@article{sarkar_mischler_randall_collier_dorman_boggess_muddiman_rao_2016, title={Identification of Epigenetic Factor Proteins Expressed in Human Embryonic Stem Cell-Derived Trophoblasts and in Human Placental Trophoblasts}, volume={15}, ISSN={["1535-3907"]}, DOI={10.1021/acs.jproteome.5b01118}, abstractNote={Human embryonic stem cells (hESCs) have been used to derive trophoblasts through differentiation in vitro. Intriguingly, mouse ESCs are prevented from differentiation to trophoblasts by certain epigenetic factor proteins such as Dnmt1, thus necessitating the study of epigenetic factor proteins during hESC differentiation to trophoblasts. We used stable isotope labeling by amino acids in cell culture and quantitative proteomics to study changes in the nuclear proteome during hESC differentiation to trophoblasts and identified changes in the expression of 30 epigenetic factor proteins. Importantly, the DNA methyltransferases DNMT1, DNMT3A, and DNMT3B were downregulated. Additionally, we hypothesized that nuclear proteomics of hESC-derived trophoblasts may be used for screening epigenetic factor proteins expressed by primary trophoblasts in human placental tissue. Accordingly, we conducted immunohistochemistry analysis of six epigenetic factor proteins identified from hESC-derived trophoblasts-DNMT1, DNMT3B, BAF155, BAF60A, BAF57, and ING5-in 6-9 week human placentas. Indeed, expression of these proteins was largely, though not fully, consistent with that observed in 6-9 week placental trophoblasts. Our results support the use of hESC-derived trophoblasts as a model for placental trophoblasts, which will enable further investigation of epigenetic factors involved in human trophoblast development.}, number={8}, journal={JOURNAL OF PROTEOME RESEARCH}, author={Sarkar, Prasenjit and Mischler, Adam and Randall, Shan M. and Collier, Timothy S. and Dorman, Karen F. and Boggess, Kim A. and Muddiman, David C. and Rao, Balaji M.}, year={2016}, month={Aug}, pages={2433–2444} }
 @misc{collier_muddiman_2012, title={Analytical strategies for the global quantification of intact proteins}, volume={43}, ISSN={["1438-2199"]}, DOI={10.1007/s00726-012-1285-z}, number={3}, journal={AMINO ACIDS}, author={Collier, Timothy S. and Muddiman, David Charles}, year={2012}, month={Sep}, pages={1109–1117} }
 @article{sarkar_collier_randall_muddiman_rao_2012, title={The subcellular proteome of undifferentiated human embryonic stem cells}, volume={12}, ISSN={["1615-9853"]}, DOI={10.1002/pmic.201100507}, abstractNote={We have characterized the subcellular proteome of human embryonic stem cells (hESCs) through MS analysis of the membrane, cytosolic, and nuclear fractions, isolated from the same sample of undifferentiated hESCs. Strikingly, 74% of all proteins identified were detected in a single subcellular fraction; we also carried out immunofluorescence studies to validate the subcellular localization suggested by proteomic analysis, for a subset of proteins. Our approach resulted in deeper proteome coverage – peptides mapping to 893, 2475, and 1185 proteins were identified in the nuclear, cytosolic, and membrane fractions, respectively. Additionally, we used spectral counting to estimate the relative abundance of all cytosolic proteins. A large number of proteins relevant to hESC biology, including growth factor receptors, cell junction proteins, transcription factors, chromatin remodeling proteins, and histone modifying enzymes were identified. Our analysis shows that components of a large number of interacting signaling pathways are expressed in hESCs. Finally, we show that proteomic analysis of the endoplasmic reticulum (ER) and Golgi compartments is a powerful alternative approach to identify secreted proteins since these are synthesized in the ER and transit through the Golgi. Taken together, our results show that systematic subcellular proteomic analysis is a valuable tool for studying hESC biology.}, number={3}, journal={PROTEOMICS}, author={Sarkar, Prasenjit and Collier, Timothy S. and Randall, Shan M. and Muddiman, David C. and Rao, Balaji M.}, year={2012}, month={Feb}, pages={421–430} }
