Protein microarray technology and ultraviolet crosslinking combined with mass spectrometry for the analysis of protein–DNA interactions

Kersten, Birgit; Possling, Alexandra; Blaesing, Franca; Mirgorodskaya, Ekaterina; Gobom, Johan; Seitz, Harald

doi:10.1016/j.ab.2004.05.008

Cited by 32 publications

(20 citation statements)

References 37 publications

(60 reference statements)

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“…ACS, the rate-limiting enzyme of ethylene biosynthesis, has been described very recently as the first plant MAPK substrate in vivo (58). Liu et al (58) found that selected isoforms of ACS including ACS-6, which we identified in our study (Table II, number 16), are substrates of MPK6. Phosphorylation of ACS led to the accumulation of the protein and to ethylene production (58).…”

Section: Discussionsupporting

confidence: 60%

“…Analytical protein microarrays consisting of antibodies or protein antigens are increasingly used to profile proteins or antibodies, respectively, in crude protein samples of interest (6,12). Using functional protein microarrays, proteins can be directly screened in vitro for a large variety of activities, including protein-protein (13,14), protein-DNA (15,16), protein-lipid (13), and protein-drug interactions (17,18), under a wide range of different conditions. Furthermore previous studies have demonstrated the suitability of protein microarrays to study protein phosphorylation by kinases, however, only in a low or medium throughput manner (17,19,20).…”

Section: Molecular and Cellular Proteomics 4:1558 -1568 2005mentioning

confidence: 99%

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High Throughput Identification of Potential Arabidopsis Mitogen-activated Protein Kinases Substrates

Feilner

Hultschig

Lee

et al. 2005

Molecular & Cellular Proteomics

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Mitogen-activated protein kinase (MAPK) cascades are universal and highly conserved signal transduction modules in eucaryotes, including plants. These protein phosphorylation cascades link extracellular stimuli to a wide range of cellular responses. However, the underlying mechanisms are so far unknown as information about phosphorylation substrates of plant MAPKs is lacking. In this study we addressed the challenging task of identifying potential substrates for Arabidopsis thaliana mitogenactivated protein kinases MPK3 and MPK6, which are activated by many environmental stress factors. For this purpose, we developed a novel protein microarray-based proteomic method allowing high throughput study of protein phosphorylation. We generated protein microarrays including 1,690 Arabidopsis proteins, which were obtained from the expression of an almost nonredundant uniclone set derived from an inflorescence meristem cDNA expression library. Microarrays were incubated with MAPKs in the presence of radioactive ATP. Using a threshold-based quantification method to evaluate the microarray results, we were able to identify 48 potential substrates of MPK3 and 39 of MPK6. 26 of them are common for both kinases. One of the identified MPK6 substrates, 1-aminocyclopropane-1-carboxylic acid synthase-6, was just recently shown as the first plant MAPK substrate in vivo, demonstrating the potential of our method to identify substrates with physiological relevance. Furthermore we revealed transcription factors, transcription regulators, splicing factors, receptors, histones, and others as candidate substrates indicating that regulation in response to MAPK signaling is very complex and not restricted to the transcriptional level. Nearly all of the 48 potential MPK3 substrates were confirmed by other in vitro methods. As a whole, our approach makes it possible to shortlist candidate substrates of mitogen-activated protein kinases as well as those of other protein kinases for further analysis. Follow-up in vivo experiments are essential to evaluate their physiological relevance.

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Section: Discussionsupporting

confidence: 60%

Section: Molecular and Cellular Proteomics 4:1558 -1568 2005mentioning

confidence: 99%

High Throughput Identification of Potential Arabidopsis Mitogen-activated Protein Kinases Substrates

Feilner

Hultschig

Lee

et al. 2005

Molecular & Cellular Proteomics

Self Cite

237

203

View full text Add to dashboard Cite

show abstract

“…The possible adverse e¡ects of spot-to-spot variability, for example, has been controlled by replicate spotting of the same analytical reagent, using four or 16 replicates. 86,87 A more signi¢cant challenge to the use of protein microarrays or other highly multiplexed assays in the clinical laboratory is that conventional procedures for daily QC of quantitative assays are likely to be impracticable for many microarray assay formats. In particular, the practice of running high-and low-range controls for each analyte becomes unwieldy when hundreds of analytes are involved.…”

