@article{hecht_scholl_walker_taylor_cliby_motsinger-reif_muddiman_2015, title={Relative Quantification and Higher-Order Modeling of the Plasma Glycan Cancer Burden Ratio in Ovarian Cancer Case-Control Samples}, volume={14}, ISSN={["1535-3907"]}, DOI={10.1021/acs.jproteome.5b00703}, abstractNote={An early-stage, population-wide biomarker for ovarian cancer (OVC) is essential to reverse its high mortality rate. Aberrant glycosylation by OVC has been reported, but studies have yet to identify an N-glycan with sufficiently high specificity. We curated a human biorepository of 82 case-control plasma samples, with 27%, 12%, 46%, and 15% falling across stages I-IV, respectively. For relative quantitation, glycans were analyzed by the individuality normalization when labeling with glycan hydrazide tags (INLIGHT) strategy for enhanced electrospray ionization, MS/MS analysis. Sixty-three glycan cancer burden ratios (GBRs), defined as the log10 ratio of the case-control extracted ion chromatogram abundances, were calculated above the limit of detection. The final GBR models, built using stepwise forward regression, included three significant terms: OVC stage, normalized mean GBR, and tag chemical purity; glycan class, fucosylation, or sialylation were not significant variables. After Bonferroni correction, seven N-glycans were identified as significant (p < 0.05), and after false discovery rate correction, an additional four glycans were determined to be significant (p < 0.05), with one borderline (p = 0.05). For all N-glycans, the vectors of the effects from stages II-IV were sequentially reversed, suggesting potential biological changes in OVC morphology or in host response.}, number={10}, journal={JOURNAL OF PROTEOME RESEARCH}, author={Hecht, Elizabeth S. and Scholl, Elizabeth H. and Walker, S. Hunter and Taylor, Amber D. and Cliby, William A. and Motsinger-Reif, Alison A. and Muddiman, David C.}, year={2015}, month={Oct}, pages={4394–4401} }
 @article{walker_taylor_muddiman_2013, title={Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT): A Novel Glycan-Relative Quantification Strategy}, volume={24}, ISSN={["1879-1123"]}, DOI={10.1007/s13361-013-0681-2}, abstractNote={The Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT) strategy for the sample preparation, data analysis, and relative quantification of N-linked glycans is presented. Glycans are derivatized with either natural (L) or stable-isotope labeled (H) hydrazide reagents and analyzed using reversed phase liquid chromatography coupled online to a Q Exactive mass spectrometer. A simple glycan ladder, maltodextrin, is first used to demonstrate the relative quantification strategy in samples with negligible analytical and biological variability. It is shown that after a molecular weight correction attributable to isotopic overlap and a post-acquisition normalization of the data to account for any systematic bias, a plot of the experimental H:L ratio versus the calculated H:L ratio exhibits a correlation of unity for maltodextrin samples mixed in different ratios. We also demonstrate that the INLIGHT approach can quantify species over four orders of magnitude in ion abundance. The INLIGHT strategy is further demonstrated in pooled human plasma, where it is shown that the post-acquisition normalization is more effective than using a single spiked-in internal standard. Finally, changes in glycosylation are able to be detected in complex biological matrices, when spiked with a glycoprotein. The ability to spike in a glycoprotein and detect change at the glycan level validates both the sample preparation and data analysis strategy, making INLIGHT an invaluable relative quantification strategy for the field of glycomics.}, number={9}, journal={JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY}, author={Walker, S. Hunter and Taylor, Amber D. and Muddiman, David C.}, year={2013}, month={Sep}, pages={1376–1384} }
