate the behavior of the iTRAQ 8-plex chemistry using MALDI-TOF/TOF instrumentation. The experimental design and data analysis approach described is simple and straightforward, which allows researchers to optimize data collection and proper analysis within a laboratory. iTRAQ reporter ion signals were normalized within each spectrum to remove peptide biases. An advantage of this approach is that missing reporter ion values can be accepted for purposes of protein identification and quantification without the need for ANOVA analysis. We investigate the distribution of reporter ion peak areas in an equimolar system and a mock biological system and provide recommendations for establishing fold-change cutoff values at the peptide level for iTRAQ data sets. These data provide a unique data set available to the community for informatics training and analysis.