Quantitative 2-D gel electrophoresis-based expression proteomics of albumin and IgG immunodepleted plasma.
Proteomic analysis of plasma is challenging because of its large dynamic range, which prevents the detection of low abundance proteins. Immunodepletion of high abundance proteins, such as albumin and IgG, has emerged as a favored technology to overcome this problem; however its suitability in quantitative expression proteomics has not yet been adequately addressed. In this study, albumin and IgG immunodepletion was evaluated by ELISAs and the reproducibility of depletion was tested with 2-DGE. Depletion of plasma resulted in removal of 62+/-1.2% of the total protein, 93+/-1.4% of the albumin (0.43 microg/microL, residual), and 94+/-1.5% of the IgG (0.21 microg/microL, residual). These results were confirmed by immunoblotting. Computerized image analysis of 2-D gels using Progenesis SameSpots software revealed an enhancement in the number of visible spots (675-1325), with 10+/-6% inter-gel variability in spot density. LC-ESI-MS/MS identification of newly resolved protein spots further validated the procedure. An innovative application of the software employed led to identification of 11 proteins lost non-specifically during depletion. This study demonstrates the effectiveness of immunodepletion of albumin and IgG in quantitative 2-DGE-based differential analysis of plasma proteins.
Seferovic
Maxim D
MD
Department of Pediatrics, University of Western Ontario, London, ON, Canada.
Krughkov
Violet
V
Pinto
Devanand
D
Han
Victor K M
VK
Gupta
Madhulika B
MB
D000418:Albumins / Q000302:isolation & purification*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000302:isolation & purification*; D040901:Proteomics; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
D000418:Albumins; D007074:Immunoglobulin G