Comprehensive characterization of the N-glycome of a therapeutic is challenging because glycans may harbor numerous modifications (e.g., phosphorylation, sulfation, sialic acids with possible O-acetylation). The current report presents a comparison of two chromatographic platforms for the comprehensive characterization of a recombinant human erythropoietin (rhEPO) N-glycome. The two platforms include a common workflow based on 2-AB-derivatization and hydrophilic interaction chromatography (HILIC) and a native N-linked glycan workflow employing high performance anion exchange (HPAE) chromatography. Both platforms were coupled to an Orbitrap mass spectrometer, and data dependent HCD fragmentation allowed confident structural elucidation of the glycans. Each platform identified glycans not revealed by the other, and both exhibited strengths and weaknesses. The reductive amination based HILIC workflow provided better throughput and sensitivity, had good isomer resolution, and revealed the presence of O-acetylated sialic acids. However, it exhibited poor performance toward phosphorylated glycans and did not reveal the presence of sulfated glycans. Furthermore, reductive amination introduced dehydration artifacts and modified the glycosylation profile in the rhEPO glycome. Conversely, HPAE provided unbiased charge classification (sialylation levels), improved isomer resolution, and revealed multiple phosphorylated and sulfated structures, but delivered lower throughput, had artifact peaks due to epimer formation, and loss of sialic acid O-acetylation. The MS based identification of phosphorylated and sulfated glycans was not possible in HILIC mode due to their poor solubility caused by the high acetonitrile concentrations employed at the beginning of the gradient. After analyzing the glycome by both approaches and determining the glycans present, a glycan library was created for site specific glycopeptide analyses. Glycopeptide analyses confirmed all the compositions annotated by the combined use of 2-AB- and native glycan workflows and provided site specific location of the glycans. These two platforms were complementary and in combination delivered a more thorough and comprehensive characterization of the rhEPO N-glycome, supporting regulatory conformance for the pharmaceutical industry.

The polyphenolic profile and antioxidant activity of peel, pomace, and juice of 'Verde Doncella', a Spanish apple cultivar, is presented. Phenolic profile of the worldwide cultivated 'Red Delicious' cultivar was used for comparison. Flavanols, hydroxycinamic acids, flavonols, phloridzin, procyanidin B2, and gallic acid were quantified by HPLC. Larger concentrations of polyphenolics were found in the peel, which is in agreement with the total phenolic content and antioxidant activity (FRAP) values. 'Verde Doncella' expressed lower concentrations of flavanols and quercetin derivates in peel, pomace, and juice when compared to 'Red Delicious'. 'Verde Doncella' was richer in p-coumaric acid and procyanidn B2 in the peel.

Background Various processing aids and fining agents are used in winemaking to help improve sensory characteristics. Some of these materials may contain or be derived from allergenic foods, such as eggs. In order to ensure food safety and that products meet regulatory compliance, it is essential to have robust and effective analytical methods to verify the removal of allergenic proteins following their use. Current methods include immunoassays (ELISA) and mass spectrometry methods, which can target either whole foods or individual proteins, and provide either quantitative data or qualitative confirmation of proteins. Mass spectrometry methods offer the potential to test for multiple proteins within a single assay to improve cost and efficiency, whereas ELISA methods typically analyze for a single protein per assay. Objectiv This study focuses on the development of a LC-MS/MS quantitative method for lysozyme in white wine and compares performance across two laboratories utilizing two different instrument platforms. Methods Lysozyme target peptides were selected by conducting bottom-up discovery proteomics. Candidate targets were evaluated using parallel reaction monitoring (PRM) or selected reaction monitoring (SRM) LC-MS/MS, depending on the instrument in each laboratory. Quantification of lysozyme was conducted using internal, stable-isotope-labeled synthetic peptide standards. Results Three of eight candidate target peptides showed performance suitable for the final quantitative method. White wine spiked with 0.1 and 0.5 ppm lysozyme demonstrated quantitative recovery of 70–120%. While the PRM method delivered better repeatability, the SRM method gave higher quantitative recovery values. Conclusion A targeted LC-MS/MS method for quantification of lysozyme in white wine has been developed and deployed on two different MS instrument platforms in two laboratories. Highlights Both SRM and PRM targeted LC-MS/MS methodologies can be used for quantification of lysozyme in white wine. This study is among the first to evaluate an MS method for food allergen quantification in multiple laboratories.

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