 @article{walker_taylor_muddiman_2013, title={The use of a xylosylated plant glycoprotein as an internal standard accounting for N-linked glycan cleavage and sample preparation variability}, volume={27}, DOI={10.1002/rcm.6579}, abstractNote={RATIONALETraditionally, free oligosaccharide internal standards are used to account for variability in glycan relative quantification experiments by mass spectrometry. However, a more suitable internal standard would be a glycoprotein, which could also control for enzymatic cleavage efficiency, allowing for more accurate quantitative experiments.METHODSHydrophobic, hydrazide N‐linked glycan reagents (both native and stable‐isotope labeled) are used to derivatize and differentially label N‐linked glycan samples for relative quantification, and the samples are analyzed by a reversed‐phase liquid chromatography chip system coupled online to a Q‐Exactive mass spectrometer. The inclusion of two internal standards, maltoheptaose (previously used) and horseradish peroxidase (HRP) (novel), is studied to demonstrate the effectiveness of using a glycoprotein as an internal standard in glycan relative quantification experiments.RESULTSHRP is a glycoprotein containing a xylosylated N‐linked glycan, which is unique from mammalian N‐linked glycans. Thus, the internal standard xylosylated glycan could be detected without interference to the sample. Additionally, it was shown that differences in cleavage efficiency can be detected by monitoring the HRP glycan. In a sample where cleavage efficiency variation is minimal, the HRP glycan performs as well as maltoheptaose.CONCLUSIONSBecause the HRP glycan performs as well as maltoheptaose but is also capable of correcting and accounting for cleavage variability, it is a more versatile internal standard and will be used in all subsequent biological studies. Because of the possible lot‐to‐lot variation of an enzyme, differences in biological matrix, and variable enzyme activity over time, it is a necessity to account for glycan cleavage variability in glycan relative quantification experiments. Copyright © 2013 John Wiley & Sons, Ltd.}, number={12}, journal={Rapid Communications in Mass Spectrometry}, author={Walker, S. H. and Taylor, A. D. and Muddiman, David}, year={2013}, pages={1354–1358} }
 @article{harton_pingali_nunnery_baker_walker_muddiman_koga_rials_urban_langan_2012, title={Evidence for Complex Molecular Architectures for Solvent-Extracted Lignins}, volume={1}, ISSN={["2161-1653"]}, DOI={10.1021/mz300045e}, abstractNote={Lignin, an abundant, naturally occurring biopolymer, is often considered "waste" and used as a simple fuel source in the paper-making process. However, lignin has emerged as a promising renewable resource for engineering materials, such as carbon fibers. Unfortunately, the molecular architecture of lignin (in vivo and extracted) is still elusive, with numerous conflicting reports in the literature, and knowledge of this structure is extremely important, not only for materials technologies, but also for production of biofuels such as cellulosic ethanol due to biomass recalcitrance. As such, the molecular structures of solvent-extracted (sulfur-free) lignins, which have been modified using various acyl chlorides, have been probed using small-angle X-ray (SAXS) and neutron (SANS) scattering in tetrahydrofuran (THF) solution along with hydrodynamic characterization using dilute solution viscometry and gel permeation chromatography (GPC) in THF. Mass spectrometry shows an absolute molecular weight ≈18-30 kDa (≈80-140 monomers), while GPC shows a relative molecular weight ∼3 kDa. A linear styrene oligomer (2.5 kDa) was also analyzed in THF using SANS. Results clearly show that lignin molecular architectures are somewhat rigid and complex, ranging from nanogels to hyperbranched macromolecules, not linear oligomers or physical assemblies of oligomers, which is consistent with previously proposed delignification (extraction) mechanisms. Future characterization using the methods discussed here can be used to guide extraction processes as well as genetic engineering technologies to convert lignin into value added materials with the potential for high positive impact on global sustainability.}, number={5}, journal={ACS MACRO LETTERS}, author={Harton, Shane E. and Pingali, Sai Venkatesh and Nunnery, Grady A. and Baker, Darren A. and Walker, S. Hunter and Muddiman, David C. and Koga, Tadanori and Rials, Timothy G. and Urban, Volker S. and Langan, Paul}, year={2012}, month={May}, pages={568–573} }