 @article{collier_randall_sarkar_rao_dean_muddiman_2011, title={Comparison of stable-isotope labeling with amino acids in cell culture and spectral counting for relative quantification of protein expression}, volume={25}, ISSN={["1097-0231"]}, DOI={10.1002/rcm.5151}, abstractNote={Protein quantification is one of the principal goals of mass spectrometry (MS)-based proteomics, and many strategies exist to achieve it. Several approaches involve the incorporation of a stable-isotope label using either chemical derivatization, enzymatically catalyzed incorporation of (18)O, or metabolic labeling in a cell or tissue culture. These techniques can be cost or time prohibitive or not amenable to the biological system of interest. Label-free techniques including those utilizing integrated ion abundance and spectral counting offer an alternative to stable-isotope-based methodologies. Herein, we present the comparison of stable-isotope labeling of amino acids in cell culture (SILAC) with spectral counting for the quantification of human embryonic stem cells as they differentiate toward the trophectoderm at three time points. Our spectral counting experimental strategy resulted in the identification of 2641 protein groups across three time points with an average sequence coverage of 30.3%, of which 1837 could be quantified with more than five spectral counts. SILAC quantification was able to identify 1369 protein groups with an average coverage of 24.7%, of which 1027 could be quantified across all time points. Within this context we further explore the capacity of each strategy for proteome coverage, variation in quantification, and the relative sensitivity of each technique to the detection of change in relative protein expression.}, number={17}, journal={RAPID COMMUNICATIONS IN MASS SPECTROMETRY}, author={Collier, Timothy S. and Randall, Shan M. and Sarkar, Prasenjit and Rao, Balaji M. and Dean, Ralph A. and Muddiman, David C.}, year={2011}, month={Sep}, pages={2524–2532} }
 @article{collier_sarkar_franck_rao_dean_muddiman_2010, title={Direct Comparison of Stable Isotope Labeling by Amino Acids in Cell Culture and Spectral Counting for Quantitative Proteomics}, volume={82}, ISSN={["1520-6882"]}, DOI={10.1021/ac101978b}, abstractNote={Numerous experimental strategies exist for relative protein quantification, one of the primary objectives of mass spectrometry based proteomics analysis. These strategies mostly involve the incorporation of a stable isotope label via either metabolic incorporation in cell or tissue culture (¹⁵N/¹⁴N metabolic labeling, stable isotope labeling by amino acids in cell culture (SILAC)), chemical derivatization (ICAT, iTRAQ, TMT), or enzymatically catalyzed incorporation (¹⁸O labeling). Also, these techniques can be cost or time prohibitive or not amenable to the biological system of interest (i.e., metabolic labeling of clinical samples, most animals, or fungi). This is the case with the quantification of fungal proteomes, which often require auxotroph mutants to fully metabolically label. Alternatively, label-free strategies for protein quantification such as using integrated ion abundance and spectral counting have been demonstrated for quantification affording over 2 orders of magnitude of dynamic range which is comparable to metabolic labeling strategies. Direct comparisons of these quantitative techniques are largely lacking in the literature but are highly warranted in order to evaluate the capabilities, limitations, and analytical variability of available quantitative strategies. Here, we present the direct comparison of SILAC to label-free quantification by spectral counting of an identical set of data from the bottom-up proteomic analysis of human embryonic stem cells, which are readily able to be quantified using both strategies, finding that both strategies result in a similar number of protein identifications. We also discuss necessary constraints for accurate quantification using spectral counting and assess the potential of this label-free strategy as a viable alternative for quantitative proteomics.}, number={20}, journal={ANALYTICAL CHEMISTRY}, author={Collier, Timothy S. and Sarkar, Prasenjit and Franck, William L. and Rao, Balaji M. and Dean, Ralph A. and Muddiman, David C.}, year={2010}, month={Oct}, pages={8696–8702} }
