@article{dinh_paudel_brochu_popowski_gracieux_cores_huang_hensley_harrell_vandergriff_et al._2020, title={Inhalation of lung spheroid cell secretome and exosomes promotes lung repair in pulmonary fibrosis}, volume={11}, ISSN={["2041-1723"]}, url={http://dx.doi.org/10.1038/s41467-020-14344-7}, DOI={10.1038/s41467-020-14344-7}, abstractNote={Abstract}, number={1}, journal={NATURE COMMUNICATIONS}, publisher={Springer Science and Business Media LLC}, author={Dinh, Phuong-Uyen C. and Paudel, Dipti and Brochu, Hayden and Popowski, Kristen D. and Gracieux, M. Cyndell and Cores, Jhon and Huang, Ke and Hensley, M. Taylor and Harrell, Erin and Vandergriff, Adam C. and et al.}, year={2020}, month={Feb} }
 @article{schroeter_blackburn_goshe_schweitzer_2019, title={Proteomic method to extract, concentrate, digest and enrich peptides from fossils with coloured (humic) substances for mass spectrometry analyses}, volume={6}, ISSN={["2054-5703"]}, DOI={10.1098/rsos.181433}, abstractNote={Humic substances are breakdown products of decaying organic matter that co-extract with proteins from fossils. These substances are difficult to separate from proteins in solution and interfere with analyses of fossil proteomes. We introduce a method combining multiple recent advances in extraction protocols to both concentrate proteins from fossil specimens with high humic content and remove humics, producing clean samples easily analysed by mass spectrometry (MS). This method includes: (i) a non-demineralizing extraction buffer that eliminates protein loss during the demineralization step in routine methods; (ii) filter-aided sample preparation (FASP) of peptides, which concentrates and digests extracts in one filter, allowing the separation of large humics after digestion; (iii) centrifugal stage tipping, which further clarifies and concentrates samples in a uniform process performed simultaneously on multiple samples. We apply this method to a moa fossil (approx. 800–1000 years) dark with humic content, generating colourless samples and enabling the detection of more proteins with greater sequence coverage than previous MS analyses on this same specimen. This workflow allows analyses of low-abundance proteins in fossils containing humics and thus may widen the range of extinct organisms and regions of their proteomes we can explore with MS.}, number={8}, journal={ROYAL SOCIETY OPEN SCIENCE}, author={Schroeter, Elena R. and Blackburn, Kevin and Goshe, Michael B. and Schweitzer, Mary H.}, year={2019}, month={Aug} }
 @article{bender_blackburn_monaghan_derbyshire_menke_zipfel_goshe_zielinski_huber_2017, title={Autophosphorylation-based Calcium (Ca2(+)) Sensitivity Priming and Ca2(+)/Calmodulin Inhibition of Arabidopsis thaliana Ca2(+)-dependent Protein Kinase 28 (CPK28)}, volume={292}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.m116.763243}, abstractNote={Plant calcium (Ca2+)-dependent protein kinases (CPKs) represent the primary Ca2+-dependent protein kinase activities in plant systems. CPKs are composed of a dual specificity (Ser/Thr and Tyr) kinase domain tethered to a calmodulin-like domain (CLD) via an autoinhibitory junction (J). Although regulation of CPKs by Ca2+ has been extensively studied, the contribution of autophosphorylation in controlling CPK activity is less well understood. Furthermore, whether calmodulin (CaM) contributes to CPK regulation, as is the case for Ca2+/CaM-dependent protein kinases outside the plant lineage, remains an open question. We therefore screened a subset of plant CPKs for CaM binding and found that CPK28 is a high affinity Ca2+/CaM-binding protein. Using synthetic peptides and native gel electrophoresis, we coarsely mapped the CaM-binding domain to a site within the CPK28 J domain that overlaps with the known site of intramolecular interaction between the J domain and the CLD. Peptide kinase activity of fully dephosphorylated CPK28 was Ca2+-responsive and was inhibited by Ca2+/CaM. Using in situ autophosphorylated protein, we expand on the known set of CPK28 autophosphorylation sites, and we demonstrate that, unexpectedly, autophosphorylated CPK28 had enhanced kinase activity at physiological concentrations of Ca2+ compared with the dephosphorylated protein, suggesting that autophosphorylation functions to prime CPK28 for Ca2+ activation and might also allow CPK28 to remain active when Ca2+ levels are low. Furthermore, CPK28 autophosphorylation substantially reduced sensitivity of the kinase to Ca2+/CaM inhibition. Overall, our analyses uncover new complexities in the control of CPK28 and provide mechanistic support for Ca2+ signaling specificity through Ca2+ sensor priming.}, number={10}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Bender, Kyle W. and Blackburn, R. Kevin and Monaghan, Jacqueline and Derbyshire, Paul and Menke, Frank L. H. and Zipfel, Cyril and Goshe, Michael B. and Zielinski, Raymond E. and Huber, Steven C.}, year={2017}, month={Mar}, pages={3988–4002} }
