2008. "“Spatial Mapping of the Neurite and Soma Proteomes Reveals a Functional Cdc42/Rac Regulatory Network”." Proceedings of the National Academy of Sciences of the United States of America 105(6):1931-1936. doi:10.1073/pnas.0706545105 Abstract Neurite extension and growth cone navigation are guided by extracellular cues that control cytoskeletal rearrangements. However, understanding the complex signaling mechanisms that mediate neuritogenesis has been limited by the inability to biochemically separate the neurite and soma for spatial proteomic and bioinformatic analyses. Here, we apply global proteome profiling in combination with a novel neurite purification methodology for comparative analysis of the soma and neurite proteomes of neuroblastoma cells. The spatial relationship of 4855 proteins were mapped revealing networks of signaling proteins that control integrins, the actin cytoskeleton, and axonal guidance in the extending neurite. Bioinformatics and functional analyses revealed a spatially compartmentalized Rac/Cdc42 signaling network that operates in conjunction with multiple GEFs and GAPs to control neurite formation. Interestingly, RNA interference experiments revealed that the different GEFs and GAPs regulate specialized functions during neurite formation including neurite growth and retraction kinetics, cytoskeletal organization, and cell polarity. Our findings provide insight into the spatial organization of signaling networks that enable neuritogenesis and provide a comprehensive system-wide profile of proteins that mediate this process including those that control Rac and Cdc42 signaling.
2007. "Applying a Targeted Label-free Approach using LC-MS AMT Tags to Evaluate Changes in Protein Phosphorylation Following Phosphatase Inhibition." Journal of Proteome Research 6(11):4489-4497. doi:10.1021/pr070068e Abstract To identify phosphoproteins regulated by the phosphoprotein phosphatase (PPP) family of S/T phosphatases, we performed a large-scale characterization of changes in protein phosphorylation on extracts from HeLa cells treated with or without calyculin A, a potent PPP enzyme inhibitor. A label-free comparative Phosphoproteomics approach using immobilized metal ion affinity chromatography and targeted tandem mass spectrometry was employed to discover and identify signatures based upon distinctive changes in abundance. Overall, 232 proteins were identified as either direct or indirect targets for PPP enzyme regulation. Most of the present identifications represent novel PPP enzyme targets at the level of both phosphorylation site and protein. These include phosphorylation sites within signaling proteins such as p120 Catenin, A Kinase Anchoring Protein 8, JunB, and Type II Phosphatidyl Inositol 4 Kinase. These data can be used to define underlying signaling pathways and events regulated by the PPP family of S/T phosphatases.
2007. "Spatial Mapping of Protein Abundances in the Mouse Brain by Voxelation Integrated with High-Throughput Liquid Chromatography ─ Mass Spectrometry." Genome Research 17(3):328-336. doi:doi:10.1101/gr.5799207 Abstract Temporally and spatially resolved mapping of protein abundance patterns within the mammalian brain is of significant interest for understanding brain function and molecular etiologies of neurodegenerative diseases; however, such imaging efforts have been greatly challenged by complexity of the proteome, throughput and sensitivity of applied analytical methodologies, and accurate quantitation of protein abundances across the brain. Here, we describe a methodology for comprehensive spatial proteome mapping that addresses these challenges by employing voxelation integrated with automated microscale sample processing, high-throughput LC system coupled with high resolution Fourier transform ion cyclotron mass spectrometer and a “universal” stable isotope labeled reference sample approach for robust quantitation. We applied this methodology as a proof-of-concept trial for the analysis of protein distribution within a single coronal slice of a C57BL/6J mouse brain. For relative quantitation of the protein abundances across the slice, an 18O-isotopically labeled reference sample, derived from a whole control coronal slice from another mouse, was spiked into each voxel sample and stable isotopic intensity ratios were used to obtain measures of relative protein abundances. In total, we generated maps of protein abundance patterns for 1,028 proteins. The significant agreement of the protein distributions with previously reported data supports the validity of this methodology, which opens new opportunities for studying the spatial brain proteome and its dynamics during the course of disease progression and other important biological and associated health aspects in a discovery-driven fashion.
2006. "AMT Tag Approach to Proteomic Characterization of Deinococcus Radiodurans and Shewanella Oneidensis ." In Microbial Proteomics, Methods of Biochemical Analysis, vol. 49, ed. I. Humphery-Smith and M. Hecker, pp. 113-134. John Wiley & Sons, Inc., Hoboken, NJ. Abstract Biology is transitioning from a largely qualitative, mostly descriptive science to a quantitative and ultimately predictive science. Advances in high throughput DNA sequencing have made increasing numbers of genome sequences available and enabled a “systems” level analysis of complex biological organisms. The ability to quantitatively measure the array of proteins, also termed the proteome, in prokaryotic cells and communities of cells is key to understanding microbial systems. This chapter focuses on the utility of the AMT tag mass spectrometric approach used to characterize the proteomes of two microbes, Deinococcus radiodurans and Shewanella oneidensis MR-1.
