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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.

SUBMITTER: de la Rosa Carrillo D

PROVIDER: S-EPMC6307719 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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

de la Rosa Carrillo Daniel D Sikorski Krzysztof K Khnykin Denis D Wu Weiwei W Lund-Johansen Fridtjof F

PloS one 20181227 12

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 p ...[more]

PMID: 30589857

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Project description:Acute leukemia is a disease pathologically manifested at both genomic and proteomic levels. Molecular genetic technologies are currently widely used in clinical research. In contrast, sensitive and high-throughput proteomic techniques for performing protein analyses in patient samples are still lacking. Here, we used a technology based on size exclusion chromatography followed by immunoprecipitation of target proteins with an antibody bead array (Size Exclusion Chromatography-Microsphere-based Affinity Proteomics, SEC-MAP) to detect hundreds of proteins from a single sample. In addition, we developed semi-automatic bioinformatics tools to adapt this technology for high-content proteomic screening of pediatric acute leukemia patients.To confirm the utility of SEC-MAP in leukemia immunophenotyping, we tested 31 leukemia diagnostic markers in parallel by SEC-MAP and flow cytometry. We identified 28 antibodies suitable for both techniques. Eighteen of them provided excellent quantitative correlation between SEC-MAP and flow cytometry (p< 0.05). Next, SEC-MAP was applied to examine 57 diagnostic samples from patients with acute leukemia. In this assay, we used 632 different antibodies and detected 501 targets. Of those, 47 targets were differentially expressed between at least two of the three acute leukemia subgroups. The CD markers correlated with immunophenotypic categories as expected. From non-CD markers, we found DBN1, PAX5, or PTK2 overexpressed in B-cell precursor acute lymphoblastic leukemias, LAT, SH2D1A, or STAT5A overexpressed in T-cell acute lymphoblastic leukemias, and HCK, GLUD1, or SYK overexpressed in acute myeloid leukemias. In addition, OPAL1 overexpression corresponded to ETV6-RUNX1 chromosomal translocation.In summary, we demonstrated that SEC-MAP technology is a powerful tool for detecting hundreds of proteins in clinical samples obtained from pediatric acute leukemia patients. It provides information about protein size and reveals differences in protein expression between particular leukemia subgroups. Forty-seven of SEC-MAP identified targets were validated by other conventional method in this study.

Dataset Information

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

Publications

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

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