Definitive Screening Design Optimization of Mass Spectrometry Parameters for Sensitive Comparison of Filter and Solid Phase Extraction Purified, INLIGHT Plasma<i>N</i>-Glycans

Hecht, Elizabeth S.; McCord, James; Muddiman, David C.

doi:10.1021/acs.analchem.5b01609

Cited by 35 publications

(43 citation statements)

References 67 publications

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“…In this case, optimized ionization conditions for the analysis of all N-glycans, including both neutral and acidic structures, were utilized. Optimized conditions for the reduction of sialic acid fragmentation of labeled glycans have been reported for glycans using the individuality normalization strategy; 43 however, in our study, a more widely applicable method was deemed appropriate for our comparisons. Additionally, despite reduced sialic acid fragmentation being observed with optimized ionization conditions used in conjunction with the INLIGHT strategy, higher sensitivity is still achieved through the sialic acid stabilizing process of permethylation.…”

Section: Resultsmentioning

confidence: 99%

Direct comparison of derivatization strategies for LC-MS/MS analysis ofN-glycans

Zhou

Veillon

Dong

et al. 2017

Analyst

106

105

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Protein glycosylation is a common post-translational modification that has significant impacts on protein folding, lifespan, conformation, distribution and function. N-glycans, which are attached to asparagine residues of proteins, are studied most often due to their compatibility with enzymatic release. Despite the ease of N-glycan release, compositional and structural complexity coupled with poor ionization efficiency during liquid chromatography mass spectrometry (LC-MS) make quantitative glycomic studies a significant challenge. To overcome these challenges, glycans are almost always derivatized prior to LC-MS analyses to impart favorable characteristics, such as improved ionization efficiency, increased LC separation efficiency and the production of more informative fragments during tandem MS. There are a number of derivatization methods available for LC-MS analysis of glycans, each of which imparts different properties that affect both glycan retention on LC columns and MS analyses. To provide guidance for the proper selection of derivatizing reagents and LC columns, herein, we describe a comprehensive assessment of 2-aminobenzamide, procainamide, aminoxyTMT, RapiFluor-MS (RFMS) labeling, reduction and reduction with permethylation for N-glycan analysis. Of the derivatization strategies examined, RFMS provided the highest MS signal enhancement for neutral glycans, while permethylation significantly enhanced the MS intensity and structural stability of sialylated glycans.

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Section: Resultsmentioning

confidence: 99%

Direct comparison of derivatization strategies for LC-MS/MS analysis ofN-glycans

Zhou

Veillon

Dong

et al. 2017

Analyst

106

105

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“… ELECTRONIC SUPPLEMENTAL MATERIAL Glossary (ESM 1) Definitive screening design tutorial (ESM 2): DOE GUI tutorial using Hecht, McCord, and Muddiman (2015) [338] (available open-source, doi: 10.1021/acs.analchem.5b01609) as a case study. Definitive screening design tutorial raw data and JMP DOE dialogue: .jmp version (ESM 3) Definitive screening design tutorial raw data: .xlsx version (ESM 4) …”

mentioning

confidence: 99%

“…Definitive screening design tutorial (ESM 2): DOE GUI tutorial using Hecht, McCord, and Muddiman (2015) [338] (available open-source, doi: 10.1021/acs.analchem.5b01609) as a case study.…”

mentioning

confidence: 99%

Optimizing Mass Spectrometry Analyses: A Tailored Review on the Utility of Design of Experiments

Hecht

Oberg

Muddiman

2016

J. Am. Soc. Mass Spectrom.

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SUMMARY 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 three 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.

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“…NOTE: The LC and MS conditions described for an Easy nLC-1000 and a Q Exactive High Field, respectively, were optimized in-house for proteomics analysis and for glycan analysis 21 . These conditions may be adapted to other ultra-high pressure-or nano-LC systems and other high-resolving power mass spectrometers but may require slight modifications.…”

Section: Ultra-high Pressure Liquid Chromatography and Mass Spectromementioning

confidence: 99%

A Quantitative Glycomics and Proteomics Combined Purification Strategy

Hecht

McCord

Muddiman

2016

JoVE

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There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define pathways, improve treatment, understand disease, and aid biomarker discovery. N-linked glycosylation is one of the most important and robust post-translational modifications on proteins and regulates critical cell functions such as signaling, adhesion, and enzymatic function.Analytical techniques to purify and analyze N-glycans have remained relatively static over the last decade. While accurate and effective, they commonly require significant expertise and resources. Though some high-throughput purification schemes have been developed, they have yet to find widespread adoption and often rely on the enrichment of glycopeptides. One promising method, developed by Thomas-Oates et al., filter aided N-glycan separation (FANGS), was qualitatively demonstrated on tissues. Herein, we adapted FANGS to plasma and coupled it to the individuality normalization when labeling with glycan hydrazide tags strategy in order to achieve accurate relative quantification by liquid chromatography mass spectrometry and enhanced electrospray ionization. Furthermore, we designed new functionality to the protocol by achieving tandem, shotgun proteomics and glycosylation site analysis on hen plasma. We showed that N-glycans purified on filter and derivatized by hydrophobic hydrazide tags were comparable in terms of abundance and class to those by solid phase extraction (SPE); the latter is considered a gold standard in the field. Importantly, the variability in the two protocols was not statistically different. Proteomic data that was collected in-line with glycomic data had the same depth compared to a standard trypsin digest. Peptide deamidation is minimized in the protocol, limiting non-specific deamidation detected at glycosylation motifs. This allowed for direct glycosylation site analysis, though the protocol can accommodate 18 O site labeling as well. Overall, we demonstrated a new in-line high-throughput, unbiased, filter based protocol for quantitative glycomics and proteomics analysis.

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Definitive Screening Design Optimization of Mass Spectrometry Parameters for Sensitive Comparison of Filter and Solid Phase Extraction Purified, INLIGHT PlasmaN-Glycans

Cited by 35 publications

References 67 publications

Direct comparison of derivatization strategies for LC-MS/MS analysis ofN-glycans

Direct comparison of derivatization strategies for LC-MS/MS analysis ofN-glycans

Optimizing Mass Spectrometry Analyses: A Tailored Review on the Utility of Design of Experiments

A Quantitative Glycomics and Proteomics Combined Purification Strategy

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