@article{lin_sun_song_chen_shi_yang_liu_tunlaya-anukit_liu_loziuk_et al._2021, title={Enzyme Complexes of Ptr4CL and PtrHCT Modulate Co-enzyme A Ligation of Hydroxycinnamic Acids for Monolignol Biosynthesis in Populus trichocarpa}, volume={12}, ISSN={["1664-462X"]}, url={http://europepmc.org/abstract/med/34691108}, DOI={10.3389/fpls.2021.727932}, abstractNote={Co-enzyme A (CoA) ligation of hydroxycinnamic acids by 4-coumaric acid:CoA ligase (4CL) is a critical step in the biosynthesis of monolignols. Perturbation of 4CL activity significantly impacts the lignin content of diverse plant species. InPopulus trichocarpa, two well-studied xylem-specific Ptr4CLs (Ptr4CL3 and Ptr4CL5) catalyze the CoA ligation of 4-coumaric acid to 4-coumaroyl-CoA and caffeic acid to caffeoyl-CoA. Subsequently, two 4-hydroxycinnamoyl-CoA:shikimic acid hydroxycinnamoyl transferases (PtrHCT1 and PtrHCT6) mediate the conversion of 4-coumaroyl-CoA to caffeoyl-CoA. Here, we show that the CoA ligation of 4-coumaric and caffeic acids is modulated by Ptr4CL/PtrHCT protein complexes. Downregulation ofPtrHCTsreduced Ptr4CL activities in the stem-differentiating xylem (SDX) of transgenicP. trichocarpa. The Ptr4CL/PtrHCT interactions were then validatedin vivousing biomolecular fluorescence complementation (BiFC) and protein pull-down assays inP. trichocarpaSDX extracts. Enzyme activity assays using recombinant proteins of Ptr4CL and PtrHCT showed elevated CoA ligation activity for Ptr4CL when supplemented with PtrHCT. Numerical analyses based on an evolutionary computation of the CoA ligation activity estimated the stoichiometry of the protein complex to consist of one Ptr4CL and two PtrHCTs, which was experimentally confirmed by chemical cross-linking using SDX plant protein extracts and recombinant proteins. Based on these results, we propose that Ptr4CL/PtrHCT complexes modulate the metabolic flux of CoA ligation for monolignol biosynthesis during wood formation inP. trichocarpa.}, journal={FRONTIERS IN PLANT SCIENCE}, author={Lin, Chien-Yuan and Sun, Yi and Song, Jina and Chen, Hsi-Chuan and Shi, Rui and Yang, Chenmin and Liu, Jie and Tunlaya-Anukit, Sermsawat and Liu, Baoguang and Loziuk, Philip L. and et al.}, year={2021}, month={Oct} }
 @article{salvato_loziuk_kiyota_daneluzzi_araujo_muddiman_mazzafera_2019, title={Label-Free Quantitative Proteomics of Enriched Nuclei from Sugarcane (Saccharum ssp) Stems in Response to Drought Stress}, volume={19}, ISSN={["1615-9861"]}, DOI={10.1002/pmic.201900004}, abstractNote={Abstract}, number={14}, journal={PROTEOMICS}, author={Salvato, Fernando and Loziuk, Philip and Kiyota, Eduardo and Daneluzzi, Gabriel Silva and Araujo, Pedro and Muddiman, David C. and Mazzafera, Paulo}, year={2019}, month={Jul} }
 @article{reis borges_salvato_loziuk_muddiman_azevedo_2019, title={Quantitative proteomic analysis of tomato genotypes with differential cadmium tolerance}, volume={26}, ISSN={["1614-7499"]}, DOI={10.1007/s11356-019-05766-y}, abstractNote={This is a report on comprehensive characterization of cadmium (Cd)-exposed root proteomes in tomato using label-free quantitative proteomic approach. Two genotypes differing in Cd tolerance, Pusa Ruby (Cd-tolerant) and Calabash Rouge (Cd-sensitive), were exposed during 4 days to assess the Cd-induced effects on root proteome. The overall changes in both genotypes in terms of differentially accumulated proteins (DAPs) were mainly associated to cell wall, redox, and stress responses. The proteome of the sensitive genotype was more responsive to Cd excess, once it presented higher number of DAPs. Contrasting protein accumulation in cellular component was observed: Cd-sensitive enhanced intracellular components, while the Cd-tolerant increased proteins of extracellular and envelope regions. Protective and regulatory mechanisms were different between genotypes, once the tolerant showed alterations of various protein groups that lead to a more efficient system to cope with Cd challenge. These findings could shed some light on the molecular basis underlying the Cd stress response in tomato, providing fundamental insights for the development of Cd-safe cultivars. Graphical abstract.}, number={25}, journal={ENVIRONMENTAL SCIENCE AND POLLUTION RESEARCH}, author={Reis Borges, Karina Lima and Salvato, Fernanda and Loziuk, Philip L. and Muddiman, David C. and Azevedo, Ricardo Antunes}, year={2019}, month={Sep}, pages={26039–26051} }
