@article{heaven_flint_randall_sosunov_wilson_barnes_goldman_muddiman_brenner_2016, title={Composition of Rosenthal Fibers, the Protein Aggregate Hallmark of Alexander Disease}, volume={15}, ISSN={["1535-3907"]}, DOI={10.1021/acs.jproteome.6b00316}, abstractNote={Alexander disease (AxD) is a neurodegenerative disorder characterized by astrocytic protein aggregates called Rosenthal fibers (RFs). We used mouse models of AxD to determine the protein composition of RFs to obtain information about disease mechanisms including the hypothesis that sequestration of proteins in RFs contributes to disease. A method was developed for RF enrichment, and analysis of the resulting fraction using isobaric tags for relative and absolute quantitation mass spectrometry identified 77 proteins not previously associated with RFs. Three of five proteins selected for follow-up were confirmed enriched in the RF fraction by immunobloting of both the AxD mouse models and human patients: receptor for activated protein C kinase 1 (RACK1), G1/S-specific cyclin D2, and ATP-dependent RNA helicase DDX3X. Immunohistochemistry validated cyclin D2 as a new RF component, but results for RACK1 and DDX3X were equivocal. None of these was decreased in the non-RF fractions compared to controls. A similar result was obtained for the previously known RF component, alphaB-crystallin, which had been a candidate for sequestration. Thus, no support was obtained for the sequestration hypothesis for AxD. Providing possible insight into disease progression, the association of several of the RF proteins with stress granules suggests a role for stress granules in the origin of RFs.}, number={7}, journal={JOURNAL OF PROTEOME RESEARCH}, author={Heaven, Michael R. and Flint, Daniel and Randall, Shan M. and Sosunov, Alexander A. and Wilson, Landon and Barnes, Stephen and Goldman, James E. and Muddiman, David C. and Brenner, Michael}, year={2016}, month={Jul}, pages={2265–2282} }
 @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} }
 @article{nepomuceno_gibson_randall_muddiman_2014, title={Accurate Identification of Deamidated Peptides in Global Proteomics Using a Quadrupole Orbitrap Mass Spectrometer}, volume={13}, ISSN={["1535-3907"]}, DOI={10.1021/pr400848n}, abstractNote={Deamidation of asparagine and glutamine residues is a common post-translational modification. Researchers often rely on mass spectrometric based proteomic techniques for the identification of these post-translational sites. Mass spectral analysis of deamidated peptides is complicated and often misassigned due to overlapping (13)C peak of the amidated form with the deamidated monoisotopic peak; these two peaks are only separated by 19.34 mDa. For proper assignment, it is inherently important to use a mass spectrometer with high mass measurement accuracy and high resolving power. Herein, mouse brain tissue lysate was prepared using filter-aided sample preparation (FASP) method and Stage Tip fractionation followed by analysis on a nanoLC coupled with a quadrupole orbitrap (Q-Exactive) mass spectrometer to accurately identify more than 5400 proteins. Mass spectral data was processed using MASCOT and ProteoIQ for accurate identification of peptides and proteins. MASCOT search values for precursor and MS/MS mass tolerances were investigated, and it was determined that data searched with greater than 5 ppm precursor mass tolerance resulted in the misassignment of deamidated peptides. Peptides that were identified with a mass measurement accuracy of ±5 ppm were correctly assigned.}, number={2}, journal={JOURNAL OF PROTEOME RESEARCH}, author={Nepomuceno, Angelito I. and Gibson, Radiance J. and Randall, Shan M. and Muddiman, David C.}, year={2014}, month={Feb}, pages={777–785} }
 @article{randall_koryakina_williams_muddiman_2014, title={Evaluating nonpolar surface area and liquid chromatography/mass spectrometry response: an application for site occupancy measurements for enzyme intermediates in polyketide biosynthesis}, volume={28}, ISSN={0951-4198}, url={http://dx.doi.org/10.1002/rcm.7051}, DOI={10.1002/rcm.7051}, abstractNote={RATIONALESite occupancy measurements using liquid chromatography/mass spectrometry (LC/MS) are reported throughout the literature. However, site occupancy quantification suffers from ionization bias between modified and unmodified peptides containing the active site. In this study, we explore the MS signal as a function of nonpolar surface area (NPSA) in order to better understand this bias in electrospray response. The correlation between hydrophobicity and LC/MS response was evaluated and applied to study enzyme intermediates in polyketide synthases.}, number={23}, journal={Rapid Communications in Mass Spectrometry}, publisher={Wiley}, author={Randall, Shan M. and Koryakina, Irina and Williams, Gavin J. and Muddiman, David C.}, year={2014}, month={Oct}, pages={2511–2522} }
