Determination of protein composition in whey fractions by molecular spectroscopy, gel electrophoresis and mass spectrometry

Abstract

Whey is becoming an important product for the dairy industry. In the last 15 years, the characterization of whey proteins (β-lactoglobulin (β-Lg), α-lactalbumin (α-La), Bovine serum albumin (BSA), Immunoglobulin (Ig), Lactoferin (LF), Glycomacropeptide (GMP) and Casein macropeptide (CMP)) has become an important issue for biochemists and dairy companies. Characterization of the protein in whey is necessary to obtain possible methods to achieve the best whey based products. This project is a comparison of three different methods for protein characterization in order to observe and compare the main proteins in whey and skimmed milk. Whey samples were generated through the microfiltration of skimmed milk where permeate is defined as native whey. During this whey sample preparation, a second type of whey was formed. This whey is the retentate generated from the ultrafiltration of permeate (native whey) and is called native whey protein concentrate from microfiltrated milk. FT-IR spectroscopy, Gel electrophoresis and Mass spectrometry based proteomics were the methods that were used in this project to characterize the milk samples concentrate. Initially sample preparation methods were developed to obtain protein samples for analysis. Fourier transform infrared spectroscopy (FT-IR) was used to evaluate differences between each sample according to the type of filtration or the concentration factor of each process. Principal component analysis (PCA) was utilized to FT-IR spectra in regions of 3043-898 cm−1 in order to identify the differences between skimmed milk, permeate and retentate proteins. Use of this method allowed analysis of the most important wavenumbers for each group of samples. PLS modelling was used to compare the data from FT-IR against other methods, by showing the correlation between samples. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to predict the presence of certain proteins in the samples based on their molecular weight. Gel scans show the distinct protein sizes in the whey and skimmed milk. The method does not allow identifying proteins but it may answer some biochemical questions. Mass spectrometry based proteomics (using LC-MS/MS) is commonly used to identify proteins in biological samples. Protein characterization by this method is based on detection of peptides following trypsination of the samples, a so called bottom-up approach. Tandem mass spectra are collected from peptides and in order to identify the proteins, the results are searched against a database of proteins derived from genomic sequences. Β-lactoglobulin, α-lactalbumin and casein were the major proteins present in my samples. The existence of BSA, lactoferin, immunoglobulin and glycomacropeptide as the minor proteins were also detected. Most of the SDS-PAGE observations were in agreement with LC-MS/MS results. The results indicate that mass spectrometry provided the most information concerning the protein content of the samples. Although depending on the research needs SDS-PAGE or FT-IR analysis may be sufficient.