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

Stuchlý, Jan; Kanderová, Veronika; Fišer, Karel; Cerna, Daniela; Holm, Anders; Wu, Weiwei; Hrušák, Ondřej; Lund-Johansen, Fridtjof; Kalina, Tomáš

doi:10.1002/cyto.a.22011

Cited by 13 publications

(15 citation statements)

References 39 publications

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“…A minimum of 50,000 microspheres were acquired from each well. The data were exported in FCS 3.0 from FACS Diva software (BD Biosciences) and further processed in the R-project environment as described previously (17).…”

Section: Methodsmentioning

confidence: 99%

“…Computational Analyses and Statistics-All computations and graph visualizations were performed in R-project/Bioconductor [packages "cluster", "flowCore", "Matrix", "igraph", "rggobi", "reshape", "ggplot2", wmtsa, available at: http://www.r-project.org/ or http://bioconductor.org/] as described earlier (17). SEC-MAP and FACS data were compared by using Pearson correlations, and p Ͻ 0.05 was considered to be statistically significant.…”

Section: Methodsmentioning

confidence: 99%

“…Both parts were separated by size exclusion chromatography (SEC) to 24 "size" fractions; 632 antibodies were used for the detection of 501 different markers in each fraction (more antibody clones against single protein were used when available). We built on our previous tools that were designed to batch-analyze the flow cytometry standard (FCS) data and generate size distribution profiles for the antibody targets (17). Additional software tool functionalities have now been developed to allow for high-content data normalization, semi-automated analysis of size distribution profiles, and differentially expressed entity discovery in multiple samples.…”

Section: Protein Entities Can Be Defined In a High-throughput Manner-mentioning

confidence: 99%

“…SEC-MAP enables the detection of hundreds of proteins in a single sample and provides essential information about protein size. Because only five to ten million cells are necessary, SEC-MAP can serve as a sensitive, sample-sparing and high-content tool for protein profiling in leukemia samples probing the relative amounts of different proteins, as well as protein size and cleavage (17). Our in-house-assembled MAP array is a set of 1152 populations of fluorescent-labeled microbeads, each carrying an antibody against a single human antigen.…”

mentioning

confidence: 99%

“…Data are analyzed with in-house R-based software. This approach permits automatic batch processing of raw flow cytometry standard (FCS) files in addition to advanced analyses including quality control steps (the minimal number of microspheres required in a population and the unimodality of the signal in the PE channel is checked) (17). We wanted to find out whether SEC-MAP can be used in the clinical laboratory to bring a biologically important information, e.g.…”

mentioning

confidence: 99%

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High-resolution Antibody Array Analysis of Childhood Acute Leukemia Cells

Kanderová

Kužílková

Stuchlý

et al. 2016

Molecular & Cellular Proteomics

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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. Acute leukemia (AL)1 is the most common childhood cancer, accounting for a quarter of all pediatric malignancies (1). Accumulated chromosomal translocations and mutations in proto-oncogenes alter proliferation, differentiation, apoptosis and death in developing hematogones, ultimately leading to the development of leukemia (2, 3). The most recent understanding of these cancer-related changes is based on molecular genetic studies that focused primarily on DNA and mRNA alterations. High-throughput molecular genetic technologies, such as mRNA expression profiling and next generation sequencing, are widely used in clinical research. These techniques can provide new classification schemes, define new prognostic subgroups and outline the background of some pathological mechanisms (2, 4, 5, 6, 7) but they cannot easily elucidate the functional consequences at the cellular level. Proteins are the principal carriers of cellular functions. Thus, From the ‡CLIP -Childhood Leukaemia Investigation Prague, Department of Paediatric Haematology and Oncology, 2

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Protein Entities Can Be Defined In a High-throughput Manner-mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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High-resolution Antibody Array Analysis of Childhood Acute Leukemia Cells

Kanderová

Kužílková

Stuchlý

et al. 2016

Molecular & Cellular Proteomics

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Precisely Encoded Barcodes Using Tetrapod CdSe/CdS Quantum Dots with a Large Stokes Shift for Multiplexed Detection

Gao

et al. 2019

Adv Funct Materials

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A serious obstacle to the construction of high‐capacity optical barcodes in suspension array technology is energy transfer, which can prompt unpredictable barcode signals, limited barcode numbers, and the need for an unfeasible number of experimental iterations. This work reports an effective and simple way to eliminate energy transfer in multicolor quantum dots (QDs)‐encoded microbeads by incorporating tetrapod CdSe/CdS QDs with a large Stokes shift (about 180 nm). Exploiting this unique feature enables the facile realization of a theoretical 7 × 7‐1 barcoding matrix combining two colors and seven intensity levels. As such, microbeads containing tetrapod CdSe/CdS QDs are demonstrated to possess a powerful encoding capacity which allows for precise barcode design. The ability of the Shirasu porous glass membrane emulsification method to easily control microbead size facilitates the establishment of a 3D barcode library of 144 distinguishable barcodes, indicating the enormous potential to enable large‐scale multiplexed detection. Moreover, when applied for the multiplexed detection of five common allergens, these barcodes exhibit superior detection performance (limit of detection: 0.01–0.02 IU mL−1) for both spiked and patient serum samples. Therefore, this new coding strategy helps to expand barcoding capacity while simultaneously reducing the technical and economic barriers to the optical encoding of microbeads for high‐throughput multiplexed detection.

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