Chromatofocusing fractionation and two‐dimensional difference gel electrophoresis for low abundance serum proteins

Abstract

The technical challenge to analysis of the serum proteome is that the serum proteins are present at unequal concentrations. A few are so dominant, such as serum albumin and immunoglobulins, that they mask detection of other proteins. Because of these high abundance proteins, current technologies, while theoretically capable of analyzing protein amounts spanning four orders of magnitude, are only able to analyze proteins ranging over two orders of magnitude and cannot analyze the lower abundance proteins that may be the next biomarkers and drug targets. To facilitate the identification of low abundance proteins, we fractionated serum samples from patients with prostate cancer and patients with benign prostate hyperplasia using anion displacement liquid chromatofocusing chromatography, which separates proteins by a pH gradient and a positively charged column. Differential expression of proteins from fractions was then determined and identified by IEF gels and 2-D DIGE. Results demonstrate improved resolution of proteins within the chosen pH gradient when compared to the unfractionated samples. Several proteins that were differentially expressed in serum from patients with prostate cancer were identified in the fractionated serum. Three of these proteins, squamous cell carcinoma antigen 1 (SCCA1), calgranulin B, and haptoglobin-related protein, are present in the serum at levels below the classical protein level of mg/mL. SCCA1 is normally expressed in serum at ng/mL levels, and calgranulin B is an intracellular protein. Our results demonstrate that the use of anion displacement liquid chromatofocusing chromatography may reduce the complexity of the serum proteome by separating proteins into distinct pH ranges, and facilitate the identification of low abundance proteins.

Comprehensive Proteomic Analysis of Human Cervical−Vaginal Fluid

“…Among the top 15 abundant CVF proteins, six proteins are known to be either non-native and/or lowabundance in serum (squamous cell carcinoma antigens, calgranulins A and B, small proline rich protein 3, fatty acidbinding protein epidermal, and mucin 5B). 20,21,53,54 Furthermore, proteins that are known to be in medium abundance in serum (complement factor C4, complement factor H, and apolipoprotein A-1) were found to be in low abundance in CVF. 20 Inspection of Table 1 suggests that 40% of the top 10 mostabundant proteins in the CVF are inflammatory response molecules.…”

Is protein overlap in two‐dimensional gels a serious practical problem?

“…Several approaches including narrow range IPG strips, sample fractionation methods [4][5][6], different sample preparation conditions, and modification of conditions during electrophoresis [7] can all help to reduce the complexity of a single 2-D gel and decrease spot overlap, but meaningful data are available without the need to incorporate additional steps.…”

Oncoproteomic Approaches in Lung Cancer Research

“…Disadvantages of 2D-PAGE include the separation of low abundant proteins and of membrane proteins. The use of fractioning methods or higher protein concentrations for less detectable proteins and the use of mild detergents to increase the solubility of membrane proteins may be a solution for the aforementioned issues (22,23). Other problems include co-migration of different proteins, the separation of a protein with different post-translational modifications, proteins with pI values below 4 or above 9, or the separation of very small or very large proteins.…”