Magnetic Bead Processor for Rapid Evaluation and Optimization of Parameters for Phosphopeptide Enrichment

Ficarro, Scott B.; Adelmant, Guillaume; Tomar, Maria N.; Zhang, Yi; Cheng, Vivian; Marto, Jarrod A.

doi:10.1021/ac9004452

Cited by 133 publications

(122 citation statements)

References 22 publications

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“…Phosphopeptides were purified from approximately 1 mg each sample. The use of IMAC/C18 columns is described in the literature (Kokubu et al, 2005;Ficarro et al, 2009;Mertins et al, 2013). Fe 31 -IMAC resin was created by taking Ni-NTA Superflow agarose resin (Qiagen, Germantown, MD) and stripping out Ni by incubating with 500 mM EDTA, pH 8 (1:1 v/v), for 5 minutes, and repeating.…”

Section: Methodsmentioning

confidence: 99%

G Protein–Coupled Receptor Endocytosis Confers Uniformity in Responses to Chemically Distinct Ligands

Tsvetanova

Trester-Zedlitz²,

Newton³

et al. 2016

Mol Pharmacol

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The ability of chemically distinct ligands to produce different effects on the same G protein-coupled receptor (GPCR) has interesting therapeutic implications, but, if excessively propagated downstream, would introduce biologic noise compromising cognate ligand detection. We asked whether cells have the ability to limit the degree to which chemical diversity imposed at the ligand-GPCR interface is propagated to the downstream signal. We carried out an unbiased analysis of the integrated cellular response elicited by two chemically and pharmacodynamically diverse b-adrenoceptor agonists, isoproterenol and salmeterol. We show that both ligands generate an identical integrated response, and that this stereotyped output requires endocytosis. We further demonstrate that the endosomal b2-adrenergic receptor signal confers uniformity on the downstream response because it is highly sensitive and saturable. Based on these findings, we propose that GPCR signaling from endosomes functions as a biologic noise filter to enhance reliability of cognate ligand detection.

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Section: Methodsmentioning

confidence: 99%

G Protein–Coupled Receptor Endocytosis Confers Uniformity in Responses to Chemically Distinct Ligands

Tsvetanova

Trester-Zedlitz²,

Newton³

et al. 2016

Mol Pharmacol

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“…SCX eluates and the flow-through samples were loaded onto a precolumn using a NanoAcquity Sample Manager and UPLC pump. Peptides were gradient eluted (1-30% B in 120 min; A ϭ 0.2 M aqueous acetic acid, B ϭ 0.2 M acetic acid in acetonitrile) at a flow rate of Ͻ 50 nL/min to an analytical column as described previously (30,31). Mass spectrometry data acquisition was performed in information dependent mode on the QSTAR Elite equipped with a Digital Picoview ESI positioning platform (New Objective, Woburn, MA) using the following settings: MS scan 300 Յ m/z Յ 1500; top eight most abundant MS/MS scans using low resolution for precursor isolation; 2.0 s. accumulation with enhance all mode; 1.8 kV ESI voltage.…”

Section: Methodsmentioning

confidence: 99%

DNA Ends Alter the Molecular Composition and Localization of Ku Multicomponent Complexes

Adelmant

Calkins

Garg

et al. 2012

Molecular & Cellular Proteomics

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The Ku heterodimer plays an essential role in non-homologous end-joining and other cellular processes including transcription, telomere maintenance and apoptosis. While the function of Ku is regulated through its association with other proteins and nucleic acids, the specific composition of these macromolecular complexes and their dynamic response to endogenous and exogenous cellular stimuli are not well understood. Here we use quantitative proteomics to define the composition of Ku multicomponent complexes and demonstrate that they are dramatically altered in response to UV radiation. The Ku heterodimer (hereafter referred to as "Ku") is composed of 70 kDa (Ku70) and 86 kDa (Ku86) subunits that associate in stoichiometric quantities and bind DNA ends with high affinity as a first step in the nonhomologous end-joining DNA repair pathway. Ku binding to double strand breaks results in recruitment of the catalytic subunit of DNA-dependent protein kinase DNA-PKcs to form the holo-enzyme DNA-PK, followed by the exonuclease Artemis and the XRCC4-like factor XLF (1). The final stage of repair requires polymerase activity and the ligation of the DNA ends by a complex composed of the x-ray repair cross-complementing protein XRCC4 and DNA-ligase-IV (2-4). In addition to its DNA-directed activities, Ku and DNA-PKcs are known to regulate other cellular functions such as RNA-transcription (5-11), processing (12, 13) and telomere maintenance (12,14).The functional diversity of Ku suggests that its cellular activities are regulated by distinct sets of molecular interactions. To date, Ku protein partners include the RNA helicase RHA and several heterogeneous ribonucleoproteins (13), the Werner Syndrome associated protein WRN (15-17), poly-(ADP-ribose) polymerase I PARP-1 (17, 18), the yin-yang transcription factor YY1 (19), histone H2AX (20), and telomerase (12,14). Binary protein-protein interactions with Ku70 were also delineated by two-hybrid screen (21), whereas mass spectrometry-based approaches have been used to identify multicomponent protein complexes that copurify with Ku (22,23). These data provide a useful inventory of Ku protein partners, although the dynamics of specific interactions in response to exogenous or endogenous stimuli remain unknown. In addition, the mechanisms by which Ku selectively engages with DNA versus RNA and whether these nucleic acid interactions are dependent on Ku protein associations, is largely unexplored. Finally, although Ku resides in distinct nuclear compartments (24,25), the relationship between Ku localization and the molecular composition of its associated macromolecular complex is unresolved.In this study, we used biochemical and proteomics approaches to delineate Ku molecular interactions, localization, and dynamic response to DNA damage. Ultraviolet (UV) irradiation resulted in the recruitment of DNA-binding proteins to the multi-component Ku complex and a concomitant loss of RNA-binding proteins. Addition of DNA ends to the Ku complex in vitro induced the loss of RNA-mediated pro...

