High-resolution antibody array analysis of proteins from primary human keratinocytes and leukocytes

Abstract

Antibody array analysis of labeled proteomes has high throughput and is simple to perform, but validation remains challenging. Here, we used differential detergent fractionation and size exclusion chromatography in sequence for high-resolution separation of biotinylated proteins from human primary keratinocytes and leukocytes. Ninety-six sample fractions from each cell type were analyzed with microsphere-based antibody arrays and flow cytometry (microsphere affinity proteomics; MAP). Monomeric proteins and multi-molecular complexes in the cytosol, cytoplasmic organelles, membranes and nuclei were resolved as discrete peaks of antibody reactivity across the fractions. The fractionation also provided a two-dimensional matrix for assessment of specificity. Thus, antibody reactivity peaks were considered to represent specific binding if the position in the matrix was in agreement with published information about i) subcellular location, ii) size of the intended target, and iii) cell type-dependent variation in protein expression. Similarities in the reactivity patterns of either different antibodies to the same protein or antibodies to similar proteins were used as additional supporting evidence. This approach provided validation of several hundred proteins and identification of monomeric proteins and protein complexes. High-resolution MAP solves many of the problems associated with obtaining specificity with immobilized antibodies and a protein label. Thus, laboratories with access to chromatography and flow cytometry can perform large-scale protein analysis on a daily basis. This opens new possibilities for cell biology research in dermatology and validation of antibodies.

Spatial-proteomics reveals phospho-signaling dynamics at subcellular resolution

“…1a ). Six extraction buffers with different detergent, salt, and chemical composition (see Methods) 23 were used sequentially to profile six distinct subcellular fractions in cell lines and tissues at both proteome and phospho-proteome level. While detergents with different solubilizing capacity have been applied earlier for chemical subcellular fractionation, an important observation made here is that better partitioning can be achieved by also varying the ionic composition of the extraction buffers.…”

Spatial-proteomics revealin-vivophospho-signaling dynamics at subcellular resolution

“…1A). Six extraction buffers with different detergent, salt and chemical composition (see Methods) 16 , were used sequentially to profile six distinct subcellular fractions in cell lines and tissues at both proteome and phospho-proteome level. In contrast to already published MS-based subcellular fractionation methods, our approach can be employed in a high-throughput manner.…”

The Antibody Society’s antibody validation webinar series

“…Like specificity (ability to correctly detect the target epitope), selectivity (ability to differentiate from similar epitopes) can also depend on the method of IgG purification (see Manufacturing specifics section below), on the choice of antigen used to generate or screen for the antibody, and on the degree of denaturation of the target protein in the assay being used. Multiplex bead-based antibody arrays 21 , 22 under both native (ELISA-like) and denatured (WB-like) conditions reveal that most tested commercial antibodies are neither selective (i.e., they cross-react with off-target proteins containing the target epitope), nor specific (i.e., they cross-react with off-target proteins not containing the target epitope). The designated target protein is usually present in the top five proteins detected, but is seldom the protein most strongly bound.…”