Targeted protein-omic methods are bridging the gap between proteomic and hypothesis-driven protein analysis approaches

Abstract

While proteomic methods have illuminated many areas of biological protein space, many fundamental questions remain with regard to systems-level relationships between mRNAs, proteins and cell behaviors. While mass spectrometric methods offer a panoramic picture of the relative expression and modification of large numbers of proteins, they are neither optimal for the analysis of predefined targets across large numbers of samples nor for assessing differences in proteins between individual cells or cell compartments. Conversely, traditional antibody-based methods are effective at sensitively analyzing small numbers of proteins across small numbers of conditions, and can be used to analyze relative differences in protein abundance and modification between cells and cell compartments. However, traditional antibody-based approaches are not optimal for analyzing large numbers of protein abundances and modifications across many samples. In this article, we will review recent advances in methodologies and philosophies behind several microarray-based, intermediate-level, 'protein-omic' methods, including a focus on reverse-phase lysate arrays and micro-western arrays, which have been helpful for bridging gaps between large- and small-scale protein analysis approaches and have provided insight into the roles that protein systems play in several biological processes.

Figure 1

Dramatized illustration of experimental setup for hypothesis-driven, “protein-omic” and proteomic approaches. (Top) Each arrow indicates an experimental condition, which may represent the stimulation, or inhibition, of components of a biological system. Grey circles indicate a protein modification measured. (Bottom) Resulting data matrix from the experimental set up. Each column represents an experimental condition and the length of the column indicates the richness of measured protein data space.

Figure 2

Four types of targeted protein-omic platforms are indicated: (a) forward phase arrays, where samples are labeled and applied to immobilized antibodies specific to certain proteins on nitrocellulose slides; (b) sandwich arrays, where captured proteins are detected by a second labeled antibody targeted to that protein; (c) reverse phase lysate arrays, where complex mixtures of proteins are printed and immobilized onto slides and then specific proteins detected with a primary antibody; and (d) micro-western arrays, where samples are printed, electrophoresed to separate proteins based on size, then transferred to nitrocellulose, and then specific proteins detected with primary antibodies. A near infrared dye-labeled secondary antibody can be used to generate fluorescent signal in (c) and (d) when used with the LI-COR Odyssey near infrared scanner. Blue ellipses denote the targeted protein of interest while green squares denote proteins that non-specifically cross-react with antibodies. The filled circles underneath each array represent the strength of each platform with respect to each criterion on the left.