Protein fingerprinting and quantification of ß-casein variants by ultra-performance liquid chromatography-high-resolution mass spectrometry.

Infant formulations are constantly evolving as novel protein ingredients are added to make them more closely mimic the protein profile of human milk; however, precise analytical methods for characterizing and quantifying the major milk proteins in such formulations are currently lacking. This article describes an ultra-performance liquid chromatography-high-resolution mass spectrometry method for intact proteins that can efficiently detect, identify, and characterize the major milk proteins and their proteoforms (phosphorylation status, degree of glycation, genetic variants among others) in ingredients and final products, with an emphasis on detecting and quantifying specific genetic variants of ß-casein in infant formulas. Method sensitivity allows detection of ß-casein A1 in A2-based infant formulas with a limit of detection of 2% (grams of ß-casein A1 per 100 g of total ß-casein). Protein glycation affects signal intensity in a linear fashion, which permits proteins to be quantified from their mass spectrometry signals after correction according to their measured glycation index. The method was validated for the quantification of ß-casein in infant formulas. Repeatability ranged from 2 to 3% and intermediate reproducibility from 5 to 9%. Calculated ß-casein amounts ranged between 77 and 110% of the values based on formulations and published protein profiles for milk. Altogether, this method can be used for general fingerprinting as well as specific characterization and quantification of individual major milk proteins in dairy-based ingredients and products.

Contribution of mesothelial cells in the expression of inflammatory-related factors in omental adipose tissue of obese subjects.

OBJECTIVE: The objective of this study was to determine the contribution of mesothelial cells, present in human omental adipose tissue (OAT) but not in the subcutaneous depot (SAT), on the expression of inflammation-related factors. DESIGN: Comparison of the expression profiles of inflammation-related genes in mesothelial cells with those in the adipocyte-enriched (AEF) and stromal vascular fractions (SVF) and localization of interleukin-18 (IL-18) expression in adipose depots. SUBJECTS: Eleven obese Caucasian female subjects undergoing gastric bypass surgery (body mass index: 43.6+/-1.3 kg/m(2); age: 41.6+/-2.3 years). MEASUREMENTS: The expression profiles of cytokine and chemokine-related genes in mesothelial cells and in cell fractions prepared from OAT were assessed by the microarray technique. The differential expression of IL-18 was confirmed by real-time PCR and the protein was localized in adipose depots by immunohistochemistry. RESULTS: Microarray data analysis demonstrated that, of the 16 cytokine and chemokine-related genes that were upregulated in mesothelial cells compared with the AEF, IL-18 was the cytokine with the highest differential expression. IL-18 expression was similar in mesothelial cells and the SVF. In both SAT and OAT, IL-18 was immunolocalized in neutrophils and mast cells, but not in macrophages nor adipocytes. This cytokine was also detected in mesothelial cells in OAT. This additional source of expression may explain the higher IL-18 expression levels in OAT than SAT (+5.9-fold). CONCLUSION: By their capacity to express inflammatory-related factors, and in particular the proinflammatory cytokine IL-18 in OAT, mesothelial cells appear as a new player in the process of low-grade inflammation associated with obesity.