Quantitative Proteomics
Protein microarrays are a promising experimental platform to yield quantitative data on changes of the proteome with high sensitivity and excellent sample capacity. Two technically different approaches are currently available to monitor protein abundance, and the turnover of protein phosphorylation in the microarray format. Both approaches employ signal detection in the near-infrared range (NIR).
Microspot Immunoassays for Quantitative Biology
Specific target proteins can be detected by employing a multiplexed microspot immunoassay. For each target protein two different antibodies recognizing spatially separated epitopes are required. Experimental procedures as well as tools for the statistic analysis of protein microarray signals and the presentation of time-resolved quantitative data were developed [1][2]. The specifically tailored software program "Quantpro" is available via http://www.dkfz.de/mga/quantpro. Current applications include the time-resolved quantification of intracellular regulation mediated by protein phosphorylation.
Reverse Phase Protein Microarrays
Samples can also be printed directly on the microarray. The detection of a specific protein, or a certain phosphorylation-site, is performed with a single, but highly specific antibody per slide. We have adapted this approach, also known as reverse phase array (RPPA) (Paweletz et al. 2001) 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 [3]. The capacity is limited to the analysis of up to 1,000 different samples per microarray. Routine applications involve analyzing the activation status of signaling pathways, for example after RNAi-based silencing experiments [4], to monitor time-resolved measurements [6], as well as protein profiling of tumor biopsy samples using a set of >80 RPPA validated antibodies [6, 7].
[1] Korf U et al, Quantitative protein microarrays for time-resolved measurements of protein phosphorylation. Proteomics, in press.
[2] Korf U et al, Antibody microarrays as an experimental platform for the analysis of signal transduction networks. Adv Biochem Eng Biotechnol. 2008, 110,153-75.
[3] Loebke C et al, Infrared-based protein detection arrays for quantitative proteomics. Proteomics 2007, 7, 558-564.
[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] Löbke C et al, Contact-spotting of protein microarrays coupled with spike-in of normalizer protein permits time-resolved analysis of ERBB receptor signaling. Proteomics 2008, 8, 1586-1594.
[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.
