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HIGHLIGHTSNovel methodology of metalloproteins determination by SEC‐ICP‐MS in human whey milk protein fractions.Two metalloproteins have detectable concentrations of Mn, Co, Cu and Se.The levels of Mn, Co, Cu and Se were within the range reported in the literature.Samples with long lactation periods still have normal levels of these metals. ABSTRACT Levels of essential metals in human breast milk (HBM) have been determined by different analytical techniques, but there is few woks about human whey milk fractions. However, the current trend lies in metalloproteomic and identification of different metalloproteins. In this sense, native separative techniques (N‐PAGE and SEC) coupled to ICP‐MS provide us with valuable information. Besides it is necessary the development of new methodologies in order to determine with accuracy and precision the profile of such metals and metalloproteins in the different whey protein fractions of HBM. Thus, the aim of this work was to develop a new method for metals and metalloproteins determination by SEC‐ICP‐MS in whey protein fractions of HBM. Human whey fractions were obtained of HBM samples by ultracentrifugation. Then, protein fractions of whey milk were separated by SEC coupled to ICP‐MS for metalloproteins and Mn, Co, Cu and Se quantification. Besides, protein profile of whey milk was determined by N‐PAGE and computer assisted image analysis. SEC‐ICP‐MS results indicated that first and second protein fractions showed detectable levels of the Mn, Co, Cu, and Se. Protein profile determined by N‐PAGE and image analysis showed that molecular weight of protein fractions ranged between 68,878–1,228.277Da. In this work, metalloproteins were analyzed by SEC coupled to ICP‐MS, with adequate sensitivity and accuracy. Our study has shown the presence of Mn, Co, Cu and Se bound to two protein fractions in whey milk of HBM. Metals levels analyzed were within the ranges reported in the literature.