 @article{walker_carlisle_muddiman_2012, title={Systematic Comparison of Reverse Phase and Hydrophilic Interaction Liquid Chromatography Platforms for the Analysis of N-Linked Glycans}, volume={84}, ISSN={["1520-6882"]}, DOI={10.1021/ac3012494}, abstractNote={Due to the hydrophilic nature of glycans, reverse phase chromatography has not been widely used as a glycomic separation technique coupled to mass spectrometry. Other approaches such as hydrophilic interaction chromatography and porous graphitized carbon chromatography are often employed, though these strategies frequently suffer from decreased chromatographic resolution, long equilibration times, indefinite retention, and column bleed. Herein, it is shown that, through an efficient hydrazone formation derivatization of N-linked glycans (~4 h of additional sample preparation time which is carried out in parallel), numerous experimental and practical advantages are gained when analyzing the glycans by online reverse phase chromatography. These benefits include an increased number of glycans detected, increased peak capacity of the separation, and the ability to analyze glycans on the identical liquid chromatography-mass spectrometry platform commonly used for proteomic analyses. The data presented show that separation of derivatized N-linked glycans by reverse phase chromatography significantly out-performs traditional separation of native or derivatized glycans by hydrophilic interaction chromatography. Furthermore, the movement to a more ubiquitous separation technique will afford numerous research groups the opportunity to analyze both proteomic and glycomic samples on the same platform with minimal time and physical change between experiments, increasing the efficiency of "multiomic" biological approaches.}, number={19}, journal={ANALYTICAL CHEMISTRY}, author={Walker, S. Hunter and Carlisle, Brandon C. and Muddiman, David C.}, year={2012}, month={Oct}, pages={8198–8206} }
 @article{walker_lilley_enamorado_comins_muddiman_2011, title={Hydrophobic Derivatization of N-linked Glycans for Increased Ion Abundance in Electrospray Ionization Mass Spectrometry}, volume={22}, ISSN={["1044-0305"]}, DOI={10.1007/s13361-011-0140-x}, abstractNote={A library of neutral, hydrophobic reagents was synthesized for use as derivatizing agents in order to increase the ion abundance of N-linked glycans in electrospray ionization mass spectrometry (ESI MS). The glycans are derivatized via hydrazone formation and are shown to increase the ion abundance of a glycan standard more than 4-fold. Additionally, the data show that the systematic addition of hydrophobic surface area to the reagent increases the glycan ion abundance, a property that can be further exploited in the analysis of glycans. The results of this study will direct the future synthesis of hydrophobic reagents for glycan analysis using the correlation between hydrophobicity and theoretical non-polar surface area calculation to facilitate the development of an optimum tag for glycan derivatization. The compatibility and advantages of this method are demonstrated by cleaving and derivatizing N-linked glycans from human plasma proteins. The ESI-MS signal for the tagged glycans are shown to be significantly more abundant, and the detection of negatively charged sialylated glycans is enhanced.}, number={8}, journal={JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY}, author={Walker, S. Hunter and Lilley, Laura M. and Enamorado, Monica F. and Comins, Daniel L. and Muddiman, David C.}, year={2011}, month={Aug}, pages={1309–1317} }
 @article{blake_walker_muddiman_hinks_beck_2011, title={Spectral Accuracy and Sulfur Counting Capabilities of the LTQ-FT-ICR and the LTQ-Orbitrap XL for Small Molecule Analysis}, volume={22}, ISSN={["1044-0305"]}, DOI={10.1007/s13361-011-0244-3}, abstractNote={Color Index Disperse Yellow 42 (DY42), a high-volume disperse dye for polyester, was used to compare the capabilities of the LTQ-Orbitrap XL and the LTQ-FT-ICR with respect to mass measurement accuracy (MMA), spectral accuracy, and sulfur counting. The results of this research will be used in the construction of a dye database for forensic purposes; the additional spectral information