Improved visualization of low abundance oral fluid proteins after triple depletion of alpha amylase, albumin and IgG

Abstract

The ability to selectively remove and elute the most abundant OF proteins visualized on the 2-DE represents an important step in the characterization of human OF. The better visualization and gel resolution achieved will improve quantification abilities in 2-DE and in tag-MS leading to better identification of disease-specific biomarkers. We further analyzed the eluted Alb and IgGs isoforms suggesting a new methodology venue for OF.

Saliva diagnostics – Current views and directions

“…There are three major methods of high-abundance protein removal 164 : enzyme-substrate absorption method used for alpha-amylase affinity removal, 165 immunodepletion method, and combinatorial peptide ligand library (CPLL). 166 Proteomic analysis of saliva is commonly used in the diagnostics of oral diseases as well as general health disorders such as oral candidiasis, 167 OSCC, 168 glossodynia, 169 head and neck squamous cell cancer, 170 Sjö gren's syndrome 171 HIV, 167 autism, 172 fibromyalgia, 173 breast cancer, 174 lung cancer, melanoma, 82 or pancreatic cancer. 26 …”

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

“…For 2-DE analysis, gels were scanned using a computerized GS-800 calibrated densitometer (Bio-Rad). Gel-spot detection was normalized to a NT OF gel, and spot expression was measured by spots O.D and matched using PDQuest software V 6.2.0 (Bio-Rad) as previously described [10]. t-Tests for spot ratios were performed between NT and TD, MD and EMD OF by calculating the t-test ratios between O.D values for each group of spots, normalized to relative portions.…”

“…Filtrate was subjected to an Alb and IgG immunodepletion column as previously described [10]. MD: OF was subjected to the sAA removal device.…”

“…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.…”