Quantification of milk fat globule membrane proteins using selected reaction monitoring mass spectrometry.
Although some of the physiological roles of milk fat globule membrane (MFGM) proteins are still unclear, there is increasing evidence that the consumption of bovine MFGM proteins has significant nutritional health benefits for humans; therefore, it may be important to be able to estimate the MFGM proteins in complex ingredients. In this study, the absolute quantification (AQUA) technique, which is typically used for the quantification of proteins in proteomic studies, was applied for the quantification of bovine MFGM proteins in butter milk protein concentrate. Six MFGM proteins (fatty acid binding protein, butyrophilin, PAS 6/7, adipophilin, xanthine oxidase, and mucin 1) were simultaneously quantified using high-resolution selected reaction monitoring mass spectrometry. Samples were rehydrated in 6.7 M urea buffer prior to dilution to 2.2 M before tryspin digestion. Direct rehydration in 2.2 M urea buffer or 2.2 M urea/20% acetonitilrile buffer reduced peptide yield digestion. Isotopically labeled peptides were used as internal standards. The coefficient of variation ranged from 5 to 15%, with a recovery of 84-105%. The limit of detection was in the range of 20-40 pg.
Fong
Bertram Y
BY
Fonterra Research Centre, Palmerston North 4412, New Zealand. bertram.fong@fonterra.com
Norris
Carmen S
CS
D000595:Amino Acid Sequence; D000818:Animals; D002079:Butter / Q000032:analysis; D000071797:Butyrophilins; D002417:Cattle; D050556:Fatty Acid-Binding Proteins / Q000032:analysis; D006017:Glycolipids / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D006023:Glycoproteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D066292:Lipid Droplets; D013058:Mass Spectrometry / Q000379:methods*; D008562:Membrane Glycoproteins / Q000032:analysis; D008565:Membrane Proteins; D008894:Milk Proteins / Q000032:analysis / Q000737:chemistry; D018396:Mucin-1 / Q000032:analysis; D010455:Peptides / Q000032:analysis; D000070780:Perilipin-2; D011786:Quality Control; D015203:Reproducibility of Results; D014357:Trypsin / Q000378:metabolism; D014969:Xanthine Oxidase / Q000032:analysis
D000071797:Butyrophilins; D050556:Fatty Acid-Binding Proteins; D006017:Glycolipids; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D008894:Milk Proteins; D018396:Mucin-1; C106215:PAS-6-7 glycoprotein, Bos taurus; C000602982:PLIN2 protein, human; D010455:Peptides; D000070780:Perilipin-2; C110367:milk fat globule; D002079:Butter; D014969:Xanthine Oxidase; D014357:Trypsin