 @article{blackburn_bustamante-marin_yin_goshe_ostrowski_2017, title={Quantitative Proteomic Analysis of Human Airway Cilia Identifies Previously Uncharacterized Proteins of High Abundance}, volume={16}, ISSN={["1535-3907"]}, DOI={10.1021/acs.jproteome.6b00972}, abstractNote={Cilia are essential to many diverse cellular processes. Although many major axonemal components have been identified and studied, how they interact to form a functional axoneme is not completely understood. To further our understanding of the protein composition of human airway cilia, we performed a semiquantitative analysis of ciliary axonemes using label-free LC/MSE, which identified over 400 proteins with high confidence. Tubulins were the most abundant proteins identified, with evidence of 20 different isoforms obtained. Twelve different isoforms of axonemal dynein heavy chain were also identified. Absolute quantification of the nontubulin components demonstrated a greater than 75-fold range of protein abundance (RSPH9;1850 fmol vs CCDC103;24 fmol), adding another level of complexity to axonemal structure. Of the identified proteins, ∼70% are known axonemal proteins. In addition, many previously uncharacterized proteins were identified. Unexpectedly, several of these, including ERICH3, C1orf87, and CCDC181, were present at high relative abundance in the cilia. RT-PCR analysis and immunoblotting confirmed cilia-specific expression for eight uncharacterized proteins, and fluorescence microscopy demonstrated unique axonemal localizations. These studies have provided the first quantitative analysis of the ciliary proteome and have identified and characterized several previously unknown proteins as major constituents of human airway cilia.}, number={4}, journal={JOURNAL OF PROTEOME RESEARCH}, author={Blackburn, Kevin and Bustamante-Marin, Ximena and Yin, Weining and Goshe, Michael B. and Ostrowski, Lawrence E.}, year={2017}, month={Apr}, pages={1579–1592} }
 @article{herring_grant_blackburn_haugh_goshe_2015, title={Development of a tandem affinity phosphoproteomic method with motif selectivity and its application in analysis of signal transduction networks}, volume={988}, ISSN={["1873-376X"]}, DOI={10.1016/j.jchromb.2015.02.017}, abstractNote={Phosphorylation is an important post-translational modification that is involved in regulating many signaling pathways. Of particular interest are the growth factor mediated Ras and phosphoinositide 3-kinase (PI3K) signaling pathways which, if misregulated, can contribute to the progression of cancer. Phosphoproteomic methods have been developed to study regulation of signaling pathways; however, due to the low stoichiometry of phosphorylation, understanding these pathways is still a challenge. In this study, we have developed a multi-dimensional method incorporating electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) with tandem IMAC/TiO2 enrichment for subsequent phosphopeptide identification by LC/MS/MS. We applied this method to PDGF-stimulated NIH 3T3 cells to provide over 11,000 unique phosphopeptide identifications. Upon motif analysis, IMAC was found to enrich for basophilic kinase substrates while the subsequent TiO2 step enriched for acidophilic kinase substrates, suggesting that both enrichment methods are necessary to capture the full complement of kinase substrates. Biological functions that were over-represented at each PDGF stimulation time point, together with the phosphorylation dynamics of several phosphopeptides containing known kinase phosphorylation sites, illustrate the feasibility of this approach in quantitative phosphoproteomic studies.}, journal={JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES}, publisher={Elsevier BV}, author={Herring, Laura E. and Grant, Kyle G. and Blackburn, Kevin and Haugh, Jason M. and Goshe, Michael B.}, year={2015}, month={Apr}, pages={166–174} }
 @article{mackenzie_schipper_england_thomas_blackburn_swartz_clark_2013, title={Lengthening the Intersubunit Linker of Procaspase 3 Leads to Constitutive Activation}, volume={52}, ISSN={["0006-2960"]}, DOI={10.1021/bi400793s}, abstractNote={The conformational ensemble of procaspase 3, the primary executioner in apoptosis, contains two major forms, inactive and active, with the inactive state favored in the native ensemble. A region of the protein known as the intersubunit linker (IL) is cleaved during maturation, resulting in movement of the IL out of the dimer interface and subsequent active site formation (activation-by-cleavage mechanism). We examined two models for the role of the IL in maintaining the inactive conformer, an IL-extension model versus a hydrophobic cluster model, and we show that increasing the length of the IL by introducing 3-5 alanines results in constitutively