2005. "Development and Evaluation of a Micro- and Nanoscale Proteomic Sample Preparation Method." Journal of Proteome Research 4(6):2397-2403. doi:10.1021/pr050160f Abstract Efficient and effective sample preparation of micro- and nano-scale (micro- and nano-gram) clinical specimens for proteomic applications is often difficult due to losses during the processing steps. Herein we describe a simple “single-tube” preparation protocol appropriate for small proteomic samples using the organic co-solvent, trifluoroethanol (TFE). TFE facilitates both protein extraction and protein denaturation without requiring a separate cleanup step, thus minimizing sample loss. The performance of the TFE method was initially evaluated by comparing to traditional detergent-based methods on relatively large scale sample processing using human breast cancer cells and mouse brain tissue. The results demonstrated that the TFE protocol provided comparable results to the traditional detergent-based protocols for larger samples (milligrams), based on both sample recovery and peptide/protein identification. The effectiveness of this protocol for micro- and nano-scale sample processing was then evaluated for the extraction of proteins/peptides and shown effective for small mouse brain tissue samples (~ 20 g total protein content) and also for samples of ~ 5 000 human breast cancer MCF-7 cells (~ 500 ng total protein content), where the detergent-based methods were ineffective due to losses during cleanup and transfer steps.
2005. "Quantitative Proteome Analysis of Human Plasma Following in vivo Lipopolysaccharide Administration using O-16/O-18 Labeling and the Accurate Mass and Time Tag Approach." Molecular & Cellular Proteomics. MCP 4(5):700-709. Abstract Identification of novel diagnostic or therapeutic biomarkers from human blood plasma would benefit significantly from quantitative measurements of the proteome constituents over a range of physiological conditions. We describe here an initial demonstration of proteome-wide quantitative analysis of human plasma. The approach utilizes post-digestion trypsin-catalyzed 16O/18O labeling, two-dimensional liquid chromatography (LC)-Fourier transform ion cyclotron resonance ((FTICR) mass spectrometry, and the accurate mass and time (AMT) tag strategy for identification and quantification of peptides/proteins from complex samples. A peptide mass and time tag database was initially generated using tandem mass spectrometry (MS/MS) following extensive multidimensional LC separations and the database serves as a ‘look-up’ table for peptide identification. The mass and time tag database contains >8,000 putative identified peptides, which yielded 938 confident plasma protein identifications. The quantitative approach was applied to the comparative analyses of plasma samples from an individual prior to and 9 hours after lipopolysaccharide (LPS) administration without depletion of high abundant proteins. Accurate quantification of changes in protein abundance was demonstrated with both 1:1 labeling of control plasma and the comparison between the plasma samples following LPS administration. A total of 429 distinct plasma proteins were quantified from the comparative analyses and the protein abundances for 28 proteins were observed to be significantly changed following LPS administration, including several known inflammatory response mediators.
2005. "Improved Proteome Coverage by Using High Efficiency Cysteinyl-peptide Enrichment: The Human Mammary Epithelial Cell Proteome." Proteomics 5(5):1263-1273. Abstract Automated multidimensional capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been increasingly applied in various large scale proteome profiling efforts. However, comprehensive global proteome analysis remains technically challenging due to issues associated with sample complexity and dynamic range of protein abundances, which is particularly apparent in mammalian biological systems. We report here the application of a high efficiency cysteinyl-peptide enrichment (CPE) approach to the global proteome analysis of human mammary epithelial cells (HMECs) which significantly improved both sequence coverage of protein identifications and the overall proteome coverage. The cysteinyl-peptides were specifically enriched by using a thiol-specific covalent resin, fractionated by strong cation exchange chromatography, and subsequently analyzed by reversed-phase capillary LC-MS/MS. An HMEC tryptic digest without CPE was also fractionated and analyzed under the same conditions for comparison. The combined analyses of HMEC tryptic digests with and without CPE resulted in a total of 14,416 confidently identified peptides covering 4,294 different proteims with an estimated 10% gene coverage of the human geome. By using the high efficiency CPE, an additional 1,096 relatively low abundance proteins were identified, resulting in 34.3% increase in proteome coverage; 1,390 protems were observed with increased sequence coverage. Comparative protein distribution analyses revealed that the CPE method is not biased by protein molecular weight, pI, gene location, cellular location, or bioloical functions. These results demonstrate that the use of the CPE approach provides improved efficiency in comprehensive proteome-wide analyses of highly complex mammalian biological systems.
2004. "Multidimensional Proteome Analysis of Human Mammary Epithelial Cells." Journal of Proteome Research 3(1):68-75. Abstract Recent multidimensional liquid chromatography MS/MS studies have contributed to the identification of large numbers of expressed proteins for numerous species. The present study couples size exclusion chromatography of intact proteins with strong cation exchange chromatography to detect tryptically digested peptides in the global protein mixture of human mammary epithelial cells (HMECs) based upon the use of very high resolution, reversed phase capillary LC–MS/MS. A total of >6,200 unique peptides were identified with high confidence covering 1,700 different proteins, out of which 93% were mapped to chromosomal locations providing an estimated 4.4% coverage of the annotated human genome based upon the National Center for Biotechnology Information (NCBI). This database provides a baseline for comparison against variations in other genetically and environmentally perturbed systems. Proteins identified were categorized based upon intracellular location and biological process with the identification of numerous receptors, regulatory proteins, and extracellular proteins, demonstrating the usefulness of this application in the global analysis of human cells for future comparative studies.
2001. "Quantitative Analysis of Bacterial and Mammalian Proteomes using a Combination of Cysteine Affinity Tags and 15N-Metabolic Labeling." Analytical Chemistry 73(9):2132-2139. Abstract N/A