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“…For proteome analysis, 2% of each concatenated fraction was dried down (lyophilization) and re-suspended in 0.1% FA in 3% ACN for liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis. The remaining 98% was processed to enrich for phosphopeptides using immobilized metal affinity chromatography as previously described (Ficarro et al 2009). Briefly, Ni-NTA-agarose beads (catalog #36113, Qiagen, Valencia, CA) were stripped with EDTA and incubated in a 10-mM FeCl 3 solution to prepare magnetic Fe 3+ -NTA-agarose beads.…”

Section: Fractionation Of Proteome and Phosphoproteome Samplesmentioning

confidence: 99%

“…The protein lysates were digested with trypsin, and the resulting peptides were subjected to fractionation via offline reverse-phase HPLC and split into aliquots for global proteome and phosphoproteome analyses. Phosphopeptides were enriched via iron metal affinity chromatography (Ficarro et al 2009), and both the global and phosphopeptide-enriched samples were analyzed by LC-MS/MS ( Figure 1A). Both data sets were analyzed using three search engines (MaxQuant/Andromeda, Spectrum Mill, and X!Tandem), and results for this combined analysis were reported for an FDR # 0.03 (see Materials and Methods).…”

Section: Lc-ms/ms-based Analysis Of Proteomic and Phosphoproteomic Rementioning

confidence: 99%

DNA Replication Stress Phosphoproteome Profiles Reveal Novel Functional Phosphorylation Sites on Xrs2 in Saccharomyces cerevisiae

et al. 2016

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In response to replication stress, a phospho-signaling cascade is activated and required for coordination of DNA repair and replication of damaged templates (intra-S-phase checkpoint) . How phospho-signaling coordinates the DNA replication stress response is largely unknown. We employed state-of-the-art liquid chromatography tandem-mass spectrometry (LC-MS/MS) approaches to generate high-coverage and quantitative proteomic and phospho-proteomic profiles during replication stress in yeast, induced by continuous exposure to the DNA alkylating agent methyl methanesulfonate (MMS) . We identified 32,057 unique peptides representing the products of 4296 genes and 22,061 unique phosphopeptides representing the products of 3183 genes. A total of 542 phosphopeptides (mapping to 339 genes) demonstrated an abundance change of greater than or equal to twofold in response to MMS. The screen enabled detection of nearly all of the proteins known to be involved in the DNA damage response, as well as many novel MMS-induced phosphorylations. We assessed the functional importance of a subset of key phosphosites by engineering a panel of phosphosite mutants in which an amino acid substitution prevents phosphorylation. In total, we successfully mutated 15 MMSresponsive phosphorylation sites in seven representative genes including APN1 (base excision repair); CTF4 and TOF1 (checkpoint and sister-chromatid cohesion); MPH1 (resolution of homologous recombination intermediates); RAD50 and XRS2 (MRX complex); and RAD18 (PRR). All of these phosphorylation site mutants exhibited MMS sensitivity, indicating an important role in protecting cells from DNA damage. In particular, we identified MMS-induced phosphorylation sites on Xrs2 that are required for MMS resistance in the absence of the MRX activator, Sae2, and that affect telomere maintenance.KEYWORDS mass spectrometry; phosphorylation; methyl methanesulfonate; DNA damage checkpoint; genetic interaction; homologous recombination; telomere; DNA damage response C ELLS utilize excision repair and DNA damage tolerance pathways without significant delay of the cell cycle to address low levels of DNA base damage (Hishida et al. 2009;Huang et al. 2013), while more extensive damage is hallmarked by the activation of additional checkpoints, prolonged cell cycle arrest, and utilization of additional repair mechanisms (Lazzaro et al. 2009). A classic example of an agent that elicits a profoundly different DNA damage response (DDR) at high and low doses is the monofunctional alkylating agent methyl methanesulfonate (MMS) (Friedberg and Friedberg 2006;Hanawalt 2015). At low doses, the MMS lesions are well tolerated by wild-type cells and do not elicit any discernible sensitivity ; however, at higher concentrations, MMS-induced DNA damage present during the S phase leads to prolonged replication fork stall, a phenomenon termed "replication stress" (Shimada et al. 2002;Zeman and Cimprich 2013). As a result of replication stress, cells synchronize into a lengthened S phase due to a kinase-media...

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Magnetic Bead Processor for Rapid Evaluation and Optimization of Parameters for Phosphopeptide Enrichment

Cited by 133 publications

References 22 publications

G Protein–Coupled Receptor Endocytosis Confers Uniformity in Responses to Chemically Distinct Ligands

G Protein–Coupled Receptor Endocytosis Confers Uniformity in Responses to Chemically Distinct Ligands

DNA Ends Alter the Molecular Composition and Localization of Ku Multicomponent Complexes

DNA Replication Stress Phosphoproteome Profiles Reveal Novel Functional Phosphorylation Sites on Xrs2 in Saccharomyces cerevisiae

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