Glycation promoted by dynamic high pressure microfluidisation pretreatment revealed by high resolution mass spectrometry

Highlights

• FTICR-MS can identify the glycation sites and determine the glycation level of BSA.

• DHPM pretreatment improved the glycation with increased glycation sites and level.

• 100 MPa DHPM pretreatment showed the most significant influence on the BSA glycation.

• Conformational changes of BSA induced by DHPM can be inferred from the glycation.

Abstract

The effect of dynamic high pressure microfluidisation (DHPM) pretreatment on the glycation of bovine serum albumin (BSA) was investigated. A detailed glycation map was obtained from high resolution mass spectrometry. Without DHPM pretreatment, only 7 glycation sites were identified, whereas the numbers were increased to 10, 11 and 11 when BSA-glucose was pretreated with DHPM at 50, 100 and 200 MPa, respectively, suggesting that DHPM pretreatment can significantly promote the Maillard reaction. Average degree of substitution per peptide molecule BSA (DSP) was used to further evaluate the glycation level under various DHPM conditions. All the DHPM pretreated samples exhibited elevated glycation level compared to the un-pretreated sample. With 100 MPa DHPM pretreatment, the protein showed the most significantly enhanced glycation extent. In addition, our results suggest that Maillard-type glycation followed by mass spectrometry analysis can be used to study the conformational changes when proteins are disturbed by external forces.

Introduction

Glycation, the first step of the Maillard reaction, which generally takes place between proteins and reducing sugars, is one of the most common and important chemical reactions occurring in food processing and storage (Jaeger, Janositz, & Knorr, 2010). Maillard-type glycation has been considered as a promising approach for modifying food ingredients, especially in protein modification for food purposes (Liu et al., 2012, Oliver et al., 2006). This reaction can be affected by many factors, such as reaction temperature, time, pH, water activity and the structure of reactants (Delgado-Andrade et al., 2010, Guan et al., 2006, Guan et al., 2010, Jaeger et al., 2010, Shi et al., 2010, van Boekel, 2001). High hydrostatic pressure processing (HPP), an external non-thermal food preservation technique, has also been found to influence the Maillard reaction. The formation and subsequent degradation of Amadori rearrangement products was accelerated by high pressure (400 MPa, 60 °C) and resulted in increased levels of intermediate and advanced reaction products (Moreno, Molina, Olano, & Lopez-Fandino, 2003). In addition, Rada-Mendoza, Villamiel, Molina, and Olano (2006) studied the impact of HPP on the lactosylation of β-lactoglobulin and found that HPP gave rise to structural changes in the protein with possible denaturation and crosslinking.

DHPM (dynamic high pressure microfluidisation) is an emerging dynamic high pressure technology. Unlike high hydrostatic pressure processing, DHPM is a collective force of high-velocity impact, high-frequency vibration, instantaneous pressure drop, intense shear, cavitation, and ultra-high pressures up to 200 MPa (Liu et al., 2009). DHPM was reported to be a potential method for modifying food biomacromolecules, including proteins (Oboroceanu et al., 2011, Shen and Tang, 2012, Tu et al., 2007, Tu et al., 2009), dietary fibre (Wan, Liu, Liu, Xiong, & Tu, 2009), enzymes (Liu, Liu, Liu, et al., 2009, Liu, Liu, Xie, et al., 2009, Liu et al., 2010) and polysaccharides (J. Chen et al., 2012). It has also been suggested that DHPM could change the structure of proteins, inducing an unfolding state and further influencing its physicochemical properties (Liu et al., 2010). Our recent study on the Maillard reaction suggests that glycation is strongly dependent on the protein tertiary structure, with much stronger reaction when the protein tertiary structure is disrupted (Huang et al., 2013). Therefore, DHPM has the potential to improve Maillard-type glycation, owing to its strong ability to cause protein structural changes.

The extent of the Maillard reaction can be evaluated by measuring the absorbance of the products (Thomsen et al., 2012), the free amino groups (Wong, Day, & Augustin, 2011) and sugar consumption (Ajandouz, Tchiakpe, Dalle Ore, Benajiba, & Puigserver, 2001) of the reaction system, the fluorescence intensity (Yaylayan et al., 1992, Yeboah et al., 1999) and also by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (Liu, Zhao, Zhao, Ren, & Yang, 2012) of the reaction products. However, all these methods can only provide changes of glycation degree at a whole protein level, without knowing what exactly happened inside the protein, in particular, the glycation sites and glycation extent at each site. Electrospray ionisation mass spectrometry (ESI-MS) and matrix-assisted laser desorption ionisation–mass spectrometry (MALDI–MS) techniques allow a detailed analysis of the nature and extent of protein modifications at a molecular level (Oliver, 2011). Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) provides the highest combination of simultaneous mass measurement accuracy, resolution, and sensitivity (Bruce, Anderson, Wen, Harkewicz, & Smith, 1999), making it an efficient tool for characterising the structure of proteins.

In this work, we aim to characterise the structural changes induced by DHPM treatment using a mass-spectrometry-based method. We firstly applied the DHPM technique to the structural perturbation of the model protein, bovine serum albumin (BSA). The structurally perturbed protein was then subjected to Maillard-type glycation. Circular dichroism (CD) spectroscopy was employed to monitor the structural changes under varying DHPM pressures. UV–Vis absorbance was used to evaluate the effect of DHPM on the extent of the BSA–glucose Maillard reaction. The structural changes were further examined by measuring glycation levels of the tryptic peptides, using high resolution mass spectrometry. The structural changes were found to be in agreement with the alterations of glycation levels measured by mass spectrometry.