Philip L. Loziuk

Understanding the role of proteolytic digestion on discovery and targeted proteomic measurements using liquid chromatography tandem mass spectrometry and design of experiments.

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.

Phosphorylation is an on/off switch for 5-hydroxyconiferaldehyde O-methyltransferase activity in poplar monolignol biosynthesis

Significance To meet environmental and developmental needs, the monolignol biosynthetic pathway for lignification in plant cell walls is regulated by complex mechanisms involving transcriptional, posttranscriptional, and metabolic controls. However, posttranslational modification by protein phosphorylation had not been demonstrated in the regulation of monolignol biosynthesis. Here, we show that reversible monophosphorylation at Ser123 or Ser125 acts as an on/off switch for the activity of 5-hydroxyconiferaldehyde O-methyltransferase 2 (PtrAldOMT2). Phosphorylation induces a loss of function of PtrAldOMT2, which directly affects metabolic flux for syringyl monolignol biosynthesis. The Ser123/Ser125 phosphorylation sites are conserved across 98% of AldOMTs from 46 diverse plant species. Protein phosphorylation provides a rapid and energetically efficient mode of regulating PtrAldOMT2 activity for syringyl monolignol biosynthesis and represents an additional level of control for this important pathway. Although phosphorylation has long been known to be an important regulatory modification of proteins, no unequivocal evidence has been presented to show functional control by phosphorylation for the plant monolignol biosynthetic pathway. Here, we present the discovery of phosphorylation-mediated on/off regulation of enzyme activity for 5-hydroxyconiferaldehyde O-methyltransferase 2 (PtrAldOMT2), an enzyme central to monolignol biosynthesis for lignification in stem-differentiating xylem (SDX) of Populus trichocarpa. Phosphorylation turned off the PtrAldOMT2 activity, as demonstrated in vitro by using purified phosphorylated and unphosphorylated recombinant PtrAldOMT2. Protein extracts of P. trichocarpa SDX, which contains endogenous kinases, also phosphorylated recombinant PtrAldOMT2 and turned off the recombinant protein activity. Similarly, ATP/Mn2+-activated phosphorylation of SDX protein extracts reduced the endogenous SDX PtrAldOMT2 activity by ∼60%, and dephosphorylation fully restored the activity. Global shotgun proteomic analysis of phosphopeptide-enriched P. trichocarpa SDX protein fractions identified PtrAldOMT2 monophosphorylation at Ser123 or Ser125 in vivo. Phosphorylation-site mutagenesis verified the PtrAldOMT2 phosphorylation at Ser123 or Ser125 and confirmed the functional importance of these phosphorylation sites for O-methyltransferase activity. The PtrAldOMT2 Ser123 phosphorylation site is conserved across 93% of AldOMTs from 46 diverse plant species, and 98% of the AldOMTs have either Ser123 or Ser125. PtrAldOMT2 is a homodimeric cytosolic enzyme expressed more abundantly in syringyl lignin-rich fiber cells than in guaiacyl lignin-rich vessel cells. The reversible phosphorylation of PtrAldOMT2 is likely to have an important role in regulating syringyl monolignol biosynthesis of P. trichocarpa.

Enzyme-Modified Carbon-Fiber Microelectrode for the Quantification of Dynamic Fluctuations of Nonelectroactive Analytes Using Fast-Scan Cyclic Voltammetry

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.

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

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.

Real-Time Chemical Measurements of Dopamine Release in the Brain

Rapid changes in extracellular dopamine concentrations in freely moving or anesthetized rats can be detected using fast-scan cyclic voltammetry (FSCV). Background-subtracted FSCV is a real-time electrochemical technique that can monitor neurochemical transmission in the brain on a subsecond timescale, while providing chemical information on the analyte. Also, this voltammetric approach allows for the investigation of the kinetics of release and uptake of molecules in the brain. This chapter describes, completely, how to make these measurements and the properties of FSCV that make it uniquely suitable for performing chemical measurements of dopaminergic neurotransmission in vivo.

Elucidation of Xylem-Specific Transcription Factors and Absolute Quantification of Enzymes Regulating Cellulose Biosynthesis in Populus trichocarpa

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.

N-linked glycosite profiling and use of Skyline as a platform for characterization and relative quantification of glycans in differentiating xylem of Populus trichocarpa

AbstractOur 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. Graphical abstractWorkflow for FANGS-INLIGHT glycosite profiling of plant xylem and monolignol proteins followed by INLIGHT tagging with semi-automated identification of glycans by light-heavy peak pairs. Finally, manual validation and relative quantification was performed in Skyline

Mechanisms of Egg Yolk Formation and Implications on Early Life History of White Perch (Morone americana)

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.

TransOmic analysis of forebrain sections in Sp2 conditional knockout embryonic mice using IR-MALDESI imaging of lipids and LC-MS/MS label-free proteomics

AbstractQuantitative 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. Graphical abstractTransOmic Analysis of Dorsolateral cortices of Sp2 conditional Knockout mouse embryos. Target tissue was extracted by laser microdissection and analyzed by LC-MS/MS label- free quantitative proteomics. In parallel, lipid imaging of these tissues was performed by conventional IR-MALDESI and Agdoped IR-MALDESI imaging. These data were then integrated to obtain insight into lipid pathways altered by presence or absence of transcription factor Specificity Protein 2

Improving wood properties for wood utilization through multi-omics integration in lignin biosynthesis

A multi-omics quantitative integrative analysis of lignin biosynthesis can advance the strategic engineering of wood for timber, pulp, and biofuels. Lignin is polymerized from three monomers (monolignols) produced by a grid-like pathway. The pathway in wood formation of Populus trichocarpa has at least 21 genes, encoding enzymes that mediate 37 reactions on 24 metabolites, leading to lignin and affecting wood properties. We perturb these 21 pathway genes and integrate transcriptomic, proteomic, fluxomic and phenomic data from 221 lines selected from ~2000 transgenics (6-month-old). The integrative analysis estimates how changing expression of pathway gene or gene combination affects protein abundance, metabolic-flux, metabolite concentrations, and 25 wood traits, including lignin, tree-growth, density, strength, and saccharification. The analysis then predicts improvements in any of these 25 traits individually or in combinations, through engineering expression of specific monolignol genes. The analysis may lead to greater understanding of other pathways for improved growth and adaptation.A systematic analysis of lignin biosynthetic genes to quantitatively understand their effect on wood properties is still lacking. Here, the authors integrate transcriptomic, proteomic, fluxomic and phenomic data to quantify the impact of perturbations of transcript abundance on lignin biosynthesis and wood properties.