 @article{collier_sarkar_rao_muddiman_2010, title={Quantitative top-down proteomics of SILAC labeled human embryonic stem cells}, volume={21}, DOI={10.1016/j.jasms.2010.01.031}, abstractNote={Human embryonic stem cells (hESCs) are self-renewing pluripotent cells with relevance to treatment of numerous medical conditions. However, a global understanding of the role of the hESC proteome in maintaining pluripotency or triggering differentiation is still largely lacking. The emergence of top-down proteomics has facilitated the identification and characterization of intact protein forms that are not readily apparent in bottom-up studies. Combined with metabolic labeling techniques such as stable isotope labeling by amino acids in cell culture (SILAC), quantitative comparison of intact protein expression under differing experimental conditions is possible. Herein, quantitative top-down proteomics of hESCs is demonstrated using the SILAC method and nano-flow reverse phase chromatography directly coupled to a linear-ion-trap Fourier transform ion cyclotron resonance mass spectrometer (nLC-LTQ-FT-ICR-MS). In this study, which to the best of our knowledge represents the first top-down analysis of hESCs, we have confidently identified 11 proteins by accurate intact mass, MS/MS, and amino acid counting facilitated by SILAC labeling. Although quantification is challenging due to the incorporation of multiple labeled amino acids (i.e., lysine and arginine) and arginine to proline conversion, we are able to quantitatively account for these phenomena using a mathematical model.}, number={6}, journal={Journal of the American Society for Mass Spectrometry}, author={Collier, T. S. and Sarkar, P. and Rao, B. and Muddiman, David}, year={2010}, pages={879–889} }
 @article{alcazar_hawkridge_collier_cousins_bhattacharya_muddiman_marin-castano_2009, title={Proteomics Characterization of Cell Membrane Blebs in Human Retinal Pigment Epithelium Cells}, volume={8}, ISSN={["1535-9484"]}, DOI={10.1074/mcp.M900203-MCP200}, abstractNote={Age-related macular degeneration (AMD) is the leading cause of legal blindness among the elderly population in the industrialized world, affecting about 14 million people in the United States alone. Smoking is a major environmental risk factor for AMD, and hydroquinone is a major component in cigarette smoke. Hydroquinone induces the formation of cell membrane blebs in human retinal pigment epithelium (RPE). Blebs may accumulate and eventually contribute first to sub-RPE deposits and then drusen formation, which is a prominent histopathologic feature in eyes with AMD. As an attempt to better understand the mechanisms involved in early AMD, we sought to investigate the proteomic profile of RPE blebs. Isolated blebs were subjected to SDS-PAGE fractionation, and in-gel trypsin-digested peptides were analyzed by LC-MS/MS that lead to the identification of a total of 314 proteins. Identified proteins were predominantly involved in oxidative phosphorylation, cell junction, focal adhesion, cytoskeleton regulation, and immunogenic processes. Importantly basigin and matrix metalloproteinase-14, key proteins involved in extracellular matrix remodeling, were identified in RPE blebs and shown to be more prevalent in AMD patients. Altogether our findings suggest, for the first time, the potential involvement of RPE blebs in eye disease and shed light on the implication of cell-derived microvesicles in human pathology.}, number={10}, journal={MOLECULAR & CELLULAR PROTEOMICS}, author={Alcazar, Oscar and Hawkridge, Adam M. and Collier, Timothy S. and Cousins, Scott W. and Bhattacharya, Sanjoy K. and Muddiman, David C. and Marin-Castano, Maria E.}, year={2009}, month={Oct}, pages={2201–2211} }