 @article{loziuk_hecht_muddiman_2017, title={N-linked glycosite profiling and use of Skyline as a platform for characterization and relative quantification of glycans in differentiating xylem of Populus trichocarpa}, volume={409}, ISSN={["1618-2650"]}, DOI={10.1007/s00216-016-9776-5}, abstractNote={Our greater understanding of the importance of N-linked glycosylation in biological systems has spawned the field of glycomics and development of analytical tools to address the many challenges regarding our ability to characterize and quantify this complex and important modification as it relates to biological function. One of the unmet needs of the field remains a systematic method for characterization of glycans in new biological systems. This study presents a novel workflow for identification of glycans using Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT™) strategy developed in our lab. This consists of monoisotopic mass extraction followed by peak pair identification of tagged glycans from a theoretical library using an in-house program. Identification and relative quantification could then be performed using the freely available bioinformatics tool Skyline. These studies were performed in the biological context of studying the N-linked glycome of differentiating xylem of the poplar tree, a widely studied model woody plant, particularly with respect to understanding lignin biosynthesis during wood formation. Through our workflow, we were able to identify 502 glycosylated proteins including 12 monolignol enzymes and 1 peroxidase (PO) through deamidation glycosite analysis. Finally, our novel semi-automated workflow allowed for rapid identification of 27 glycans by intact mass and by NAT/SIL peak pairing from a library containing 1573 potential glycans, eliminating the need for extensive manual analysis. Implementing Skyline for relative glycan quantification allowed for improved accuracy and precision of quantitative measurements over current processing tools which we attribute to superior algorithms correction for baseline variation and MS1 peak filtering.}, number={2}, journal={ANALYTICAL AND BIOANALYTICAL CHEMISTRY}, author={Loziuk, Philip L. and Hecht, Elizabeth S. and Muddiman, David C.}, year={2017}, month={Jan}, pages={487–497} }
 @article{hecht_loziuk_muddiman_2017, title={Xylose Migration During Tandem Mass Spectrometry of N-Linked Glycans}, volume={28}, ISSN={["1879-1123"]}, DOI={10.1007/s13361-016-1588-5}, abstractNote={Understanding the rearrangement of gas-phase ions via tandem mass spectrometry is critical to improving manual and automated interpretation of complex datasets. N-glycan analysis may be carried out under collision induced (CID) or higher energy collision dissociation (HCD), which favors cleavage at the glycosidic bond. However, fucose migration has been observed in tandem MS, leading to the formation of new bonds over four saccharide units away. In the following work, we report the second instance of saccharide migration ever to occur for N-glycans. Using horseradish peroxidase as a standard, the beta-1,2 xylose was observed to migrate from a hexose to a glucosamine residue on the (Xyl)Man3GlcNac2 glycan. This investigation was followed up in a complex N-linked glycan mixture derived from stem differentiating xylem tissue, and the rearranged product ion was observed for 75% of the glycans. Rearrangement was not favored in isomeric glycans with a core or antennae fucose and unobserved in glycans predicted to have a permanent core-fucose modification. As the first empirical observation of this rearrangement, this work warrants dissemination so it may be searched in de novo sequencing glycan workflows. Graphical Abstract ᅟ.}, number={4}, journal={JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY}, author={Hecht, Elizabeth S. and Loziuk, Philip L. and Muddiman, David C.}, year={2017}, month={Apr}, pages={729–732} }
