Quantitative Proteomics

We have established reverse phase protein arrays (RPPA) as a targeted proteomics approach. This technique yields quantitative data on changes of the proteome with high sensitivity and excellent sample capacity. We employ RPPA for the analysis of clinical samples as well as for samples derived by in vitro experimentation, mostly in the field of human breast cancer and cancer metastasis.

Reverse Phase Protein Microarrays

RPPA principle
© U Korf et al. Expert Opin. Drug Discov. (2008) 3 (2)

In RPPA, samples are printed directly on solid-phase carriers. The detection of a specific protein, or a certain phosphorylation site, is carried out with a single, highly specific antibody per slide. We have adapted this approach initially published by Paweletz et al. [1] to fluorescence detection in the near infrared (NIR) range to permit protein profiling from as little as only 20,000 cells with sensitivity in the fg-range [2, 3]. This way, less than 100 up to a few 1,000 different samples can be analyzed in parallel. Routine applications involve analyzing the activation status of signaling pathways, for example after RNAi-based silencing experiments [4], identification of protein networks regulated by miRNAs [5], profiling of tumor biopsy samples [6, 7], and the characterization of drug resistance mechanisms [8]. We have also contributed to advancing the RPPA technology by introducing the "RPPanalyzer" as tool for data analysis [9] as well as protocols for antibody validation [10] and automated cell culture experimentation [8].

Protocols for RPPA as well as for other protein microarray applications are published in "Protein microarrays".

The 4th Global RPPA workshop will take place October 24-25, 2014 in Paris, France.

[1] Paweletz CP, Charboneau L, Bichsel VE, Simone NL, Chen T, Gillespie JW, et al. Reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front. Oncogene 2001, 20,1981-9.

[2] Loebke C et al, Infrared-based protein detection arrays for quantitative proteomics. Proteomics 2007, 7, 558-564.

[3] Korf U, Loebke C, Sultmann H, Poustka A. Infrared-based protein detection arrays for quantitative proteomics. Expert Opin Drug Discov. 2008, 3, 273-283.

[4] Sahin O et al, Combinatorial RNAi strategy: The next generation of quantitative protein network analysis. Proc Natl Acad Sci U S A 2007, 104, 6579-6584. 

[5] Uhlmann S, Mannsperger H, Zhang JD, Horvat EA, Schmidt C, Küblbeck M, Henjes F, Ward A, Tschulena U, Zweig K, Korf U, Wiemann S & Sahin Ö (2012). Global microRNA level regulation of EGFR-driven cell-cycle protein network in breast cancer. Mol. Syst. Biol. 8, 570.

[6] Haller F et al, Loss of chromosome 9p triggers inactivation of retinoblastoma protein RB in gastrointestinal stromal tumors (GISTs) as cause of increased E2F1-dependent gene transcription and amplified cell proliferation. J. Pathol. 2008, 213, 253-62.

[7] Haller F et al, Increased KIT signalling with up-regulation of cyclin D correlates to accelerated proliferation and shorter disease-free survival in gastrointestinal stromal tumours (GISTs) with KIT exon 11 deletions. J Pathol. 2008, 216, 225-35.

[8] Henjes F, Bender C, von der Heyde S, Braun L, Mannsperger HA, Schmidt C, Wiemann S, Hasmann M, Aulmann S, Beissbarth T, Korf U (2012). Drugging EGFR is required for targeted therapies of ERBB2 positive breast cancer with high level expression of EGFR. Oncogenesis 2012, 1, e16.

[9] Mannsperger H, Gade S, Beissbarth T, Korf U. RPPanalyzer: Analysis of reverse phase protein array data. Bioinformatics 2010, 26, 2202-3.

[10] Mannsperger HA, Uhlmann S, Wiemann S, Sahin O, Korf U. RNAi-based validation of antibodies reverse phase protein arrays. Proteome Sci. 2010, 8, 69.

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