Simultaneous quantification of α-lactalbumin and β-casein in human milk using ultra-performance liquid chromatography with tandem mass spectrometry based on their signature peptides and winged isotope internal standards.
In recent years, there is an increasing need to measure the concentration of individual proteins in human milk, instead of total human milk proteins. Due to lack of human milk protein standards, there are only few quantification methods established. The objective of the present work was to develop a simple and rapid quantification method for simultaneous determination of α-lactalbumin and β-casein in human milk using signature peptides according to a modified quantitative proteomics strategy. The internal standards containing the signature peptide sequences were synthesized with isotope-labeled amino acids. The purity of synthesized peptides as standards was determined by amino acid analysis method and area normalization method. The contents of α-lactalbumin and β-casein in human milk were measured according to the equimolar relationship between the two proteins and their corresponding signature peptides. The method validation results showed a satisfied linearity (R(2)>0.99) and recoveries (97.2-102.5% for α-lactalbumin and 99.5-100.3% for β-casein). The limit of quantification for α-lactalbumin and β-casein was 8.0mg/100g and 1.2mg/100g, respectively. CVs for α-lactalbumin and β-casein in human milk were 5.2% and 3.0%. The contents of α-lactalbumin and β-casein in 147 human milk samples were successfully determined by the established method and their contents were 205.5-578.2mg/100g and 116.4-467.4mg/100g at different lactation stages. The developed method allows simultaneously determination of α-lactalbumin and β-casein in human milk. The quantitative strategy based on signature peptide should be applicable to other endogenous proteins in breast milk and other body fluids.
Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051, China.
Qi
Q
Chen
Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051, China.
Jingshun
J
Zhang
Zhejiang University of Technology, Hangzhou 310014, China.
Xing
X
Ke
Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051, China.
Shiyun
S
Lai
Department of Food Science & Nutrition and APCNS Center of Nutrition and Food Safety, Zhejiang University, Hangzhou 310058, China.
Duo
D
Li
Waters Corporation, Milford, MA 01757, USA.
Jinchuan
J
Yang
Zhejiang University of Technology, Hangzhou 310014, China. Electronic address: mowm@zjut.edu.cn.
Weimin
W
Mo
Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051, China. Electronic address: renyiping@263.net.
Yiping
Y
Ren
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D002247:Carbon Isotopes; D002364:Caseins / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007768:Lactalbumin / Q000302:isolation & purification*; D057230:Limit of Detection; D008895:Milk, Human / Q000737:chemistry; D009587:Nitrogen Isotopes; D010446:Peptide Fragments / Q000032:analysis*; D012015:Reference Standards; D013194:Staining and Labeling / Q000379:methods; D053719:Tandem Mass Spectrometry
D000596:Amino Acids; D002247:Carbon Isotopes; D002364:Caseins; D009587:Nitrogen Isotopes; D010446:Peptide Fragments; D007768:Lactalbumin
Human milk; Mass spectrometry; Signature peptide; α-Lactalbumin; β-Casein