Proteomic and phosphoproteomic comparison of human ES and iPS cells

Phanstiel, Douglas H.; Brumbaugh, Justin; Wenger, Craig D.; Tian, Shulan; Probasco, Mitchell D.; Bailey, Derek J.; Swaney, Danielle L.; Tervo, Mark A.; Bolin, Jennifer M.; Ruotti, Victor; Stewart, Ron; Thomson, James A.; Coon, Joshua J.

doi:10.1038/nmeth.1699

Cited by 251 publications

(286 citation statements)

References 48 publications

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“…3A). Interestingly, three independent studies revealed that RbBP5 S350 is phosphorylated in vivo (Christensen et al 2010;Phanstiel et al 2011;Shiromizu et al 2013). To determine the impact of RbBP5 phosphorylation on WRAD formation, we ectopically expressed constructs corresponding to either wild-type RbBP5 or an RbBP5 S350A mutant in fusion with a Flag tag in HEK293 cells.…”

Section: Disruption Of Ash2l/rbbp5 Interaction Impairs Mll1 Enzymaticmentioning

confidence: 99%

A phosphorylation switch on RbBP5 regulates histone H3 Lys4 methylation

et al. 2015

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The methyltransferase activity of the trithorax group (TrxG) protein MLL1 found within its COMPASS (complex associated with SET1)-like complex is allosterically regulated by a four-subunit complex composed of WDR5, RbBP5, Ash2L, and DPY30 (also referred to as WRAD). We report structural evidence showing that in WRAD, a concave surface of the Ash2L SPIa and ryanodine receptor (SPRY) domain binds to a cluster of acidic residues, referred to as the D/E box, in RbBP5. Mutational analysis shows that residues forming the Ash2L/RbBP5 interface are important for heterodimer formation, stimulation of MLL1 catalytic activity, and erythroid cell terminal differentiation. We also demonstrate that a phosphorylation switch on RbBP5 stimulates WRAD complex formation and significantly increases KMT2 (lysine [K] methyltransferase 2) enzyme methylation rates. Overall, our findings provide structural insights into the assembly of the WRAD complex and point to a novel regulatory mechanism controlling the activity of the KMT2/COMPASS family of lysine methyltransferases.Supplemental material is available for this article.Received October 27, 2014; revised version accepted December 15, 2014. The methyltransferase activity of the trithorax group (TrxG) protein MLL1 as well as the other members of the KMT2 (lysine [K] methyltransferase 2) family found within COMPASS (complex associated with SET1) catalyzes the site-specific methylation of the e-amine of Lys4 (K4) of histone H3 (Shilatifard 2012). While these enzymes share the ability to methylate the same residue on histone H3, the catalytic activity of these enzymes is linked to different biological processes. MLL1/MLL2 di/trimethylate H3K4 (H3K4me2/3) and regulate Hox gene expression during embryonic development (Yu et al. 1995;Dou et al. 2006). MLL3/MLL4 regulate adipogenesis ) and primarily monomethylate H3K4 (H3K4me1) at both enhancer (Herz et al. 2012;Hu et al. 2013) and promoter (Cheng et al. 2014) regions, while SET1A/B are the primary H3K4 trimethyltransferases (Wu et al. 2008). However, despite divergence in catalytic activity and functional roles, enzymes of the KMT2/COMPASS family must assemble into multisubunit complexes to carry out their biological functions.Our molecular understanding of the protein complexes involved in H3K4 methylation stems from the isolation of COMPASS from Saccharomyces cerevisiae (Miller et al. 2001;Roguev et al. 2001;Krogan et al. 2002;Dehe et al. 2006). These studies demonstrated that regulatory subunits found within COMPASS and mammalian COMPASS-like complexes play key roles in stabilizing the enzyme and stimulating its methyltransferase activity as well as targeting the protein complex to specific genomic loci (Couture and Skiniotis 2013). While each of these multisubunit protein complexes contains unique subunits, each member of the KMT2 family associates with a common set of four evolutionarily conserved regulatory proteins; namely, WDR5, RbBP5, Ash2L, and DPY30 (WRAD) (Couture and Skiniotis 2013). The foursubunit complex directly binds ...

