An approach to remove alpha amylase for proteomic analysis of low abundance biomarkers in human saliva

Abstract

Proteomic characterization of human whole saliva for the identification of disease-specific biomarkers is guaranteed to be an easy-to-use and powerful diagnostic tool for defining the onset, progression and prognosis of human systemic diseases and, in particular, oral diseases. The high abundance of proteins, mainly alpha amylase, hampers the detection of low abundant proteins appearing in the disease state and therefore should be removed. In the present study a 2-DE was used to analyze human whole saliva following the removal of alpha amylase by affinity adsorption to potato starch. After alpha amylase removal whole saliva was analyzed by SDS-PAGE showing at least sixfold removal efficiency and by an alpha amylase activity assay showing 97% reduced activity. MS identification of the captured alpha amylase after elution demonstrated specific removal; 2-DE analysis showed the selective removal of alpha amylase and consequently increased gel resolution. MS identification of protein spots in the 60 kDa area revealed 15 proteins, which were masked before alpha amylase removal. In conclusion, treatment of human whole saliva with an alpha amylase removal device increases gel resolution and enables a higher protein sample for analysis.

Comparison of diverse affinity based high-abundance protein depletion strategies for improved bio-marker discovery in oral fluids

“…This is generally not the case in EMD that either removes or not remove proteins. None CPLL depletion methods capability to elute the depleted proteins from the depletion column is feasible as we previously reported [9,10]. This may be important in diseases in which one of the depleted proteins is essential for diagnosis.…”

“…We have shown that their removal contributes significantly to the detection and visibility of proteins expressed at lower levels in OF [9,10]. Furthermore, recent developments suggest the use of combinatorial peptide ligand library (CPLL) for removing high abundance proteins resulting in concentrated low abundance proteins, proved its feasibility to increase protein identification in OF by a factor of 2 [2].…”

“…We therefore hypothesized that further protein removal and increased low-abundance protein detection can be achieved by the depletion of a panel of medium-abundance proteins, similar to that developed for serum proteins [12,13]. We therefore modified a 20-serum-proteins immunodepletion kit for OF and used it as an addition to our previously published methods for sAA [9], Alb and IgG depletion [10]. Specifically, we evaluated and compared the performance of four affinity-depletion treatments: triple depletion (TD) of sAA , Alb and IgG, multiple depletion (MD) of sAA and a panel of 20 other proteins, enriched multiple depletion (EMD) which combines the two depletion approaches (TD + MD) and CPLL which use hexapeptide libraries to concentrate low abundance proteins.…”

The Use of Electrophoresis for the Study of Saliva Involvement in Ingestive Behavior

“…This is even more pertinent considering studies aimed at detecting physiological biomarkers, since most of them are present in saliva at low amounts. The protein α-amylase contributes to almost 60% of total salivary proteome [68] and, many times, its depletion is necessary. Salivary α-amylase depletion can be achieved through elution of samples from starch columns to reduce this protein amounts specifically [64,69].…”

Novel Isozyme-Specific Quantitation Method for Alpha-Amylases in Human Plasma Revealed Possible AMY2B Induction by Alpha-Amylase Inhibitor, CS-1036

“…Therefore, we developed a method to enrich α-amylase from human plasma and established a protocol using potato starch affinity based on Deutsch et al's study [26]. Several conditions of the sample preparation for LC-MRM-MS analysis were optimized.…”