 @article{parker_oh_moazami_pierce_loziuk_dean_muddiman_2016, title={Examining ubiquitinated peptide enrichment efficiency through an epitope labeled protein}, volume={512}, ISSN={["1096-0309"]}, DOI={10.1016/j.ab.2016.08.017}, abstractNote={Ubiquitination is a dynamic process that is responsible for regulation of cellular responses to stimuli in a number of biological systems. Previous efforts to study this post-translational modification have focused on protein enrichment; however, recent research utilizes the presence of the di-glycine (Gly-Gly) remnants following trypsin digestion to immuno-enrich ubiquitinated peptides. Monoclonal antibodies developed to the cleaved ubiquitin modification epitope, (tert-butoxycarbonyl) glycylglycine (Boc-Gly-Gly-NHS)1, are used to identify the Gly-Gly signature. Here, we have successfully generated the Boc-Gly-Gly-NHS modification and showed that when conjugated to a lysine containing protein, such as lysozyme, it can be applied as a standard protein to examine ubiquitinated peptide enrichment within a complex background.}, journal={ANALYTICAL BIOCHEMISTRY}, author={Parker, J. and Oh, Y. and Moazami, Y. and Pierce, J. G. and Loziuk, P. L. and Dean, R. A. and Muddiman, D. C.}, year={2016}, month={Nov}, pages={114–119} }
 @article{loziuk_meier_johnson_ghashghaei_muddiman_2016, title={TransOmic analysis of forebrain sections in Sp2 conditional knockout embryonic mice using IR-MALDESI imaging of lipids and LC-MS/MS label-free proteomics}, volume={408}, ISSN={1618-2642 1618-2650}, url={http://dx.doi.org/10.1007/s00216-016-9421-3}, DOI={10.1007/s00216-016-9421-3}, abstractNote={Quantitative methods for detection of biological molecules are needed more than ever before in the emerging age of "omics" and "big data." Here, we provide an integrated approach for systematic analysis of the "lipidome" in tissue. To test our approach in a biological context, we utilized brain tissue selectively deficient for the transcription factor Specificity Protein 2 (Sp2). Conditional deletion of Sp2 in the mouse cerebral cortex results in developmental deficiencies including disruption of lipid metabolism. Silver (Ag) cationization was implemented for infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) to enhance the ion abundances for olefinic lipids, as these have been linked to regulation by Sp2. Combining Ag-doped and conventional IR-MALDESI imaging, this approach was extended to IR-MALDESI imaging of embryonic mouse brains. Further, our imaging technique was combined with bottom-up shotgun proteomic LC-MS/MS analysis and western blot for comparing Sp2 conditional knockout (Sp2-cKO) and wild-type (WT) cortices of tissue sections. This provided an integrated omics dataset which revealed many specific changes to fundamental cellular processes and biosynthetic pathways. In particular, step-specific altered abundances of nucleotides, lipids, and associated proteins were observed in the cerebral cortices of Sp2-cKO embryos.}, number={13}, journal={Analytical and Bioanalytical Chemistry}, publisher={Springer Science and Business Media LLC}, author={Loziuk, Philip and Meier, Florian and Johnson, Caroline and Ghashghaei, H. Troy and Muddiman, David C.}, year={2016}, month={Mar}, pages={3453–3474} }
