@article{de leoz_duewer_fung_liu_yau_potter_staples_furuki_frenkel_hu_et al._2019, title={NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods}, volume={19}, ISSN={1535-9476 1535-9484}, url={http://dx.doi.org/10.1074/mcp.RA119.001677}, DOI={10.1074/mcp.RA119.001677}, abstractNote={A broad-based interlaboratory study of glycosylation profiles of a reference and modified IgG antibody involving 103 reports from 76 laboratories. Graphical Abstract Highlights A broad-based interlaboratory study of the glycosylation of a reference antibody: NISTmAb. 103 reports were received from 76 diverse laboratories worldwide. Analysis involved two samples, the NISTmAb and an enzymatically modified sample, enabling within-lab separation of random and systematic errors using the “Youden two-sample” method. Consensus values were derived and similar performance across all experimental methods was noted. Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.}, number={1}, journal={Molecular & Cellular Proteomics}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={De Leoz, Maria Lorna A. and Duewer, David L. and Fung, Adam and Liu, Lily and Yau, Hoi Kei and Potter, Oscar and Staples, Gregory O. and Furuki, Kenichiro and Frenkel, Ruth and Hu, Yunli and et al.}, year={2019}, month={Oct}, pages={11–30} }
 @article{kottke_lee_jonke_seneviratne_hecht_muddiman_torres_fedorov_2017, title={DRILL: An Electrospray Ionization-Mass Spectrometry Interface for Improved Sensitivity via Inertial Droplet Sorting and Electrohydrodynamic Focusing in a Swirling Flow}, volume={89}, ISSN={["1520-6882"]}, DOI={10.1021/acs.analchem.7b01555}, abstractNote={We describe the DRILL (dry ion localization and locomotion) device, which is an interface for electrospray ionization (ESI)-mass spectrometry (MS) that exploits a swirling flow to enable the use of inertial separation to prescribe different fates for electrosprayed droplets based on their size. This source adds a new approach to charged droplet trajectory manipulation which, when combined with hydrodynamic drag forces and electric field forces, provides a rich range of possible DRILL operational modes. Here, we experimentally demonstrate sensitivity improvement obtained via vortex-induced inertial sorting of electrosprayed droplets/ions: one possible mode of DRILL operation. In this mode, DRILL removes larger droplets while accelerating the remainder of the ESI plume, producing a high velocity stream of gas-enriched spray with small, highly charged droplets and ions and directing it toward the MS inlet. The improved signal-to-noise ratio (10-fold enhancement) in the detection of angiotensin I is demonstrated using the DRILL interface coupled to ESI-MS along with an improved limit of detection (10-fold enhancement, 100 picomole) in the detection of angiotensin II. The utility of DRILL has also been demonstrated by liquid chromatography (LC)-MS: a stable isotope labeled peptide cocktail was spiked into a complex native tissue extract and quantified by unscheduled multiple reaction monitoring on a TSQ Vantage. DRILL demonstrated improved signal strength (up to a 700-fold) for 8 out of 9 peptides and had no effects on the peak shape of the transitions.}, number={17}, journal={ANALYTICAL CHEMISTRY}, author={Kottke, Peter A. and Lee, Jurt Y. and Jonke, Alex P. and Seneviratne, Chinthaka A. and Hecht, Elizabeth S. and Muddiman, David C. and Torres, Matthew P. and Fedorov, Andrei G.}, year={2017}, month={Sep}, pages={8981–8987} }
 @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{hecht_mccord_muddiman_2016, title={A quantitative glycomics and proteomics combined purification strategy}, number={109}, journal={Jove-Journal of Visualized Experiments}, author={Hecht, E. S. and McCord, J. P. and Muddiman, D. C.}, year={2016} }
 @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} }
 @misc{hecht_oberg_muddiman_2016, title={Optimizing Mass Spectrometry Analyses: A Tailored Review on the Utility of Design of Experiments}, volume={27}, ISSN={["1879-1123"]}, DOI={10.1007/s13361-016-1344-x}, abstractNote={Mass spectrometry (MS) has emerged as a tool that can analyze nearly all classes of molecules, with its scope rapidly expanding in the areas of post-translational modifications, MS instrumentation, and many others. Yet integration of novel analyte preparatory and purification methods with existing or novel mass spectrometers can introduce new challenges for MS sensitivity. The mechanisms that govern detection by MS are particularly complex and interdependent, including ionization efficiency, ion suppression, and transmission. Performance of both off-line and MS methods can be optimized separately or, when appropriate, simultaneously through statistical designs, broadly referred to as "design of experiments" (DOE). The following review provides a tutorial-like guide into the selection of DOE for MS experiments, the practices for modeling and optimization of response variables, and the available software tools that support DOE implementation in any laboratory. This review comes 3 years after the latest DOE review (Hibbert DB, 2012), which provided a comprehensive overview on the types of designs available and their statistical construction. Since that time, new classes of DOE, such as the definitive screening design, have emerged and new calls have been made for mass spectrometrists to adopt the practice. Rather than exhaustively cover all possible designs, we have highlighted the three most practical DOE classes available to mass spectrometrists. This review further differentiates itself by providing expert recommendations for experimental setup and defining DOE entirely in the context of three case-studies that highlight the utility of different designs to achieve different goals. A step-by-step tutorial is also provided.}, number={5}, journal={JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY}, author={Hecht, Elizabeth S. and Oberg, Ann L. and Muddiman, David C.}, year={2016}, month={May}, pages={767–785} }
 @article{hecht_mccord_muddiman_2015, title={Definitive Screening Design Optimization of Mass Spectrometry Parameters for Sensitive Comparison of Filter and Solid Phase Extraction Purified, INLIGHT Plasma N-Glycans}, volume={87}, ISSN={["1520-6882"]}, DOI={10.1021/acs.analchem.5b01609}, abstractNote={High-throughput, quantitative processing of N-linked glycans would facilitate large-scale studies correlating the glycome with disease and open the field to basic and applied researchers. We sought to meet these goals by coupling filter-aided-N-glycan separation (FANGS) to the individuality normalization when labeling with glycan hydrazide tags (INLIGHT) for analysis of plasma. A quantitative comparison of this method was conducted against solid phase extraction (SPE), a ubiquitous and trusted method for glycan purification. We demonstrate that FANGS-INLIGHT purification was not significantly different from SPE in terms of glycan abundances, variability, functional classes, or molecular weight distributions. Furthermore, to increase the depth of glycome coverage, we executed a definitive screening design of experiments (DOE) to optimize the MS parameters for glycan analyses. We optimized MS parameters across five N-glycan responses using a standard glycan mixture, translated these to plasma and achieved up to a 3-fold increase in ion abundances.}, number={14}, journal={ANALYTICAL CHEMISTRY}, author={Hecht, Elizabeth S. and McCord, James P. and Muddiman, David C.}, year={2015}, month={Jul}, pages={7305–7312} }
 @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} }