2-DE and MALDI-TOF MS-based identification of bovine whey proteins in milk collected soon after parturition

Abstract

Bovine milk is widely consumed by humans and is a primary ingredient of dairy foods. Proteomic approaches have the potential to elucidate complex milk proteins and have been used to study milk of various species. Here, we performed a proteomic analysis using 2-dimensional electrophoresis (2-DE) and matrix assisted laser desorption ionization-time of flight mass spectrometer (MALDI-TOF MS) to identify whey proteins in bovine milk obtained soon after parturition (bovine early milk). The major casein proteins were removed, and the whey proteins were analyzed with 2-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The whey proteins (2 mg) were separated by pI and molecular weight across pH ranges of 3.0 - 10.0 and 4.0 - 7.0. The 2-DE gels held about 300 to 700 detectable protein spots. We randomly picked 12 and nine spots that were consistently expressed in the pH 3.0 - 10.0 and pH 4.0 - 7.0 ranges, respectively. Following MALDI-TOF MS analysis, the 21 randomly selected proteins included proteins known to be present in bovine milk, such as albumin, lactoferrin, serum albumin precursor, T cell receptor, polymeric immunoglobulin receptor, pancreatic trypsin inhibitor, aldehyde oxidase and microglobulin. These proteins have major functions in immune responses, metabolism and protein binding. In summary, we herein identified both known and novel whey proteins present in bovine early milk, and our sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed their expression pattern.

Fig. 1. 2-dimensional polyacrylamide gel electrophoresis (2-D PAGE) gel pattern of bovine whey proteins in early milk obtained soon after parturition. The proteins were visualized by Coomassie blue staining. The first dimension was pH 3.0 - 10.0 non-linear immobilized pH gradient (IPG), and the second dimension was a 6 - 200 kDa separation in an 8 - 16% gradient gel. A: Stained gel. B: Image-analyzed gel. SDS-PAGE, sulfate-polyacrylamide gel electrophoresis.

Fig. 2. 2-dimensional polyacrylamide gel electrophoresis (2-D PAGE) gel pattern of bovine whey proteins of early milk. The first dimension was pH 4.0 - 7.0 non-linear immobilized pH gradient (IPG), and the second dimension was a 6 - 200 kDa separation in an 8 - 16% gradient gel. Proteins were stained with Coomassie blue. A: Stained gel. B: Image-analyzed gel. SDS-PAGE, sulfate-polyacrylamide gel electrophoresis.

Fig. 3. Twelve (A) and nine protein spots (B) corresponding to bovine whey proteins in 2-dimensional polyacrylamide gel electrophoresis (2-D PAGE) were randomly selected from the pH 3.0 - 10.0 (A) and pH 4.0 - 7.0 (B) nonlinear immobilized pH gradient (IPG) strips, respectively, and subjected to matrix assisted laser desorption ionization–time of flight mass spectrometer (MALDI-TOF MS). SDS-PAGE, sulfate-polyacrylamide gel electrophoresis.

Fig. 4. Functional categorization of 21 matrix assisted laser desorption ionization–time of flight mass spectrometer (MALDI-TOF MS)-identified proteins in bovine early milk. A number of the proteins are included in multiple categories.

Table 1. Identifcation of bovine whey proteins selected from the pH 3.0 - 10.0 range.

Table 2. Identifcation of bovine whey proteins in the pH 4.0 - 7.0 range.