 @article{lin_wang_li_chen_liu_loziuk_song_williams_muddiman_sederoff_et al._2015, title={4-Coumaroyl and Caffeoyl Shikimic Acids Inhibit 4-Coumaric Acid: Coenzyme A Ligases and Modulate Metabolic Flux for 3-Hydroxylation in Monolignol Biosynthesis of Populus trichocarpa}, volume={8}, ISSN={["1752-9867"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84925201417&partnerID=MN8TOARS}, DOI={10.1016/j.molp.2014.12.003}, abstractNote={Downregulation of 4-coumaric acid:coenzyme A ligase (4CL) can reduce lignin content in a number of plant species. In lignin precursor (monolignol) biosynthesis during stem wood formation in Populus trichocarpa, two enzymes, Ptr4CL3 and Ptr4CL5, catalyze the coenzyme A (CoA) ligation of 4-coumaric acid to 4-coumaroyl-CoA and caffeic acid to caffeoyl-CoA. CoA ligation of 4-coumaric acid is essential for the 3-hydroxylation of 4-coumaroyl shikimic acid. This hydroxylation results from sequential reactions of 4-hydroxycinnamoyl-CoA:shikimic acid hydroxycinnamoyl transferases (PtrHCT1 and PtrHCT6) and 4-coumaric acid 3-hydroxylase 3 (PtrC3H3). Alternatively, 3-hydroxylation of 4-coumaric acid to caffeic acid may occur through an enzyme complex of cinnamic acid 4-hydroxylase 1 and 2 (PtrC4H1 and PtrC4H2) and PtrC3H3. We found that 4-coumaroyl and caffeoyl shikimic acids are inhibitors of Ptr4CL3 and Ptr4CL5. 4-Coumaroyl shikimic acid strongly inhibits the formation of 4-coumaroyl-CoA and caffeoyl-CoA. Caffeoyl shikimic acid inhibits only the formation of 4-coumaroyl-CoA. 4-Coumaroyl and caffeoyl shikimic acids both act as competitive and uncompetitive inhibitors. Metabolic flux in wild-type and PtrC3H3 downregulated P. trichocarpa transgenics has been estimated by absolute protein and metabolite quantification based on liquid chromatography–tandem mass spectrometry, mass action kinetics, and inhibition equations. Inhibition by 4-coumaroyl and caffeoyl shikimic acids may play significant regulatory roles when these inhibitors accumulate.}, number={1}, journal={MOLECULAR PLANT}, author={Lin, Chien-Yuan and Wang, Jack P. and Li, Quanzi and Chen, Hsi-Chuan and Liu, Jie and Loziuk, Philip and Song, Jina and Williams, Cranos and Muddiman, David C. and Sederoff, Ronald R. and et al.}, year={2015}, month={Jan}, pages={176–187} }
 @article{loziuk_parker_li_lin_wang_li_sederoff_chiang_muddiman_2015, title={Elucidation of Xylem-Specific Transcription Factors and Absolute Quantification of Enzymes Regulating Cellulose Biosynthesis in Populus trichocarpa}, volume={14}, ISSN={["1535-3907"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84942916917&partnerID=MN8TOARS}, DOI={10.1021/acs.jproteome.5b00233}, abstractNote={Cellulose, the main chemical polymer of wood, is the most abundant polysaccharide in nature.1 The ability to perturb the abundance and structure of cellulose microfibrils is of critical importance to the pulp and paper industry as well as for the textile, wood products, and liquid biofuels industries. Although much has been learned at the transcript level about the biosynthesis of cellulose, a quantitative understanding at the proteome level has yet to be established. The study described herein sought to identify the proteins directly involved in cellulose biosynthesis during wood formation in Populus trichocarpa along with known xylem-specific transcription factors involved in regulating these key proteins. Development of an effective discovery proteomic strategy through a combination of subcellular fractionation of stem differentiating xylem tissue (SDX) with recently optimized FASP digestion protocols, StageTip fractionation, as well as optimized instrument parameters for global proteomic analysis using the quadrupole-orbitrap mass spectrometer resulted in the deepest proteomic coverage of SDX protein from P. trichocarpa with 9,146 protein groups being identified (1% FDR). Of these, 20 cellulosic/hemicellulosic enzymes and 43 xylem-specific transcription factor groups were identified. Finally, selection of surrogate peptides led to an assay for absolute quantification of 14 cellulosic proteins in SDX of P. trichocarpa.}, number={10}, journal={JOURNAL OF PROTEOME RESEARCH}, author={Loziuk, Philip L. and Parker, Jennifer and Li, Wei and Lin, Chien-Yuan and Wang, Jack P. and Li, Quanzi and Sederoff, Ronald R. and Chiang, Vincent L. and Muddiman, David C.}, year={2015}, month={Oct}, pages={4158–4168} }
