Dataset Information

Online trypsin digestion coupled with LC-MS/MS for detecting of A1 and A2 types of β-casein proteins in pasteurized milk using biomarker peptides.

ABSTRACT: Bovine A1-or A2-type β-caseins have attracted a growing interest due to their variation in beta-casomorphin-7 (BCM-7) formation, which may affect health. In the present work, identification and quantification of A1 and A2 types of β-casein proteins at the peptide level was achieved for the first time. An automated and online immobilized trypsin digestion system was employed for high throughput digesting of proteins into peptides. Tryptic peptides were separated and analyzed subsequently by liquid chromatography coupled to mass spectrometry platform. Two specific peptides ranging from the position of 49 to 97 in the peptide chain were selected for the identification and quantification of A1 and A2 β-casein, which covered the different amino acids between them. Synthetic isotopically labeled winged peptides were used for absolute quantification. Compared with traditional in-solution digestion, online digestion shortens digestion times from 2 to 24 h to 4 min. The limits of quantification (LOQ) of A1 and A2 β-casein in pasteurized milk are 0.8 and 2.4 µg/g, respectively. To further demonstrate the applicability of the proposed method, commercial pasteurized milk tests were performed with satisfactory results.

Supplementary information

The online version contains supplementary material available at 10.1007/s13197-022-05376-6.

SUBMITTER: Guo D

PROVIDER: S-EPMC9304457 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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Online trypsin digestion coupled with LC-MS/MS for detecting of A1 and A2 types of β-casein proteins in pasteurized milk using biomarker peptides.

Guo Dehua D Deng Xiaojun X Gu Shuqing S Chen Niannian N Zhang Xiaomei X Wang Shuo S

Journal of food science and technology 20220227 8

Bovine A1-or A2-type β-caseins have attracted a growing interest due to their variation in beta-casomorphin-7 (BCM-7) formation, which may affect health. In the present work, identification and quantification of A1 and A2 types of β-casein proteins at the peptide level was achieved for the first time. An automated and online immobilized trypsin digestion system was employed for high throughput digesting of proteins into peptides. Tryptic peptides were separated and analyzed subsequently by liqui ...[more]

PMID: 35872738

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Project description:BackgroundCows' milk generally contains two types of β-casein, A1 and A2 types. Digestion of A1 type can yield the peptide β-casomorphin-7, which is implicated in adverse gastrointestinal effects of milk consumption, some of which resemble those in lactose intolerance. This study aimed to compare the effects of milk containing A1 β-casein with those of milk containing only A2 β-casein on inflammation, symptoms of post-dairy digestive discomfort (PD3), and cognitive processing in subjects with self-reported lactose intolerance.MethodsForty-five Han Chinese subjects participated in this double-blind, randomized, 2 × 2 crossover trial and consumed milk containing both β-casein types or milk containing only A2 β-casein. Each treatment period was 14 days with a 14-day washout period at baseline and between treatment periods. Outcomes included PD3, gastrointestinal function (measured by smart pill), Subtle Cognitive Impairment Test (SCIT), serum/fecal laboratory biomarkers, and adverse events.ResultsCompared with milk containing only A2 β-casein, the consumption of milk containing both β-casein types was associated with significantly greater PD3 symptoms; higher concentrations of inflammation-related biomarkers and β-casomorphin-7; longer gastrointestinal transit times and lower levels of short-chain fatty acids; and increased response time and error rate on the SCIT. Consumption of milk containing both β-casein types was associated with worsening of PD3 symptoms relative to baseline in lactose tolerant and lactose intolerant subjects. Consumption of milk containing only A2 β-casein did not aggravate PD3 symptoms relative to baseline (i.e., after washout of dairy products) in lactose tolerant and intolerant subjects.ConclusionsConsumption of milk containing A1 β-casein was associated with increased gastrointestinal inflammation, worsening of PD3 symptoms, delayed transit, and decreased cognitive processing speed and accuracy. Because elimination of A1 β-casein attenuated these effects, some symptoms of lactose intolerance may stem from inflammation it triggers, and can be avoided by consuming milk containing only the A2 type of beta casein.Trial registrationClinicalTrials.gov/NCT02406469.

Project description:The high toxic abrin from the plant Abrus precatorius is a type II ribosome-inactivating protein toxin with a human lethal dose of 0.1-1.0 µg/kg body weight. Due to its high toxicity and the potential misuse as a biothreat agent, it is of great importance to developing fast and reliable methods for the identification and quantification of abrin in complex matrices. Here, we report rapid and efficient acetonitrile (ACN)- and ultrasound-assisted on-bead trypsin digestion method combined with HPLC-MS/MS for the quantification of abrin isoforms in complex matrices. Specific peptides of abrin isoforms were generated by direct ACN-assisted trypsin digestion and analyzed by HPLC-HRMS. Combined with in silico digestion and BLASTp database search, fifteen marker peptides were selected for differential detection of abrin isoforms. The abrin in milk and plasma was enriched by immunomagnetic beads prepared by biotinylated anti-abrin polyclonal antibodies conjugated to streptavidin magnetic beads. The ultrasound-assisted on-bead trypsin digestion method was carried out under the condition of 10% ACN as denaturant solvent, the entire digestion time was further shortened from 90 min to 30 min. The four peptides of T3Aa,b,c,d, T12Aa, T15Ab, and T9Ac,d were chosen as quantification for total abrin, abrin-a, abrin-b, and abrin-c/d, respectively. The absolute quantification of abrin and its isoforms was accomplished by isotope dilution with labeled AQUA peptides and analyzed by HPLC-MS/MS (MRM). The developed method was fully validated in milk and plasma matrices with quantification limits in the range of 1.0-9.4 ng/mL for the isoforms of abrin. Furthermore, the developed approach was applied for the characterization of abrin isoforms from various fractions from gel filtration separation of the seeds, and measurement of abrin in the samples of biotoxin exercises organized by the Organization for the Prohibition of Chemical Weapons (OPCW). This study provided a recommended method for the differential identification of abrin isoforms, which are easily applied in international laboratories to improve the capabilities for the analysis of biotoxin samples.

Dataset Information

Online trypsin digestion coupled with LC-MS/MS for detecting of A1 and A2 types of β-casein proteins in pasteurized milk using biomarker peptides.

Supplementary information

Publications

Online trypsin digestion coupled with LC-MS/MS for detecting of A1 and A2 types of β-casein proteins in pasteurized milk using biomarker peptides.

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