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Section: Disruption Of Ash2l/rbbp5 Interaction Impairs Mll1 Enzymaticmentioning

confidence: 99%

A phosphorylation switch on RbBP5 regulates histone H3 Lys4 methylation

et al. 2015

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“…In regards to their pluripotency, ability to form the three embryonic germ layers, and overall global gene expression programs [2][3][4], the two stem cell classes appear to be very similar [3,5]. Nevertheless, others have observed subtle differences between their RNA levels, DNA methylation, protein expression and protein phosphorylation [6]. Some of these differences are attributed to the current limitations of the various reprogramming processes used in the generation of iPSCs [7,8] and may contribute to the variable efficiencies with which hiPSCs can undergo differentiation [9].…”

Section: Introductionmentioning

confidence: 99%

Fourier transform infrared microspectroscopy reveals unique phenotypes for human embryonic and induced pluripotent stem cell lines and their progeny

Cao

McNaughton

et al. 2013

Journal of Biophotonics

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Journal of BIOPHOTONICSFourier transform infrared (FTIR) microspectroscopy was employed to elucidate the macromolecular phenotype of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) and their differentiated progeny. Undifferentiated hESCs and hiPSC lines were found to be not clearly distinguishable from each other. However, although both hESC and hiPSC variants appeared to undergo similar changes during differentiation in terms of cell surface antigens, the derived cell types from all cell lines could be discriminated using FTIR spectroscopy. We foresee a possible future role for FTIR microspectroscopy as a powerful and objective investigative and quality control tool in regenerative medicine.Bright field image of human embryonic stem cells in culture.

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“…The method of stable isotope labeling has greatly propelled large-scale, quantitative analysis (26)(27)(28)(29)(30)(31). While generally robust, these techniques can yield spotty data for certain peptide and protein groups-mainly those present at low abundances.…”

Section: Resultsmentioning

confidence: 99%

Instant spectral assignment for advanced decision tree-driven mass spectrometry

Bailey

Rose

McAlister

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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We have developed and implemented a sequence identification algorithm (inSeq) that processes tandem mass spectra in real-time using the mass spectrometer's (MS) onboard processors. The inSeq algorithm relies on accurate mass tandem MS data for swift spectral matching with high accuracy. The instant spectral processing technology takes ∼16 ms to execute and provides information to enable autonomous, real-time decision making by the MS system. Using inSeq and its advanced decision tree logic, we demonstrate (i) realtime prediction of peptide elution windows en masse (∼3 min width, 3,000 targets), (ii) significant improvement of quantitative precision and accuracy (~3x boost in detected protein differences), and (iii) boosted rates of posttranslation modification site localization (90% agreement in real-time vs. offline localization rate and an approximate 25% gain in localized sites). The decision tree logic enabled by inSeq promises to circumvent problems with the conventional data-dependent acquisition paradigm and provides a direct route to streamlined and expedient targeted protein analysis.T he shotgun sequencing method has rapidly evolved over the past two decades (1, 2). In this strategy eluting peptide cations have their mass-to-charge (m∕z) values measured in the MS 1 scan. Then precursor m∕z values are selected for a series of sequential tandem MS events (MS 2 ). This succession is cycled for the duration of the analysis. The process, called data-dependent acquisition (DDA), is at the very core of shotgun analysis and has not changed for over 15 y, however, MS hardware has. Major improvements in MS sensitivity, scan rate, mass accuracy, and resolution have been achieved. Orbitrap hybrid systems, for example, routinely achieve low ppm mass accuracy with MS/MS repetition rates of 5-10 Hz (3, 4). Constant operation of such systems generates hundreds of thousands of spectra in hours. These MS 2 spectra are then mapped to sequence using database search algorithms (5-7).The DDA sampling strategy offers an elegant simplicity and has proven highly useful for discovery-driven proteomics. Of recent years, however, emphasis has shifted from identification to quantification-often with certain targets in mind. In this context, faults in the DDA approach have become increasingly evident. There are two primary limitations of the DDA approach: First, is poor run-to-run reproducibility and, second, is the inability to effectively target peptides of interest (8). Hundreds of peptides often coelute so that low-level signals often are selected in one run and not the next, and selecting m∕z peaks to sequence by abundance certainly does not offer the opportunity to inform the system of preselected targets.Several DDA add-ons and alternatives have been examined. Sampling depth, for example, can be increased by preventing selection of an m∕z value identified in a prior technical replicate (PAnDA) (9). Irreproducibility can be somewhat countered by informing the DDA algorithm of the precursor m∕z values of desired targets (inclu...

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Proteomic and phosphoproteomic comparison of human ES and iPS cells

Cited by 251 publications

References 48 publications

A phosphorylation switch on RbBP5 regulates histone H3 Lys4 methylation

A phosphorylation switch on RbBP5 regulates histone H3 Lys4 methylation

Fourier transform infrared microspectroscopy reveals unique phenotypes for human embryonic and induced pluripotent stem cell lines and their progeny

Instant spectral assignment for advanced decision tree-driven mass spectrometry

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