 @article{schilling_loziuk_muddiman_daniels_reading_2015, title={Mechanisms of Egg Yolk Formation and Implications on Early Life History of White Perch (Morone americana)}, volume={10}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0143225}, abstractNote={The three white perch (Morone americana) vitellogenins (VtgAa, VtgAb, VtgC) were quantified accurately and precisely in the liver, plasma, and ovary during pre-, early-, mid-, and post-vitellogenic oocyte growth using protein cleavage-isotope dilution mass spectrometry (PC-IDMS). Western blotting generally mirrored the PC-IDMS results. By PC-IDMS, VtgC was quantifiable in pre-vitellogenic ovary tissues and VtgAb was quantifiable in pre-vitellogenic liver tissues however, neither protein was detected by western blotting in these respective tissues at this time point. Immunohistochemistry indicated that VtgC was present within pre-vitellogenic oocytes and localized to lipid droplets within vitellogenic oocytes. Affinity purification coupled to tandem mass spectrometry using highly purified VtgC as a bait protein revealed a single specific interacting protein (Y-box binding protein 2a-like [Ybx2a-like]) that eluted with suramin buffer and confirmed that VtgC does not bind the ovary vitellogenin receptors (LR8 and Lrp13). Western blotting for LR8 and Lrp13 showed that both receptors were expressed during vitellogenesis with LR8 and Lrp13 expression highest in early- and mid-vitellogenesis, respectively. The VtgAa within the ovary peaked during post-vitellogenesis, while VtgAb peaked during early-vitellogenesis in both white perch and the closely related striped bass (M. saxatilis). The VtgC was steadily accumulated by oocytes beginning during pre-vitellogenesis and continued until post-vitellogenesis and its composition varies widely between striped bass and white perch. In striped bass, the VtgC accounted for 26% of the vitellogenin-derived egg yolk, however in the white perch it comprised only 4%. Striped bass larvae have an extended developmental window and these larvae have yolk stores that may enable them to survive in the absence of food for twice as long as white perch after hatch. Thus, the VtgC may play an integral role in providing nutrients to late stage fish larvae prior to the onset of exogenous feeding and its composition in the egg yolk may relate to different early life histories among this diverse group of animals.}, number={11}, journal={PLOS ONE}, author={Schilling, Justin and Loziuk, Philip L. and Muddiman, David C. and Daniels, Harry V. and Reading, Benjamin J.}, year={2015}, month={Nov} }
 @article{loziuk_sederoff_chiang_muddiman_2014, title={Establishing ion ratio thresholds based on absolute peak area for absolute protein quantification using protein cleavage isotope dilution mass spectrometry}, volume={139}, ISSN={["1364-5528"]}, DOI={10.1039/c4an00567h}, abstractNote={Relative abundance values and their associated variability are dynamic and dependent on absolute abundance.}, number={21}, journal={ANALYST}, author={Loziuk, Philip L. and Sederoff, Ronald R. and Chiang, Vincent L. and Muddiman, David C.}, year={2014}, pages={5439–5450} }
