Antibody Array Analysis with Label-based Detection and Resolution of Protein Size

Abstract

Elements from DNA microarray analysis, such as sample labeling and micro-spotting of capture reagents, have been successfully adapted to multiplex measurements of soluble cytokines. Application in cell biology is hampered by the lack of mono-specific antibodies and the fact that many proteins occur in complexes. Here, we incorporated a principle from Western blotting and resolved protein size as an additional parameter. Proteins from different cellular compartments were labeled and separated by size exclusion chromatography into 20 fractions. All were analyzed with replicate antibody arrays. The elution profiles of all antibody targets were compiled to color maps that resemble Western blots with bands of antibody reactivity across the size separation range (670 -10 kDa). A new solid phase designed for processing in microwell plates was developed to handle the large number of samples. Antibodies were bound to protein G-coupled microspheres surface-labeled with 300 combinations of four fluorescent dyes. Fluorescence from particle color codes and the protein label were measured by high-speed flow cytometry. Cytoplasmic protein kinases were detected as bands near predictable elution points. For proteins with atypical elution characteristics or multiple contexts, two or more antibodies were used as internal references of specificity. Membrane proteins eluted near the void volume, and additional bands corresponding to intracellular forms were detected for several targets. Elution profiles of cyclin-dependent kinases (cdks), cyclins, and cyclindependent kinase inhibitors, were compatible with their occurrence in complexes that vary with the cell cycle phase and subcellular localization. A two-dimensional platform circumvents the need for mono-specific capture antibodies and extends the utility of antibody array analysis to studies of protein complexes. Molecular & Cellular Proteomics 8:245-257, 2009.

An automated analysis of highly complex flow cytometry‐based proteomic data

“…The reactivity peaks on the line plots parallel bands on western blots. Typically, a protein is present as 1-3 protein entities (16,17). Our aim is to analyze Size-MAP proteomic data at the protein entity level.…”

“…The cells were seeded onto 24-well plates at a density of 10 3 10 6 cells/well in the presence or absence of imatinib mesylate (10 lM) or solvent alone. After 24 h at 378C under 5% CO 2 , the cells were washed twice in ice-cold PBS and processed as previously described (16).…”

“…Suspension microsphere-based arrays were produced using maleimide-derivatives of fluorescent dyes (Invitrogen, Carlsbad, CA) as described previously (16,17), with more extensive color-coding: two levels of Alexa Fluor 750 (Ax750), five levels of Alexa Fluor 488 (Ax488), and Alexa Fluor 647 (Ax647) and four levels of Pacific blue (PacB) and Pacific Orange (PacO) labeling intensities were produced. Different capture antibodies were attached to 1,006 out of 1,152 colorcoded microspheres, and the full spectrum of microspheres was mixed to form a microsphere array and stored frozen.…”

“…The principles of size exclusion chromatography-resolved microsphere-based affinity proteomics (Size-MAP) recently introduced by Wu et al (16) and Holm et al (17) are based on whole-cell lysis, biotinylation of cell lysates, size-exclusion chromatography, and microsphere-based protein array analysis via specific antibodies bound to color-coded microspheres. Size-MAP is a novel concept that overcomes several critical constraints of antibody arrays that have been developed thus far: its combination with size exclusion chromatography provides information about protein sizes and the presence of protein complexes that resembles a multiplexed western blot.…”

Lysate Microarrays Enable High-throughput, Quantitative Investigations of Cellular Signaling

“…Several different immunoaffinity-based methods have been used in systems-level studies to determine the amounts, subcellular locations, and PTM levels of proteins in complex biological samples. Antibody-based technologies that are compatible with multiplexing include flow cytometry (1), microsphere-based assays (2,3), immunocytochemistry coupled with automated microscopy (4), miniaturized Western blotting (5), and antibody microarray-based methods such as direct-detection microarrays (6,7), sandwich-style microarrays (8 -11), and "reverse-phase" or lysate microarrays (12,13). Compared with their low-throughput counterparts, highthroughput technologies are often constrained by smaller sample sizes, lack of separation steps, and an inability to tailor the assay to each antibody.…”