Detection of tyrosine phosphorylated proteins by combination of immunoaffinity enrichment, two-dimensional difference gel electrophoresis and fluorescent Western blotting

Lind, Sara Bergström; Hagner-McWhirter, Åsa; Elfineh, Lioudmila; Molin, Magnus; Jorsback, Anneli; Öhman, Johan; Pettersson, Ulf

doi:10.1016/j.bbrc.2010.09.104

Cited by 8 publications

(4 citation statements)

References 15 publications

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“…Compared to Figure 2A when only the 4G10 antibody was used to visualize all pTyr proteins, fewer signals were observed when the 4G10 was combined with another antibody for detection of a specific phosphorylated protein. Imatinib treatment of K562 cells is a well characterized model system [16,17] and it was chosen since reference data was available. Data from a complementary technique is very valuable when implementing novel approaches as this paper describes.…”

Section: Resultsmentioning

confidence: 99%

Tyrosine phosphorylation profiling via in situproximity ligation assay

et al. 2014

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BackgroundTyrosine phosphorylation (pTyr) is an important cancer relevant posttranslational modification since it regulates protein activity and cellular localization. By controlling cell growth and differentiation it plays an important role in tumor development. This paper describes a novel approach for detection and visualization of a panel of pTyr proteins in tumors using in situ proximity ligation assay.MethodsK562 leukemia cells were treated with tyrosine kinase and/or phosphatase inhibitors to induce differences in pTyr levels and mimic cells with different malignant properties. Cells were then probed with one antibody against the pTyr modification and another probe against the detected protein, resulting in a detectable fluorescent signal once the probes were in proximity.ResultsTotal and protein specific pTyr levels on ABL, SHC, ERK2 and PI3K proteins were detected and samples of control and treated cells were distinguished at the pTyr level using this novel approach. Promising results were also detected for formalin fixed and paraffin embedded cells in the micro array format.ConclusionsThis application of in situ proximity ligation assay is valuable in order to study the pTyr modification of a panel of proteins in large data sets to validate mass spectrometric data and to be combined with tissue microarrays. The approach offers new opportunities to reveal the pTyr signatures in cells of different malignant properties that can be used as biomarker of disease in the future.

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Section: Resultsmentioning

confidence: 99%

Tyrosine phosphorylation profiling via in situproximity ligation assay

et al. 2014

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“…Using MS‐based approaches, thousands of phosphorylations can be monitored in a sample, [ 22,23 ] but enrichment steps are needed during sample preparation to separate off high abundant native peptides that mask the detection of modified peptides. [ 24–26 ] A variety of different enrichment strategies are used, [ 24,27,28 ] and among them, metal oxides such as titanium dioxide (TiO 2 ) and zirconium dioxide are preferred due to their great tolerance to buffers and detergents. The combination of these enrichment procedures with sensitive and high‐resolution MS enables the identification and characterization of a high number of proteins and modifications simultaneously.…”

Section: Introductionmentioning

confidence: 99%

Phosphorylation Time‐Course Study of the Response during Adenovirus Type 2 Infection

et al. 2020

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PTMs such as phosphorylations are usually involved in signal transduction pathways. To investigate the temporal dynamics of phosphoproteome changes upon viral infection, a model system of IMR‐90 cells infected with human adenovirus type 2 (Ad2) is used in a time‐course quantitative analysis combining titanium dioxide (TiO2) particle enrichment and SILAC‐MS. Quantitative data from 1552 phosphorylated sites clustered the highly altered phosphorylated sites to the signaling by rho family GTPases, the actin cytoskeleton signaling, and the cAMP‐dependent protein kinase A signaling pathways. Their activation is especially pronounced at early time post‐infection. Changes of several phosphorylated sites involved in the glycolysis pathway, related to the activation of the Warburg effect, point at virus‐induced energy production. For Ad2 proteins, 32 novel phosphorylation sites are identified and as many as 52 phosphorylated sites on 17 different Ad2 proteins are quantified, most of them at late time post‐infection. Kinase predictions highlighted activation of PKA, CDK1/2, MAPK, and CKII. Overlaps of kinase motif sequences for viral and human proteins are observed, stressing the importance of phosphorylation during Ad2 infection.

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“…Some authors used chemifluorescence to reveal the antigenic map [ 55 , 63 , 64 ]. By contrast, others have used fluorescent probes for protein labeling before the electrophoresis steps [ 65 – 67 ]. Goeb et al [ 59 ] use a fluorescent dye for staining a landmark map in order to confirm the localization of antigenic spots as potential autoimmune targets.…”

Section: Discussionmentioning

confidence: 99%

An Optimized Fluorescence-Based Bidimensional Immunoproteomic Approach for Accurate Screening of Autoantibodies

et al. 2015

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Serological proteome analysis (SERPA) combines classical proteomic technology with effective separation of cellular protein extracts on two-dimensional gel electrophoresis, western blotting, and identification of the antigenic spot of interest by mass spectrometry. A critical point is related to the antigenic target characterization by mass spectrometry, which depends on the accuracy of the matching of antigenic reactivities on the protein spots during the 2D immunoproteomic procedures. The superimposition, based essentially on visual criteria of antigenic and protein spots, remains the major limitation of SERPA. The introduction of fluorescent dyes in proteomic strategies, commonly known as 2D-DIGE (differential in-gel electrophoresis), has boosted the qualitative capabilities of 2D electrophoresis. Based on this 2D-DIGE strategy, we have improved the conventional SERPA by developing a new and entirely fluorescence-based bi-dimensional immunoproteomic (FBIP) analysis, performed with three fluorescent dyes. To optimize the alignment of the different antigenic maps, we introduced a landmark map composed of a combination of specific antibodies. This methodological development allows simultaneous revelation of the antigenic, landmark and proteomic maps on each immunoblot. A computer-assisted process using commercially available software automatically leads to the superimposition of the different maps, ensuring accurate localization of antigenic spots of interest.

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Detection of tyrosine phosphorylated proteins by combination of immunoaffinity enrichment, two-dimensional difference gel electrophoresis and fluorescent Western blotting

Cited by 8 publications

References 15 publications

Tyrosine phosphorylation profiling via in situproximity ligation assay

Tyrosine phosphorylation profiling via in situproximity ligation assay

Phosphorylation Time‐Course Study of the Response during Adenovirus Type 2 Infection

An Optimized Fluorescence-Based Bidimensional Immunoproteomic Approach for Accurate Screening of Autoantibodies

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