Tandem affinity purification of functional TAP-tagged proteins from human cells

Gregáň, Juraj; Riedel, Christian G.; Petronczki, Mark; Čipák, Luboš; Rumpf, Cornelia; Poser, Ina; Buchholz, Frank; Mechtler, Karl; Nasmyth, Kim

doi:10.1038/nprot.2007.172

Cited by 59 publications

(46 citation statements)

References 24 publications

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“…The AP-MS samples of primary T cells were analyzed using a slightly different methodological approach. Proteins were precipitated using a methanol-chloroform procedure (29) and were digested tryptically, as previously described (30). Peptides were separated and analyzed in an LTQ Velos ion trap mass spectrometer (Thermo Fisher, Bremen, Germany).…”

Section: Mass Spectrometrymentioning

confidence: 99%

Association of CD147 and Calcium Exporter PMCA4 Uncouples IL-2 Expression from Early TCR Signaling

Supper

Schiller

Paster

et al. 2016

The Journal of Immunology

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The Ig superfamily member CD147 is upregulated following T cell activation and was shown to serve as a negative regulator of T cell proliferation. Thus, Abs targeting CD147 are being tested as new treatment strategies for cancer and autoimmune diseases. How CD147 mediates immunosuppression and whether association with other coreceptor complexes is needed have remained unknown. In the current study, we show that silencing of CD147 in human T cells increases IL-2 production without affecting the TCR proximal signaling components. We mapped the immunosuppressive moieties of CD147 to its transmembrane domain and Ig-like domain II. Using affinity purification combined with mass spectrometry, we determined the domain specificity of CD147 interaction partners and identified the calcium exporter plasma membrane calcium ATPase isoform 4 (PMCA4) as the interaction partner of the immunosuppressive moieties of CD147. CD147 does not control the proper membrane localization of PMCA4, but PMCA4 is essential for the CD147-dependent inhibition of IL-2 expression via a calcium-independent mechanism. In summary, our data show that CD147 interacts via its immunomodulatory domains with PMCA4 to bypass TCR proximal signaling and inhibit IL-2 expression

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Section: Mass Spectrometrymentioning

confidence: 99%

Association of CD147 and Calcium Exporter PMCA4 Uncouples IL-2 Expression from Early TCR Signaling

Supper

Schiller

Paster

et al. 2016

The Journal of Immunology

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“…Human protein complexes were isolated from HeLa cells by immunoaffinity chromatography basically as described by Gregan et al 42 Briefly, proteins eluted from affinity beads with 50-200 µL of the buffer containing 100 mM glycine in 100 mM Tris, pH 8.0, were digested insolution with trypsin at the enzyme concentration of 16 ng/µL at 39 °C overnight and tryptic peptides concentrated off-line on a UltraMicroSpin-C18 (The Nest Group, Southborough MA) cartridge. 43 …”

Section: Protein Samplesmentioning

confidence: 99%

Separating the Wheat from the Chaff: Unbiased Filtering of Background Tandem Mass Spectra Improves Protein Identification

et al. 2008

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Only a small fraction of spectra acquired in LC-MS/MS runs matches peptides from target proteins upon database searches. The remaining, operationally termed background, spectra originate from a variety of poorly controlled sources and affect the throughput and confidence of database searches. Here, we report an algorithm and its software implementation that rapidly removes background spectra, regardless of their precise origin. The method estimates the dissimilarity distance between screened MS/MS spectra and unannotated spectra from a partially redundant background library compiled from several control and blank runs. Filtering MS/MS queries enhanced the protein identification capacity when searches lacked spectrum to sequence matching specificity. In sequencesimilarity searches it reduced by, on average, 30-fold the number of orphan hits, which were not explicitly related to background protein contaminants and required manual validation. Removing high quality background MS/MS spectra, while preserving in the data set the genuine spectra from target proteins, decreased the false positive rate of stringent database searches and improved the identification of low-abundance proteins.

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“…We are presently utilizing tandem affinity purification (TAP) and mass spectrometry technology platform to probe the components of interacting proteins associated with DRR1. TAP method is a useful tool that allows rapid purification of native protein complexes under their natural level (33)(34)(35)(36)(37). Such investigation will be informative to elucidate the function of DRR1 in regulating cell cycle.…”

Section: Discussionmentioning

confidence: 99%

Induction of tumor inhibition and apoptosis by a candidate tumor suppressor gene DRR1 on 3p21.1

Liu

Zhao²,

Bai³

et al. 2009

Oncol Rep

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Abstract. Down-regulated in renal cell carcinoma gene (DRR1) is one of the candidate tumor suppressor genes (TSGs) on human 3p21.1. This study was performed to validate the expression status of DRR1 gene in cancer cells and the expression pattern of the protein in clinical specimens of human lung cancer and to examine its potential as a molecular target for treatment of lung cancer in vivo. DRR1 expression was analyzed in 7 human lung cancer cell lines. DRR1 protein expression was also examined in clinical nonsmall cell lung cancer (NSCLC) specimens. Furthermore, effects of DRR1 re-expression on A549 cells in vitro and A549 xenograft tumors in nude mice were evaluated. Loss of DRR1 mRNA expression was detected in 6 of the 7 human cancer cell lines, the exception was the renal cancer cell line OS-RC-2. DRR1 protein expression was absent in 15 of 20 (75%) human NSCLC specimens by immunostaining. Transfection of DRR1 gene into DRR1-negative-expressing A549 cells resulted in significant cell growth suppression and apoptosis. Plasmids containing DRR1 cDNA complexed with DOTAP:Chol liposomes were administered intravenously via tail vein to nude mice bearing A549 xenograft tumors resulting in tumor growth inhibition and elevation of apoptosis compared with the controls. DRR1 is a potent growth suppressor of NSCLC, acting through apoptosis pathway in vivo and it may be a potential therapeutic gene for human lung cancer.

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Tandem affinity purification of functional TAP-tagged proteins from human cells

Cited by 59 publications

References 24 publications

Association of CD147 and Calcium Exporter PMCA4 Uncouples IL-2 Expression from Early TCR Signaling

Association of CD147 and Calcium Exporter PMCA4 Uncouples IL-2 Expression from Early TCR Signaling

Separating the Wheat from the Chaff: Unbiased Filtering of Background Tandem Mass Spectra Improves Protein Identification

Induction of tumor inhibition and apoptosis by a candidate tumor suppressor gene DRR1 on 3p21.1

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