Development and Interlaboratory Evaluation of an LC-MS/MS Method for the Quantification of Lysozyme in Wine Across Independent Instrument Platforms.

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 ELISA and MS methods, which can target either whole foods or individual proteins, and provide either quantitative data or qualitative confirmation of proteins. MS 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. OBJECTIVE: This study focuses on the development of a LC-tandem MS (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.

Parallel Reaction Monitoring Mass Spectrometry Method for Detection of Both Casein and Whey Milk Allergens from a Baked Food Matrix.

Milk allergy is among the most common food allergies present in early childhood, which in some cases may persist into adulthood as well. Proteins belonging to both casein and whey fractions of milk can trigger an allergic response in susceptible individuals. Milk is present as an ingredient in many foods, and it can also be present as casein- or whey-enriched milk-derived ingredients. As whey proteins are more susceptible to thermal processing than caseins, conventional methods often posed a challenge in accurate detection of whey allergens, particularly from a processed complex food matrix. In this study, a targeted mass spectrometry method has been developed to detect the presence of both casein and whey allergens from thermally processed foods. A pool of 19 candidate peptides representing four casein proteins and two whey proteins was identified using a discovery-driven target selection approach from various milk-derived ingredients. These target peptides were evaluated by parallel reaction monitoring of baked cookie samples containing known amounts of nonfat dry milk (NFDM). The presence of milk could be detected from baked cookies incurred with NFDM at levels as low as 1 ppm using seven peptides representing α-, ß-, and κ-casein proteins and three peptides representing a whey protein, ß-lactoglobulin, by this consensus PRM method.

Thiol based mechanism internalises interacting partners to outer dense fibers in sperm.

The sperm tail outer dense fibres (ODFs) contribute passive structural role in sperm motility. The level of disulphide cross-linking of ODFs and their structural thickness determines flagellar bending curvature and motility. During epididymal maturation, proteins are internalized to modify ODF disulphide cross-linking and enable motility. Sperm thiol status is further altered during capacitation in female tract. This suggests that components in female reproductive tract acting on thiol/disulphides could be capable of modulating the tail stiffness to facilitate modulation of the sperm tail rigidity and waveform en route to fertilization. Understanding the biochemical properties and client proteins of ODFs in reproductive tract fluids will help bridge this gap. Using recombinant ODF2 (aka Testis Specific Antigen of 70 kDa) as bait, we identified client proteins in male and female reproductive fluids. A thiol-based interaction and internalization indicates sperm can harness reproductive tract fluids for proteins that interact with ODFs and likely modulate the tail stiffness en route to fertilization.

Temozolomide-modulated glioma proteome: role of interleukin-1 receptor-associated kinase-4 (IRAK4) in chemosensitivity.

The current treatment for glioblastoma includes temozolomide (TMZ) chemotherapy, yet the mechanism of action of TMZ is not thoroughly understood. Here, we investigated the TMZ-induced changes in the proteome of the glioma-derived cell line (U251) by 2D DIGE. We found 95 protein spots to be significantly altered in their expression after TMZ treatment. MS identified four upregulated spots: aspartyl tRNA synthetase glutathione synthetase, interleukin-1 receptor-associated kinase-4 (IRAK4), and breast carcinoma amplified sequence-1 and one downregulated spot: optineurin. TMZ-induced regulation of these five genes was validated by RT-qPCR and Western blot analysis. RNAi-mediated knockdown of IRAK4, an important mediator of Toll-like receptors signaling and chemoresistance, rendered the glioma cells resistant to TMZ. High levels of IRAK4 induced upon TMZ treatment resulted in IRAK1 downregulation and inhibition of NFkB pathway. Endogenous IRAK4 protein, but not transcript levels in glioma cell lines, correlated with TMZ sensitivity. Thus, we have identified several TMZ-modulated proteins and discovered an important novel role for IRAK4 in determining TMZ sensitivity of glioma cells through its ability to inhibit Toll-like receptor signaling and NFkB pathway.

Recombinant E. coli expressing Vitreoscilla haemoglobin prefers aerobic metabolism under microaerobic conditions: a proteome-level study.

Vitreoscilla haemoglobin (VHb) expression in heterologous host was shown to enhance growth and oxygen utilization capabilities under oxygen-limited conditions. The exact mechanism by which VHb enhances the oxygen utilization under oxygen-limiting conditions is still unknown. In order to understand the role of VHb in promoting oxygen utilization, changes in the total protein profile of E. coli expressing the vgb gene under its native promoter was analysed. Two-dimensional difference gel electrophoresis (2D DIGE) was employed to quantify the differentially expressed proteins under oxygen-limiting conditions. Overexpression of proteins involved in aerobic metabolic pathways and suppression of proteins involved in non-oxidative metabolic pathways shown in this study indicates that the cells expressing VHb prefer aerobic metabolic pathways even under oxygen limitation. Under these conditions, the expression levels of proteins involved in central metabolic pathways, cellular adaptation and cell division were also found to be altered. These results imply that Vitreoscilla haemoglobin expression alters aerobic metabolism specifically, in addition to altering proteins involved in other pathways, the significance of which is not clear as of now.