Discontinuous pH gradient-mediated separation of TiO2-enriched phosphopeptides

Maudsley, Stuart

doi:10.1016/j.ab.2010.10.003

Cited by 21 publications

(19 citation statements)

References 34 publications

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“…Label swapping of the Ctrl siRNA and miR-519 siRNA groups with the two label states, medium (K4 and R6) and heavy (K8 and R10), was used to reduce artifactual label bias. SILAC was carried out, as previously described (42,43), by using HeLa cells (300 g from the Ctrl siRNA group and 300 g from the miR-519 group).…”

Section: Methodsmentioning

confidence: 99%

Growth Inhibition by miR-519 via Multiple p21-Inducing Pathways

Abdelmohsen

Srikantan

Tominaga

et al. 2012

Molecular and Cellular Biology

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eThe microRNA miR-519 robustly inhibits cell proliferation, in turn triggering senescence and decreasing tumor growth. However, the molecular mediators of miR-519-elicited growth inhibition are unknown. Here, we systematically investigated the influence of miR-519 on gene expression profiles leading to growth cessation in HeLa human cervical carcinoma cells. By analyzing miR-519-triggered changes in protein and mRNA expression patterns and by identifying mRNAs associated with biotinylated miR-519, we uncovered two prominent subsets of miR-519-regulated mRNAs. One subset of miR-519 target mRNAs encoded DNA maintenance proteins (including DUT1, EXO1, RPA2, and POLE4); miR-519 repressed their expression and increased DNA damage, in turn raising the levels of the cyclin-dependent kinase (cdk) inhibitor p21. The other subset of miR-519 target mRNAs encoded proteins that control intracellular calcium levels (notably, ATP2C1 and ORAI1); their downregulation by miR-519 aberrantly elevated levels of cytosolic [Ca 2؉ ] storage in HeLa cells, similarly increasing p21 levels in a manner dependent on the Ca 2؉ -activated kinases CaMKII and GSK3␤. The rises in levels of DNA damage, the Ca 2؉ concentration, and p21 levels stimulated an autophagic phenotype in HeLa and other human carcinoma cell lines. As a consequence, ATP levels increased, and the level of activity of the AMP-activated protein kinase (AMPK) declined, further contributing to the elevation in the abundance of p21. Our results indicate that miR-519 promotes DNA damage, alters Ca 2؉ homeostasis, and enhances energy production; together, these processes elevate the expression level of p21, promoting growth inhibition and cell survival.

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Section: Methodsmentioning

confidence: 99%

Growth Inhibition by miR-519 via Multiple p21-Inducing Pathways

Abdelmohsen

Srikantan

Tominaga

et al. 2012

Molecular and Cellular Biology

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“…Another approach how to minimize the level of nonspecifi cally bound peptides is chemical derivatization before the enrichment, e.g., methylesterifi cation of peptides (Ficarro et al 2002, Simon et al 2008. A variety of pH conditions during the binding or the elution step have been employed to optimize the protocol for enrichment on MOAC resins (Simon et al 2008 , Park andMaudsley 2011 ).…”

Section: Metal Oxide/hydroxide Affi Nity Chromatography (Moac)mentioning

confidence: 99%

Enrichment strategies for phosphoproteomics: state-of-the-art

Šalovská¹,

Tichý²,

Řezáčová³

et al. 2012

Reviews in Analytical Chemistry

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Protein phosphorylation is a key regulator in many biological processes, such as homeostasis, cellular signaling and communication, transcriptional and translational regulation, and apoptosis. The defects in this tightly controlled reversible posttranslational modifi cation have been described to contribute to genesis and progression of various diseases, emphasizing the importance of a systematic research of this phenomenon. Although considerable effort has been devoted to improving the analysis of phosphorylation by mass spectrometry, which is currently the method of choice to study protein phosphorylation, the detection and identifi cation of phosphorylation sites remains challenging because of the low abundance and low ionization effi cacy of phosphoproteins in comparison with nonphosphorylated proteins. To overcome this obstacle, different enrichment strategies for phosphorylated peptides/proteins have been established and optimized for subsequent mass spectrometry analysis. In this review, we will give an overview of the methods currently available for the enrichment of phosphorylated proteins and peptides including immunoprecipitation, chemical derivatization and affi nity enrichment techniques.

