Protease‐resistant streptavidin for interaction proteomics

Rafiee, Mahmoud-Reza; Sigismondo, Gianluca; Kalxdorf, Mathias; Förster, Laura; Brügger, Britta; Béthune, Julien; Krijgsveld, Jeroen

doi:10.15252/msb.20199370

Cited by 52 publications

(65 citation statements)

References 32 publications

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“…Another volume of BW buffer was added, samples were transferred to tubes, incubated for 5 min in 70 °C, then left it in RT for 15 min to slowly cool down. Samples were transferred to 15 mL tubes, mixed with BW buffer to the final volume of 10 mL and 100 µL of streptavidin beads (New England Biolabs, Ipswich, MA, USA; pre-prepared by blocking for the MS experiment, as described in [ 37 ]) was added to each of the tubes. Samples were incubated in RT overnight on a rotator wheel.…”

Section: Methodsmentioning

confidence: 99%

Chromatin-Directed Proteomics Identifies ZNF84 as a p53-Independent Regulator of p21 in Genotoxic Stress Response

Strzeszewska-Potyrała

Staniak

Czarnecka‐Herok

et al. 2021

Cancers

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The p21WAF1/Cip1 protein, encoded by CDKN1A, plays a vital role in senescence, and its transcriptional control by the tumour suppressor p53 is well-established. However, p21 can also be regulated in a p53-independent manner, by mechanisms that still remain less understood. We aimed to expand the knowledge about p53-independent senescence by looking for novel players involved in CDKN1A regulation. We used a chromatin-directed proteomic approach and identified ZNF84 as a novel regulator of p21 in various p53-deficient cell lines treated with cytostatic dose of doxorubicin. Knock-down of ZNF84, an as-yet un-characterized protein, inhibited p21 gene and protein expression in response to doxorubicin, it attenuated senescence and was associated with enhanced proliferation, indicating that ZNF84-deficiency can favor senescence bypass. ZNF84 deficiency was also associated with transcriptomic changes in genes governing various cancer-relevant processes e.g., mitosis. In cells with ZNF84 knock-down we discovered significantly lower level of H2AX Ser139 phosphorylation (γH2AX), which is triggered by DNA double strand breaks. Intriguingly, we observed a reverse correlation between the level of ZNF84 expression and survival rate of colon cancer patients. In conclusion, ZNF84, whose function was previously not recognized, was identified here as a critical p53-independent regulator of senescence, opening possibilities for its targeting in novel therapies of p53-null cancers.

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Section: Methodsmentioning

confidence: 99%

Chromatin-Directed Proteomics Identifies ZNF84 as a p53-Independent Regulator of p21 in Genotoxic Stress Response

Strzeszewska-Potyrała

Staniak

Czarnecka‐Herok

et al. 2021

Cancers

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“…PDB was then performed upon addition of exogenous biotin for 10 min (ultraID fusions) or 24h (BioID fusions) to four biological replicate samples. The resulting biotinylated proteins were processed for MS analysis after their capture with trypsin-resistant streptavidin beads 17 and on-beads trypsin digestion (Figure 7A). Peptide identification was performed with the MaxQuant software 18 and the relative enrichment of each protein across samples was quantified by label-free quantification (LFQ) 19 using the proDA (probabilistic drop out analysis) package to infer for each protein its mean LFQ intensity and associated variance, taking into account the missing values 20 .…”

Section: Ultraid Supports Pdb-ms Within a 10 Min Timeframe In Authentic Experimental Conditionsmentioning

confidence: 99%

“…High performance streptavidin sepharose beads (Cytiva, #17-5113-01, Lot 10280314) were chemically modified to yield resistance against tryptic digestion as described in ref. 17 . 40 μL modified streptavidin-sepharose beads equilibrated in RIPA buffer were used per pulldown.…”

Section: Streptavidin Affinity Pulldowns and Sample Preparation For Ms Analysismentioning

confidence: 99%

ultraID: a compact and efficient enzyme for proximity-dependent biotinylation in living cells

Zhao

Bitsch

Kubitz

et al. 2021

Preprint

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Proximity-dependent biotinylation (PDB) combined with mass spectrometry analysis has established itself as a key technology to study protein-protein interactions in living cells. A widespread approach, BioID, uses an abortive variant of the E. coli BirA biotin protein ligase, a quite bulky enzyme with slow labeling kinetics. To improve PDB versatility and speed, various enzymes have been developed by different approaches. Here we present a novel small-size engineered enzyme: ultraID. We show its practical use to probe the interactome of Argonaute-2 after a 10 min labeling pulse and expression at physiological levels. Moreover, using ultraID, we provide a membrane-associated interactome of coatomer, the coat protein complex of COPI vesicles. To date, ultraID is the smallest and most efficient biotin ligase available for PDB and offers the possibility of investigating interactomes at a high temporal resolution.

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“…Trapped proteins are typically digested in situ for subsequent MS analysis. To reduce cross contamination, a protease‑resistant streptavidin has recently been developed (Rafiee et al, 2020). BioID, a promiscuous biotin ligase fusion (Roux, Kim, Raida, & Burke, 2012), has been developed to label neighboring proteins in cells (proximity labeling) with biotin for interactomics studies.…”

Section: Key Modes Of Separationmentioning

confidence: 99%

The separation sciences, the front end to proteomics: An historical perspective

Nice

2020

Biomedical Chromatography

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It is now over 25 years since the term proteomics (analysis of the entire protein complement of a cell, tissue, or organism under a specific, defined set of conditions) was originally coined. Since then, the field has advanced rapidly and there are now more than 135,500 publications addressing the field. With current instrumentation it is possible to detect over 10,000 protein forms in a single experiment. The separation of proteins and peptides has been a key component of many of these studies for both sample concentration and enrichment and to reduce the complexity of the samples under analysis, allowing deeper mining of the individual proteomes. In this review, the roles of chromatography, electrophoresis, and other allied techniques in the advancement of the field will be investigated. Key technologies will be presented, and examples given of their application showing how the field has now advanced to a stage where it is enhancing our understanding of the human biology underlying health and disease, and clinical translation, supporting precision/personalized medicine, is now feasible. Clearly the separation sciences have played a key role in many of these advances.

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Protease‐resistant streptavidin for interaction proteomics

Cited by 52 publications

References 32 publications

Chromatin-Directed Proteomics Identifies ZNF84 as a p53-Independent Regulator of p21 in Genotoxic Stress Response

Chromatin-Directed Proteomics Identifies ZNF84 as a p53-Independent Regulator of p21 in Genotoxic Stress Response

ultraID: a compact and efficient enzyme for proximity-dependent biotinylation in living cells

The separation sciences, the front end to proteomics: An historical perspective

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