Profiling without Immunoaffinity Depletion
Applied Biomics’ strategy focuses on the low-abundance, low molecular weight (LMW) proteins in blood. In general, only the LMW proteins and peptides are small enough to leak from the surrounding tissues into blood to produce diagnostic traces. Furthermore, many signaling molecules transmitted through the bloodstream are small proteins, often below 40 kDa. The LMW portion of the blood proteome is a treasure trove of diagnostic information.
In order to specifically detect LMW protein biomarkers in blood and compare their relative abundance between disease samples and normal controls, Applied Biomics optimized 2-D gel analysis by adopting three technology improvements. First, plasma/serum samples are denatured under conditions that are designed to effectively solubilize small proteins and peptides, but not the larger proteins that are over 45 kDa. Under such conditions, the LMW proteins can be efficiently focused in the isoelectric focusing (IEF) strips (GE Healthcare). High molecular weight proteins and high-abundance proteins (most of which are greater than 45 kDa) are poorly absorbed into the IEF strips.
Furthermore, high percentage (>13.5%) SDS-PAGE gels are used for the second-dimension separation, thereby maximizing resolution of the LMW proteins. This has resulted in a significantly increased number of detectable proteins at the LMW range of the plasma/serum proteome (Figure 2). Because the samples are thoroughly denatured, LMW proteins are completely dissociated from the carrier proteins so that no loss of LMW proteins will result from the treatment.
Second, 2-D differential in-gel electrophoresis (2-D DIGE), rather than regular 2-D gel electrophoresis, is used for the detection and quantification of protein differences between test and control samples. Each sample is separately labeled with a distinct fluorescent CyDye (Cy2, Cy3, or Cy5). The labeled samples are mixed together and resolved on a single 2-D gel, and the corresponding protein spot patterns are visualized by multichannel scanning of the same gel. The resulting images are analyzed both visually and by using software such as DeCyder (GE Healthcare).
Fluorescent labeling increases the detection sensitivity over standard colorimetric staining methods. More protein spots can be detected and analyzed: using the optimized protocol for LMW plasma proteins, Coomassie brilliant blue staining typically has a detection limit in the range of 300 protein spots on a 13x15 cm gel, silver staining approximately 1,200, whereas fluorescent labeling with CyDyes typically allows for the detection of more than 2,000 protein spots.
Post-electrophoretic staining with fluorescent dyes (such as SYPRO Ruby) displays a similar detection sensitivity to that of CyDye-labeling. However, post-electrophoretic processing increases the possibility of protein losses particularly in the LMW range.
Most critical is the increased confidence with 2-D DIGE that differences in spot fluorescence intensity are purely attributable to biological and not experimental variation, as realized by the complete elimination of gel-to-gel variability through using a single gel for the comparison of different plasma samples. Because every protein spot has its own internal control, small differences in protein abundance (as low as 15%) between samples can be detected with greater than 95% statistical confidence.
Third, using 2-D DIGE technology allows several plasma samples (each labeled with a different fluorescent CyDye) to be mixed and partitioned simultaneously. For example, centrifugation of the mixture in a spin-style concentrator with a defined molecular weight cut-off size can proportionally remove high molecular weight proteins from each sample. The co-partitioning process is intrinsically more reliable than partitioning in parallel, as the latter will inevitably introduce sample-to-sample variability during the partitioning steps and compromise quantitative accuracy.
Figure 3 shows an example that demonstrates the potential of these strategies for biomarker discovery. It is now possible to analyze the plasma proteome from multiple species in a potentially rapid and large-scale capacity for biomarker discovery, drug target discovery, and toxicology studies.