Two‐dimensional electrophoretic analysis of human milk‐fat‐globule membrane proteins with attention to apolipoprotein E patterns

Abstract

Two-dimensional (2-D; Iso Dalt method) electrophoresis was done on lipid membrane fractions from human milk samples collected in polypropylene tubes at home by the mother. The samples represent milk from two weeks to ten months post partum. Identification was made by immunoassay of Western blots. Apolipoprotein E was not easily seen on silver-stained gels because concentration is small and spots overlap another protein. Blot assays show a more complex pattern than 34 kDa plasma apo E with molecular size isoforms at 36 kDa, 34 kDa, 20 kDa and 15 kDa. One sample was negative for apo E. Apo E plays an important role in lipid transport in tissue and in the brain. It is synthesized in a variety of tissues by specific cells. Milk may reflect several sources.

Protein and lipid composition of bovine milk-fat-globule membrane

“…Six minor proteins (polymeric Ig receptor protein, apolipoprotein E, apolipoprotein A1, 71 kDa heat-shock cognate protein, peptidylprolyl isomerase A (cyclophilin or rotamase) and clusterin), which have not been previously reported as being present in bovine MFGM, were identified. The human homologues of these six proteins have been reported in human MFGM (Bertino et al, 2002;Fortunato et al, 2003;Goldfarb, 1997). The 71 kDa heatshock cognate protein and Apolipoprotein A1 were found as proteins precipitated on the solution IEF membrane during fractionation of the MFGM (Table 2).…”

“…More recent studies have been focused on specific major MFGM proteins such as periodic acid schiff (PAS) 6/7 (lactadherin), Cluster of differentiation (CD) 36 and adipophilin and the post-translational modifications of these proteins (Heid, Schno¨lzer, & Keenan, 1996;Kanno, Hwangbo, & Azuma, 1995;Seok, Shimoda, Azuma, & Kanno, 2001 MFGM, with a focus on proteins (Bertino et al, 2002;Cavaletto, Giuffrida, & Conti, 2004;Cavaletto et al, 1999;Fortunato et al, 2003;Goldfarb, 1997;Iman, Laurence, & Neville, 1981Quaranta et al, 2001;). As a result, many proteins identified in human MFGM have yet to be reported in bovine MFGM.…”

Human proteome enhancement: High-recovery method and improved two-dimensional map of colostral fat globule membrane proteins

“…Solution B (Table 1) was used as sample solution to solubilize the precipitated proteins. Solutions A, B and C were taken from [18], [19] and [11], respectively, with slight modifications.…”

“…A higher quantity of proteins extracted from the fat and a higher resolution were observed, because of less interference from fat during the IEF step. Following this modified procedure, the solubilizer used to obtain the map of the existing MFGM proteins (solution C [11] in Table 1) was also tried after lipid extraction: the protein yield was lower, but resolution improved (Fig. 1D); the procedure also appeared to be selective for proteins with a molecular mass of about 30 kDa.…”

“…Among the limiting factors for membrane proteins analysis by 2-DE, the most difficult problems to solve are those linked to the hydrophobicity, which limits protein solubilization. That is probably why only one 2-DE map of the human MFGMPs has been reported to date, where a rather small number of proteins were identified, and only by immunoassay [11]. The aim of this study was to further knowledge of human milk protein composition, especially of the bioactive compounds, so as to produce a standard reference as close as possible to reality, the so-called "gold standard", for the production of formula milk.…”