active procaspases. Active site labeling and subsequent analyses by mass spectrometry show that the full-length zymogen is enzymatically active. We also show that minor populations of alternately cleaved procaspase result from processing at D169 when the normal cleavage site, D175, is unavailable. Importantly, the alternately cleaved proteins have little to no activity, but increased flexibility of the linker increases the exposure of D169. The data show that releasing the strain of the short IL, in and of itself, is not sufficient to populate the active conformer of the native ensemble. The IL must also allow for interactions that stabilize the active site, possibly from a combination of optimal length, flexibility in the IL, and specific contacts between the IL and interface. The results provide further evidence that substantial energy is required to shift the protein to the active conformer. As a result, the activation-by-cleavage mechanism dominates in the cell.}, number={36}, journal={BIOCHEMISTRY}, author={MacKenzie, Sarah H. and Schipper, Joshua L. and England, Erika J. and Thomas, Melvin E., III and Blackburn, Kevin and Swartz, Paul and Clark, A. Clay}, year={2013}, month={Sep}, pages={6219–6231} }
 @article{menegatti_ward_naik_kish_blackburn_carbonell_2013, title={Reversible cyclic peptide libraries for the discovery of affinity ligands}, volume={85}, DOI={10.1021/ac401954k}, abstractNote={A novel strategy is presented for the identification of cyclic peptide ligands from combinatorial libraries of reversible cyclic depsipeptides. A method for the solid-phase synthesis of individual cyclic depsipeptides and combinatorial libraries of these compounds is proposed, which employs lactic acid (Lact) and the dipeptide ester (Nα-Ac)-Ser(Ala)- as linkers for dilactonization. Upon alkaline treatment of the beads selected by screening a model library, the cyclic depsipeptides are linearized and released from the solid support to the liquid phase, to be sequenced via single-step tandem mass spectrometry (MS/MS). The protocol presented for library synthesis provides for wide structural diversity. Two model sequences, VVWVVK and AAWAAR, were chosen to present different structural examples for depsipeptide libraries and demonstrate the process of sequence determination by mass spectrometry. Further, a case study using the IgG binding cyclic depsipeptide cyclo[(Nα-Ac)-S(A)-RWHYFK-Lact-E] is presented to demonstrate the process of library screening and sequence determination on the selected beads. Finally, a method is shown for synthesis of the irreversible cyclic peptide corresponding to the proposed depsipeptide structure, to make the ligand stable to the aqueous acid and alkaline conditions encountered in affinity chromatographic applications. The cyclic peptide ligand was synthesized on a poly(methacrylate) resin and used for chromatographic binding of the target IgG.}, number={19}, journal={Analytical Chemistry}, author={Menegatti, S. and Ward, K. L. and Naik, A. D. and Kish, W. S. and Blackburn, R. K. and Carbonell, R. G.}, year={2013}, pages={9229–9237} }
 @article{chien_blackburn_liu_goshe_2012, title={Proteomic and Phosphoproteomic Analysis of Chicken Embryo Fibroblasts Infected with Cell Culture-Attenuated and Vaccine Strains of Marek's Disease Virus}, volume={11}, ISSN={["1535-3907"]}, DOI={10.1021/pr300471y}, abstractNote={Vaccination is an effective strategy to reduce the loss of chickens in the poultry industry caused by Marek's Disease (MD), an avian lymphoproliferative disease. The vaccines currently used are from attenuated serotype 1 Marek's disease virus (MDV) or naturally nononcogenic MDV strains. To prepare for future immunity breaks, functional genomic and proteomic studies have been used to better understand the underlying mechanisms of MDV pathogenicity and the effects induced by the vaccine viruses. In this study, a combined approach of quantitative GeLC-MSE and qualitative ERLIC/IMAC/LC-MS/MS analysis were used to identify abundance changes of proteins and the variations of phosphorylation status resulting from the perturbations due to infection with an attenuated oncogenic virus strain (Md11/75C) and several nononcogenic virus strains (CVI988, FC126 and 301B) in vitro. Using this combined approach, several signal transduction pathways mapped by the identified proteins were found to be altered at both the level of protein abundance and phosphorylation. On the basis of this study, a kinase-dependent pathway to regulate phosphorylation of 4E-BP1 to modulate assembly of the protein translation initiation complex was revealed. The differences of 4E-BP1 phosphorylation patterns as well as the measured abundance changes among several other proteins that regulate host transcriptional and translational activities across the virus strains used in this study provide new insight for future functional and biochemical characterization of specific proteins involved in MDV pathogenesis.}, number={12}, journal={JOURNAL OF PROTEOME RESEARCH}, author={Chien, Ko-yi and Blackburn, Kevin and Liu, Hsiao-Ching and Goshe, Michael B.}, year={2012}, month={Dec}, pages={5663–5677} }