 @article{lugo-morales_loziuk_corder_toups_roberts_mccaffrey_sombers_2013, title={Enzyme-Modified Carbon-Fiber Microelectrode for the Quantification of Dynamic Fluctuations of Nonelectroactive Analytes Using Fast-Scan Cyclic Voltammetry}, volume={85}, ISSN={["1520-6882"]}, DOI={10.1021/ac4017852}, abstractNote={Neurotransmission occurs on a millisecond time scale, but conventional methods for monitoring nonelectroactive neurochemicals are limited by slow sampling rates. Despite a significant global market, a sensor capable of measuring the dynamics of rapidly fluctuating, nonelectroactive molecules at a single recording site with high sensitivity, electrochemical selectivity, and a subsecond response time is still lacking. To address this need, we have enabled the real-time detection of dynamic glucose fluctuations in live brain tissue using background-subtracted, fast-scan cyclic voltammetry. The novel microbiosensor consists of a simple carbon fiber surface modified with an electrodeposited chitosan hydrogel encapsulating glucose oxidase. The selectivity afforded by voltammetry enables quantitative and qualitative measurements of enzymatically generated H2O2 without the need for additional strategies to eliminate interfering agents. The microbiosensors possess a sensitivity and limit of detection for glucose of 19.4 ± 0.2 nA mM(-1) and 13.1 ± 0.7 μM, respectively. They are stable, even under deviations from physiological normoxic conditions, and show minimal interference from endogenous electroactive substances. Using this approach, we have quantitatively and selectively monitored pharmacologically evoked glucose fluctuations with unprecedented chemical and spatial resolution. Furthermore, this novel biosensing strategy is widely applicable to the immobilization of any H2O2 producing enzyme, enabling rapid monitoring of many nonelectroactive enzyme substrates.}, number={18}, journal={ANALYTICAL CHEMISTRY}, author={Lugo-Morales, Leyda Z. and Loziuk, Philip L. and Corder, Amanda K. and Toups, J. Vincent and Roberts, James G. and McCaffrey, Katherine A. and Sombers, Leslie A.}, year={2013}, month={Sep}, pages={8780–8786} }
 @article{loziuk_wang_li_sederoff_chiang_muddiman_2013, title={Understanding the Role of Proteolytic Digestion on Discovery and Targeted Proteomic Measurements Using Liquid Chromatography Tandem Mass Spectrometry and Design of Experiments}, volume={12}, ISSN={["1535-3907"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84890109136&partnerID=MN8TOARS}, DOI={10.1021/pr4008442}, abstractNote={Workflows in bottom-up proteomics have traditionally implemented the use of proteolysis during sample preparation; enzymatic digestion is most commonly performed using trypsin. This results in the hydrolysis of peptide bonds forming tryptic peptides, which can then be subjected to LC-MS/MS analysis. While the structure, specificity, and kinetics of trypsin are well characterized, a lack of consensus and understanding has remained regarding fundamental parameters critical to obtaining optimal data from a proteomics experiment. These include the type of trypsin used, pH during digestion, incubation temperature as well as enzyme-to-substrate ratio. Through the use of design of experiments (DOE), we optimized these parameters, resulting in deeper proteome coverage and a greater dynamic range of measurement. The knowledge gained from optimization of a discovery-based proteomics experiment was applied to targeted LC-MS/MS experiments using protein cleavage-isotope dilution mass spectrometry for absolute quantification. We demonstrated the importance of these digest parameters with respect to our limit of detection as well as our ability to acquire more accurate quantitative measurements. Additionally, we were able to quantitatively account for peptide decay observed in previous studies, caused by nonspecific activity of trypsin. The tryptic digest optimization described here has eliminated this previously observed peptide decay as well as provided a greater understanding and standardization for a common but critical sample treatment used across the field of proteomics.}, number={12}, journal={JOURNAL OF PROTEOME RESEARCH}, author={Loziuk, Philip L. and Wang, Jack and Li, Quanzi and Sederoff, Ronald R. and Chiang, Vincent L. and Muddiman, David C.}, year={2013}, month={Dec}, pages={5820–5829} }