will increase the confidence in the identification of unknown dyes found in fibers at crime scenes. Initial LTQ-Orbitrap XL data showed MMAs greater than 3 ppm and poor spectral accuracy. Modification of several Orbitrap installation parameters (e.g., deflector voltage) resulted in a significant improvement of the data. The LTQ-FT-ICR and LTQ-Orbitrap XL (after installation parameters were modified) exhibited MMA ≤ 3 ppm, good spectral accuracy (χ2 values for the isotopic distribution ≤ 2), and were correctly able to ascertain the number of sulfur atoms in the compound at all resolving powers investigated for AGC targets of 5.00 × 105 and 1.00 × 106.}, number={12}, journal={JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY}, author={Blake, Samantha L. and Walker, S. Hunter and Muddiman, David C. and Hinks, David and Beck, Keith R.}, year={2011}, month={Dec}, pages={2269–2275} }
 @article{walker_budhathoki-uprety_novak_muddiman_2011, title={Stable-Isotope Labeled Hydrophobic Hydrazide Reagents for the Relative Quantification of N-Linked Glycans by Electrospray Ionization Mass Spectrometry}, volume={83}, ISSN={["1520-6882"]}, url={http://dx.doi.org/10.1021/ac201376q}, DOI={10.1021/ac201376q}, abstractNote={This study presents the development of stable-isotope labeled hydrophobic, hydrazide reagents for the relative quantification of N-linked glycans. The P2GPN "light" ((12)C) and "heavy" ((13)C(6)) pair are used to differentially label two N-linked glycan samples. The samples are combined 1:1, separated using HILIC, and then mass differentiated and quantified using mass spectrometry. These reagents have several benefits: (1) impart hydrophobic character to the glycans affording an increase in electrospray ionization efficiency and MS detection; (2) indistinguishable chromatographic, MS, and MS/MS performance of the "light" and "heavy" reagents affording relative quantification; and (3) analytical variability is significantly reduced due to the two samples being mixed together after sample preparation. Obtaining these analytical benefits only requires ~4 h of sample preparation time. It is shown that these reagents are capable of quantifying changes in glycosylation in simple mixtures, and the analytical variability of the reagents in pooled plasma samples is shown to be less than ±30%. Additionally, the incorporation of an internal standard allows one to account for the difference in systematic error between the two samples due to the samples being processed in parallel and not mixed until after derivatization.}, number={17}, journal={ANALYTICAL CHEMISTRY}, author={Walker, S. Hunter and Budhathoki-Uprety, Januka and Novak, Bruce M. and Muddiman, David C.}, year={2011}, month={Sep}, pages={6738–6745} }
 @article{walker_papas_comins_muddiman_2010, title={Interplay of Permanent Charge and Hydrophobicity in the Electrospray Ionization of Glycans}, volume={82}, ISSN={["1520-6882"]}, DOI={10.1021/ac101227a}, abstractNote={The analysis of N-linked glycans by mass spectrometry (MS) has been characterized by low signal-to-noise ratios and high limits of detection due to their hydrophilicity and lack of basic sites able to be protonated. As a result, every step in glycan sample preparation must be thoroughly optimized in order to minimize sample loss, contamination, and analytical variability. Importantly, properties of glycans and their derivatized counterparts must be thoroughly studied in order to exploit certain characteristics for enhancing MS analysis. Herein, the effectiveness of the incorporation of a permanent charge is studied and determined to hamper glycan analysis. Also, a procedure for glycan hydrazone formation is optimized and outlined where a large number of variables were simultaneously analyzed using a fractional factorial design (FFD) in order to determine which conditions affected the reaction efficiency of the hydrazone formation reaction. Finally, the hydrophobic tagging of glycans is shown to be a viable opportunity to further increase the ion abundance of glycans in MS.}, number={15}, journal={ANALYTICAL CHEMISTRY}, author={Walker, S. Hunter and Papas, Brian N. and Comins, Daniel L. and Muddiman, David C.}, year={2010}, month={Aug}, pages={6636–6642} }