 @article{blackburn_cheng_williamson_goshe_2010, title={Data-independent liquid chromatography/mass spectrometry (LC/MSE) detection and quantification of the secreted Apium graveolens pathogen defense protein mannitol dehydrogenase}, volume={24}, DOI={10.1002/rcm.4476}, abstractNote={Abstract}, number={7}, journal={Rapid Communications in Mass Spectrometry}, author={Blackburn, K. and Cheng, F. Y. and Williamson, J. D. and Goshe, M. B.}, year={2010}, pages={1009–1016} }
 @article{blackburn_mbeunkui_mitra_mentzel_goshe_2010, title={Improving Protein and Proteome Coverage through Data-Independent Multiplexed Peptide Fragmentation}, volume={9}, ISSN={["1535-3907"]}, DOI={10.1021/pr100144z}, abstractNote={Performance differences in protein and proteome characterization achieved by data-independent acquisition (DIA) LC/MS(E) and data-dependent acquisition (DDA) LC/MS/MS approaches were investigated. LC/MS(E) is a novel mode of generating product ion data for all coeluting precursors in parallel as opposed to LC/MS/MS where coeluting precursors must be serially fragmented one at a time. During LC/MS(E) analysis, alternating MS scans of "normal" and "elevated" collision energy are collected at regular intervals, providing nearly a 100% duty cycle for precursor detection and fragmentation because all precursors are fragmented across their full chromatographic elution profile. This is in contrast to DDA-based MS/MS where serial selection of precursor ions is biased toward interrogation and detection of the highest abundance sample components by virtue of the intensity-driven interrogation scheme employed. Both modes of acquisition were applied to a simple four-protein standard mixture with a 16-fold dynamic range in concentration, an in-gel digest of the Arabidopsis thaliana protein FLS2 purified by immunoprecipitation, and a solution-digested tomato leaf proteome sample. Dramatic improvement for individual protein sequence coverage was obtained for all three samples analyzed by the DIA approach, particularly for the lowest abundance sample components. In many instances, precursors readily detected and identified during DIA were either interrogated by MS/MS during DDA at inopportune points in their chromatographic elution profiles resulting in poor quality product ion spectra or not interrogated at all. Detailed evaluation of both DDA and DIA raw data and timing of the MS-to-MS/MS switching events clearly revealed the fundamental limitations of serial MS/MS interrogation and the advantages of parallel fragmentation by DIA for more comprehensive protein identification and characterization which holds promise for enhanced isoform and post-translational modification analysis.}, number={7}, journal={JOURNAL OF PROTEOME RESEARCH}, author={Blackburn, Kevin and Mbeunkui, Flaubert and Mitra, Srijeet K. and Mentzel, Tobias and Goshe, Michael B.}, year={2010}, month={Jul}, pages={3621–3637} }
 @article{blackburn_goshe_2009, title={Mass Spectrometry Bioinformatics: Tools for Navigating the Proteomics Landscape}, volume={5}, ISSN={["1875-6727"]}, DOI={10.2174/157341109787846135}, abstractNote={Central to the successful implementation of a proteomics pipeline are appropriate bioinformatics tools to provide a level of automation and standardization to the qualitative identification of proteins, quantitation of protein changes, data capture and storage, and integration with other data platforms. Many of these efforts are in various stages of maturity; however, the identification, or recognition, of peptides/proteins from mass spectrometry data, arguably the most developed area, continues to remain challenging due to the ever increasing size of proteomic datasets. Confident peptide and protein identification, including assignment of any post-translational modifications, is a necessary prerequisite for any proteomic study aimed at elucidating biological and physiological responses. This review includes discussions of bioinformatic approaches for the qualitative identification of peptides/proteins from mass spectrometry data as well as the software tools and the analytical considerations required for analysis. Issues related to placing a proteomics dataset in a larger biological context which includes splice variants, variations in databases, and neglected proteomes are also discussed. Keywords: Mass spectrometry, Peptide identification, Product ion spectrum, Database searching, Proteomics, Bioinformatics}, number={2}, journal={CURRENT ANALYTICAL CHEMISTRY}, author={Blackburn, Kevin and Goshe, Michael B.}, year={2009}, month={Apr}, pages={131–143} }