Section: Quality Controlmentioning

confidence: 99%

Current perspectives in protein array technology

et al. 2006

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AddressesThis article reviews post-2000 trends in the development of two-dimensional protein microarrays and nanoarrays. Progress in array manufacture, assay design and applications are considered, with an emphasis on issues surrounding the implementation of arrays in clinical diagnostics. These include the effect of factors in the pre-analytical phase (quality of the reagents, sample integrity, etc.), and those in the analytical phase that contribute to inaccuracy and imprecision of an array-based assay. Important requirements for the quality control and quality assurance of protein microarray assays as they move from the research environment into routine clinical application are also discussed.

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“…4, C and D). In FBP, the peptides RKARGT-GELTQLLNS (aa [22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] and GTIFGIYRKKSTDEP (aa 130 -147) were detected by overlay screens (Fig. 4, C and D).…”

Section: Fig 2-continuedmentioning

confidence: 99%

Identification of VCP/p97, Carboxyl Terminus of Hsp70-interacting Protein (CHIP), and Amphiphysin II Interaction Partners Using Membrane-based Human Proteome Arrays

Grelle

Kostka

Otto

et al. 2006

Molecular & Cellular Proteomics

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Proteins mediate their biological function through interactions with other proteins. Therefore, the systematic identification and characterization of protein-protein interactions have become a powerful proteomic strategy to understand protein function and comprehensive cellular regulatory networks. For the screening of valosin-containing protein, carboxyl terminus of Hsp70-interacting protein (CHIP), and amphiphysin II interaction partners, we utilized a membrane-based array technology that allows the identification of human protein-protein interactions with crude bacterial cell extracts. Many novel interaction pairs such as valosin-containing protein/autocrine motility factor receptor, CHIP/caytaxin, or amphiphysin II/DLP4 were identified and subsequently confirmed by pull-down, two-hybrid and co-immunoprecipitation experiments. In addition, assays were performed to validate the interactions functionally. CHIP e.g. was found to efficiently polyubiquitinate caytaxin in vitro, suggesting that it might influence caytaxin degradation in vivo. Using peptide arrays, we also identified the binding motifs in the proteins DLP4, XRCC4, and fructose-1,6-bisphosphatase, which are crucial for the association with the Src homology 3 domain of amphiphysin II. Together these studies indicate that our human proteome array technology permits the identification of protein-protein interactions that are functionally involved in neurodegenerative disease processes, the degradation of protein substrates, and the transport of membrane vesicles. Molecular & Cellular Proteomics 5:234 -244, 2006.As protein-protein interactions (PPIs) 1 are central to most biological processes, their systematic identification is considered a key strategy for uncovering the complex organization principles of functional cellular networks (1-3). A number of experimental and computational techniques have been developed to determine all the potential and actual PPIs in selected model organisms (4 -8) and humans (9 -11).Because protein arrays allow the parallel detection of addressable elements in a single experiment (12-18), they have been recognized as a promising technology for the identification of PPIs (19,20) or other specific biochemical activities (20 -23), and much effort has been devoted to their development in recent years. Zhu et al. (20) e.g. printed 5,800 purified yeast proteins onto coated glass slides and used them successfully in proof-of-principle experiments for the detection of novel calmodulin-and phospholipid-interacting proteins. Protein arrays were also used efficiently for identifying novel targets of protein kinases (24 -26). In principle, the production of protein arrays and their utilization for systematic large scale interaction and activity screens has proven viable. One major drawback, however, has been the necessity to use purified proteins. Protein purification is usually difficult, time-consuming, and expensive. For array-based studies, especially high throughput systematic screening (19), technologies are needed that permit...

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Protein microarray technology and ultraviolet crosslinking combined with mass spectrometry for the analysis of protein–DNA interactions

Cited by 32 publications

References 37 publications

High Throughput Identification of Potential Arabidopsis Mitogen-activated Protein Kinases Substrates

High Throughput Identification of Potential Arabidopsis Mitogen-activated Protein Kinases Substrates

Current perspectives in protein array technology

Identification of VCP/p97, Carboxyl Terminus of Hsp70-interacting Protein (CHIP), and Amphiphysin II Interaction Partners Using Membrane-based Human Proteome Arrays

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