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“…a A tryptic digest of a cytosol fraction of Xenopus eggs without Ca 2+ -stimulation (4 mg) was applied to an automated phosphopeptide purification system. Phosphopeptides bound on a titania column were eluted with 1-step or 4-steps under Condition B in Table I method for the determination of multiply phosphorylated peptides (16). A pH gradient of between 9.2 and 11.3 is applied for this method.…”

Section: Discussionmentioning

confidence: 99%

Exploring the phosphoproteome profiles duringXenopusegg activation by calcium stimulation using a fully automated phosphopeptide purification system

Kanno

Horigome

2015

J Biochem

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To explore the phosphoproteome profiles during Xenopus egg activation by Ca 2+ -stimulation, an automated phosphopeptide purification system involving a titania column was improved by introducing 4-step elution with phosphate buffers. The number of detected phosphopeptides in the tryptic digest of a Xenopus egg cytosol fraction on mass spectrometry (MS) was increased 1.5-fold and the percentage of multiply phosphorylated peptides increased from 17 to 24% with introduction of the 4-step elution method. Phosphopeptides were purified by the improved method from tryptic digests of cytosol fractions of Xenopus eggs without and with a Ca 2+ -stimulus, and then, analysed by MS. One thousand three hundred and seventy-five and 994 phosphopeptides were reproducibly detected on duplicate MS, respectively. They included 818 and 437 phosphopeptides specific to each digest, respectively. A method involving isobaric tags for relative and absolute quantitation (iTRAQ) was also applied to compare the phosphorylation levels in Xenopus eggs without and with a Ca 2+ -stimulus, the ratios for 112 phosphopeptides in tryptic digests of these egg cytosol fractions being obtained. It was suggested from all the results that the phosphorylation sites and levels change during Xenopus egg activation for many known and unknown sites on structural proteins, signalling related proteins, cell cyclerelated proteins and others.Keywords: egg/phosphoproteome/proteome/titania/ Xenopus.Abbreviations: 4EBP, eukaryotic translation initiation factor 4E binding protein; iTRAQ, isobaric tags for relative and absolute quantitation; LC MS/MS, nano-flow liquid chromatography-linear ion trap/ Orbitrap tandem mass spectrometry; MAPK, mitogen-activated protein kinase; MS, mass spectrometry; mTOR, mammalian target of rapamycin; m/z, massto-charge ratio; ODS, octadecylsilica; TFA, trifluoroacetic acid.Xenopus eggs have been used in studies on egg maturation, egg activation and other mechanisms, which facilitated the progress of developmental biology (1, 2). It has been shown that signalling pathways including protein phosphorylation and dephosphorylation participate greatly in these regulation mechanisms (1, 3, 4). To clarify the complicated regulation mechanism of egg activation, comprehensive analysis of protein phosphorylation during egg activation is a powerful means. The cell cycle of Xenopus eggs is arrested at the second meiotic metaphase. So, Xenopus eggs without Ca 2+ -stimulation show meiotic metaphase properties. When eggs are activated by Ca 2+ -stimulation to mimic fertilization, they show synthetic phase properties. So, we can analyse the changes of protein phosphorylation during activation of Xenopus eggs by comparison of the phosphorylation in Ca 2+ -unstimulated and Ca 2+ -stimulated eggs (5). In this study, therefore, we obtained phosphoproteome profiles of Xenopus egg cytosol fractions without and with Ca 2+ -stimulation as a basis for understanding the egg activation mechanism.To obtain high quality phosphoproteome data, i) determination of a ...

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Discontinuous pH gradient-mediated separation of TiO2-enriched phosphopeptides

Cited by 21 publications

References 34 publications

Growth Inhibition by miR-519 via Multiple p21-Inducing Pathways

Growth Inhibition by miR-519 via Multiple p21-Inducing Pathways

Enrichment strategies for phosphoproteomics: state-of-the-art

Exploring the phosphoproteome profiles duringXenopusegg activation by calcium stimulation using a fully automated phosphopeptide purification system

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