 @article{randall_cardasis_muddiman_2013, title={Factorial Experimental Designs Elucidate Significant Variables Affecting Data Acquisition on a Quadrupole Orbitrap Mass Spectrometer}, volume={24}, ISSN={["1044-0305"]}, DOI={10.1007/s13361-013-0693-y}, abstractNote={Instrument parameter values for a quadrupole Orbitrap mass spectrometer were optimized for performing global proteomic analyses. Fourteen factors were evaluated for their influence on data-dependent acquisition with an emphasis on both the rate of sequencing and spectral quality by maximizing two individually tested response variables (unique peptides and protein groups). Of the 14 factors, 12 factors were assigned significant contrast values (P < 0.05) for both response variables. Fundamentally, when optimizing parameters, a balance between spectral quality and duty cycle needs to be reached in order to maximize proteome coverage. This is especially true when using a data-dependent approach for sequencing complex proteomes. For example, maximum ion injection time, automatic gain control settings, and minimum threshold settings for triggering MS/MS isolation and activation all heavily influence ion signal, the number of spectra collected, and spectral quality. To better assess the effect these parameters have on data acquisition, all MS/MS data were parsed according to ion abundance by calculating the percent of the AGC target reached for each MS/MS event and then compared with successful peptide-spectrum matches. This proved to be an effective approach for understanding the effect of ion abundance on successful peptide-spectrum matches and establishing minimum ion abundance thresholds for triggering MS/MS isolation and activation.}, number={10}, journal={JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY}, author={Randall, Shan M. and Cardasis, Helene L. and Muddiman, David C.}, year={2013}, month={Oct}, pages={1501–1512} }
 @article{sarkar_randall_muddiman_rao_2012, title={Targeted Proteomics of the Secretory Pathway Reveals the Secretome of Mouse Embryonic Fibroblasts and Human Embryonic Stem Cells}, volume={11}, ISSN={["1535-9484"]}, DOI={10.1074/mcp.m112.020503}, abstractNote={Proteins endogenously secreted by human embryonic stem cells (hESCs) and those present in hESC culture medium are critical regulators of hESC self-renewal and differentiation. Current MS-based approaches for identifying secreted proteins rely predominantly on MS analysis of cell culture supernatants. Here we show that targeted proteomics of secretory pathway organelles is a powerful alternate approach for interrogating the cellular secretome. We have developed procedures to obtain subcellular fractions from mouse embryonic fibroblasts (MEFs) and hESCs that are enriched in secretory pathway organelles while ensuring retention of the secretory cargo. MS analysis of these fractions from hESCs cultured in MEF conditioned medium (MEF-CM) or MEFs exposed to hESC medium revealed 99 and 129 proteins putatively secreted by hESCs and MEFs, respectively. Of these, 53 and 62 proteins have been previously identified in cell culture supernatants of MEFs and hESCs, respectively, thus establishing the validity of our approach. Furthermore, 76 and 37 putatively secreted proteins identified in this study in MEFs and hESCs, respectively, have not been reported in previous MS analyses. The identification of low abundance secreted proteins via MS analysis of cell culture supernatants typically necessitates the use of altered culture conditions such as serum-free medium. However, an altered medium formulation might directly influence the cellular secretome. Indeed, we observed significant differences between the abundances of several secreted proteins in subcellular fractions isolated from hESCs cultured in MEF-CM and those exposed to unconditioned hESC medium for 24 h. In contrast, targeted proteomics of secretory pathway organelles does not require the use of customized media. We expect that our approach will be particularly valuable in two contexts highly relevant to hESC biology: obtaining a temporal snapshot of proteins secreted in response to a differentiation trigger, and identifying proteins secreted by cells that are isolated from a heterogeneous population.}, number={12}, journal={MOLECULAR & CELLULAR PROTEOMICS}, author={Sarkar, Prasenjit and Randall, Shan M. and Muddiman, David C. and Rao, Balaji M.}, year={2012}, month={Dec}, pages={1829–1839} }
 @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={Abstract}, 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 18O, 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. Copyright © 2011 John Wiley & Sons, Ltd.}, 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} }