Food Protein Glycation Studied By Mass Spectrometry-based Proteomics

In this study, mass spectrometry-based proteomics was used, including Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FTICR-MS), LTQ-MS" and hydrogen/deuterium exchange mass spectrometry (HDX-MS) to study the effect of protein structure and external processing on the protein glycation and the conformation of modified protein. A series of proteomics, including the extraction and SDS-PAGE, in gel digestion and ultra high liquid chromatography combined with mass spectrometry in separation and identification of egg white protein and the glycation extent and glycation sites of each component. The results indicated:(1) The disruption of disulfide bond induced protein structure disruption could increase the glycation extent of ovalbumin, the glycation extent in dry state was much stronger than that in solution because of the high reactants concentration and energy transfer rate. The glycated peptides were increased from 6 to 11 in dry state and 1 to 2 in solution. The glycation sites were main lysines. A closer look at the tertiary structure of ovalbumin, the glycation sites were affected by many factors, including protein conformation, the hydrogen bonds and the neighborhood amino acids. The emulsifying and foaming properties were promoted after being reduced and glycaion.(2) Ovalbumin was reacted with four common reducing sugars during food processing in solution with high temperature. By measuring the physiochemical properties, it is found that the ovalbumin-glucose and ovalbumin-maltose system exhibited strong browning intensity, reducing power and DPPH scavenging activity. The ovalbumin-glucose and ovalbumin-maltose presented strong TEAC value, and it was an efficient way of preparing antioxidants. The antioxidant activity is closely related to the browning extent of the ovalbumin-sugar system, the deeper of the solution color, and the stronger of the antioxidant activity. Different ovalbumin-sugar mixture exhibited diverse thermal properties, the thermal stability could be changed by conjugation with sugar.(3) Ovalbumin was reacted with glucose and was partially glycated. The glycation extent and glycation sites were identified by high resolution mass spectrometry coupled with tandem mass spectrometry after pepsin digestion. The conformational change was studied by hydrogen-deuterium exchange-mass spectrometry. The results indicated that most of the peptides of ovalbumin were protected after glycation, which meaned the protein structure tended to be stable. The conformation of both the glycated and unglycated area was changed, indicating that glycation not only could induce the local conformational change but also the distal conformation.(4) DHPM pretreatment could promote that protein glycation, increasing the glycation extent as well as the glycation sites. DHPM pretreatment at 100MPa showed the most significant impact on the BSA glycation. The glycation sites were increased from 7 to 11 and most peptides exhibited the strongest glycation extent at 100MPa. The glycation coupled with high resolution mass spectrometry could probe the protein conformational change induced by high pressure. DHPM induced protein unfolding and the promoted glycation indicated that the DHPM provided an efficient way of glycation controlling during food processing.(5) The conformation change of BSA induced by DHPM was studied by hydrogen-deuterium exchange-mass spectrometry. The results indicated that the structure of BSA was disrupted by DHPM. The structure mainly tended to be unfolding. Domain II and III tended to be unfolding while domain I was protected and tended to be stable. Different pressure induced BSA to different unfolding extent and a maximum unfolding extent was obtained at 100MPa. The unfolding extent is related to the glycation extent of each peptide, which is in agreement with the glycation sites and extent of the last chapter.(6) The egg white contains 47 types of protein and the molecular range is from 4248 to 275408 Da. Some proteins were existed as different types or by binding with small molecules, indicating that the protein in natural egg white protein has various modification. Part of the egg white protein components was glycated during heating with glucose and they presented different glycation extent. In the major components, the ovotransferrin was easily glycated whereas the lysozyme was difficult to get glycated. The glycation sites were main lysines.