 @article{schweitzer_avci_collier_goodwin_2008, title={Microscopic, chemical and molecular methods for examining fossil specimens}, volume={7}, ISSN={["1777-571X"]}, DOI={10.1016/j.crpv.2008.02.005}, abstractNote={Advances in technology over the past two decades have resulted in unprecedented access to data from biological specimens. These data have expanded our understanding of physical characteristics, physiological, cellular and subcellular processes, and evolutionary relationships at the molecular level and beyond. Paleontological and archaeological sciences have recently begun to apply these technologies to fossil and subfossil representatives of extinct organisms. Data derived from multidisciplinary, non-traditional techniques can be difficult to decipher, and without a basic understanding of the type of information provided by these methods, their usefulness for fossil studies may be overlooked. This review describes some of these powerful new analytical tools, the data that may be accessible through their use, advantages and limitations, and how they can be applied to fossil material to elucidate characteristics of extinct organisms and their paleoecological environments. Des avancées technologiques intervenues au cours des deux dernières décades ont permis d’accéder à des données sans précédent sur des échantillons biologiques. Ces données ont élargi notre compréhension des caractéristiques physiques, des processus physiologiques cellulaires et subcellulaires, ainsi que des relations évolutives au niveau de la molécule et au-delà. Les sciences paléontologiques et archéologiques se sont récemment orientées vers l’application de ces technologies à des représentants fossiles ou subfossiles d’organismes disparus. Les bénéfices de ces techniques, qui sont du domaine de multiples disciplines, sont difficiles à déchiffrer et, sans une connaissance de base du type d’information fourni par ces méthodes, leur utilité pour l’étude des fossiles peut ne pas être bien mesurée. Cet article décrit certains de ces nouveaux outils analytiques puissants, les données qu’ils permettent d’obtenir, leurs avantages et leurs limites, et comment ils peuvent être appliqués au matériel fossile pour élucider les caractéristiques d’organismes disparus et leurs environnements paléoécologiques.}, number={2-3}, journal={Comptes Rendus Palevol}, author={Schweitzer, M.H. and Avci, R. and Collier, T. and Goodwin, M.B.}, year={2008}, month={Apr}, pages={159–184} }
 @article{collier_hawkridge_georgianna_payne_muddiman_2008, title={Top-down identification and quantification of stable isotope labeled proteins from Aspergillus flavus using online nano-flow reversed-phase liquid chromatography coupled to a LTQ-FTICR mass spectrometer}, volume={80}, ISSN={["1520-6882"]}, DOI={10.1021/ac800254z}, abstractNote={Online liquid chromatography-mass spectrometric (LC-MS) analysis of intact proteins (i.e., top-down proteomics) is a growing area of research in the mass spectrometry community. A major advantage of top-down MS characterization of proteins is that the information of the intact protein is retained over the vastly more common bottom-up approach that uses protease-generated peptides to search genomic databases for protein identification. Concurrent to the emergence of top-down MS characterization of proteins has been the development and implementation of the stable isotope labeling of amino acids in cell culture (SILAC) method for relative quantification of proteins by LC-MS. Herein we describe the qualitative and quantitative top-down characterization of proteins derived from SILAC-labeled Aspergillus flavus using nanoflow reversed-phase liquid chromatography directly coupled to a linear ion trap Fourier transform ion cyclotron resonance mass spectrometer (nLC-LTQ-FTICR-MS). A. flavus is a toxic filamentous fungus that significantly impacts the agricultural economy and human health. SILAC labeling improved the confidence of protein identification, and we observed 1318 unique protein masses corresponding to 659 SILAC pairs, of which 22 were confidently identified. However, we have observed some limiting issues with regard to protein quantification using top-down MS/MS analyses of SILAC-labeled proteins. The role of SILAC labeling in the presence of competing endogenously produced amino acid residues and its impact on quantification of intact species are discussed in detail.}, number={13}, journal={ANALYTICAL CHEMISTRY}, author={Collier, Timothy S. and Hawkridge, Adam M. and Georgianna, D. Ryan and Payne, Gary A. and Muddiman, David C.}, year={2008}, month={Jul}, pages={4994–5001} }