Phosphopeptide-selective Column-switching RP-HPLC with a Titania Precolumn

Kuroda, Ikuma; Shintani, Yukihiro; Motokawa, Masanori; Abe, Shuzo; Furuno, Masahiro

doi:10.2116/analsci.20.1313

Cited by 99 publications

(60 citation statements)

References 16 publications

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“…phy (21)(22)(23)(24) and immunoprecipitation (36), respectively. Prior to LC-MS/MS analysis, all iTRAQ ® labeled samples were prefractionated offline on a TSKGel Amide 80 HILIC HPLC column.…”

Section: Resultsmentioning

confidence: 99%

“…Phosphopeptide Enrichment-TiO 2 chromatography was used to enrich samples for phosphopeptides as described previously (21)(22)(23)(24) with minor modifications. Sample (130 g) was resuspended in 80% acetonitrile (ACN) (v/v), 5% (v/v) trifluoroacetic acid (TFA), 1 M glycolic acid in a polypropylene tube containing 1 mg TiO 2 beads and incubated with constant shaking for 30 min.…”

Section: Methodsmentioning

confidence: 99%

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Modulation of Protein Phosphorylation, N-Glycosylation and Lys-Acetylation in Grape (Vitis vinifera) Mesocarp and Exocarp Owing to Lobesia botrana Infection

Melo‐Braga

Verano‐Braga

León

et al. 2012

Molecular & Cellular Proteomics

112

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Grapevine (Vitis vinifera) is an economically important fruit crop that is subject to many types of insect and pathogen attack. To better elucidate the plant response to Lobesia botrana pathogen infection, we initiated a global comparative proteomic study monitoring steady-state protein expression as well as changes in N-glycosylation, phosphorylation, and Lys-acetylation in control and infected mesocarp and exocarp from V. vinifera cv Italia. A multi-parallel, large-scale proteomic approach employing iTRAQ labeling prior to three peptide enrichment techniques followed by tandem mass spectrometry led to the identification of a total of 3059 proteins, 1135 phosphorylation sites, 323 N-linked glycosylation sites and 138 Lysacetylation sites. Of these, we could identify changes in abundance of 899 proteins. The occupancy of 110 phosphorylation sites, 10 N-glycosylation sites and 20 Lysacetylation sites differentially changed during L. botrana infection. Sequence consensus analysis for phosphorylation sites showed eight significant motifs, two of which containing up-regulated phosphopeptides (X-G-S-X and S-X-X-D) and two containing down-regulated phosphopeptides (R-X-X-S and S-D-X-E) in response to

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Modulation of Protein Phosphorylation, N-Glycosylation and Lys-Acetylation in Grape (Vitis vinifera) Mesocarp and Exocarp Owing to Lobesia botrana Infection

Melo‐Braga

Verano‐Braga

León

et al. 2012

Molecular & Cellular Proteomics

112

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“…However, one of the drawbacks in the tyrosine-phosphorylated protein enrichment methods by antiphosphotyrosine antibodies is the lack of information about phosphorylation sites in the identified proteins (43). A number of phosphopeptide enrichment methods including immobilized metal affinity chromatography (IMAC) (44,45), titanium dioxide (TiO 2 )-based phosphopeptide enrichment (46,47), strong cation exchange (SCX) chromatography (48,49), and antiphosphotyrosine antibody-based enrichment of tyrosine phosphorylated peptides (50) have been developed to pinpoint the phosphorylation sites in the phosphoproteome. These enrichment methods have also been combined with the SILAC strategy to quantitate phosphorylation changes in various biological systems.…”

Section: Thymic Stromal Lymphopoietin (Tslp)mentioning

confidence: 99%

TSLP Signaling Network Revealed by SILAC-Based Phosphoproteomics

Zhong

Kim

Chaerkady³

et al. 2012

Molecular & Cellular Proteomics

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Thymic stromal lymphopoietin (TSLP) is a cytokine that plays diverse roles in the regulation of immune responses. TSLP requires a heterodimeric receptor complex consisting of IL-7 receptor ␣ subunit and its unique TSLP receptor (gene symbol CRLF2) to transmit signals in cells. Abnormal TSLP signaling (e.g. overexpression of TSLP or its unique receptor TSLPR) contributes to the development of a number of diseases including asthma and leukemia. However, a detailed understanding of the signaling pathways activated by TSLP remains elusive. In this study, we performed a global quantitative phosphoproteomic analysis of the TSLP signaling network using stable isotope labeling by amino acids in cell culture. By employing titanium dioxide in addition to antiphosphotyrosine antibodies as enrichment methods, we identified 4164 phosphopeptides on 1670 phosphoproteins. Using stable isotope labeling by amino acids in cell culture-based quantitation, we determined that the phosphorylation status of 226 proteins was modulated by TSLP stimulation. Our analysis identified activation of several members of the Src and Tec families of kinases including Btk, Lyn, and Tec by TSLP for the first time. In addition, we report TSLP-induced phosphorylation of protein phosphatases such as Ptpn6 (SHP-1) and Ptpn11 (Shp2), which has also not been reported previously. Co-immunoprecipitation assays showed that Shp2 binds to the adapter protein Gab2 in a TSLP-dependent manner. This is the first demonstration of an inducible protein complex in TSLP signaling. A kinase inhibitor screen revealed that pharmacological inhibition of PI-3 kinase, Jak family kinases, Src family kinases or Btk suppressed TSLP-dependent cellular proliferation making them candidate therapeutic targets in diseases resulting from aberrant TSLP signaling. Our study is the first phosphoproteomic analysis of the TSLP signaling pathway that greatly expands our understanding of TSLP signaling and provides novel therapeutic targets for TSLP/ TSLPR-associated diseases in humans. Molecular & Cellular Proteomics 11: 10.1074/mcp.M112.017764, 1-22, 2012. Thymic stromal lymphopoietin (TSLP)1 is an IL-7-like fourhelix bundle cytokine that was originally identified as a growth factor from the conditioned medium of the Z210R.1 thymic stromal cell line to support B-cell development in the absence of IL-7 (1, 2). TSLP mediates its effects through a heterodimeric receptor complex consisting of IL-7R␣ and a unique TSLP receptor (TSLPR, also known as CRLF2) (3, 4). In humans, epithelial cells are the major source of TSLP (5), which acts on a number of distinctive cell types. TSLP released from airway epithelial cells synergizes with IL-1 and TNF to stimulate the production of high levels of Th2 cytokines by mast cells (6). TSLP can promote the maturation of dendritic cells, thereby driving the Type 2 differentiation of T helper cells (7). Studies have also shown that TSLP enhances proliferation of CD4ϩ and CD8ϩ T cells activated by T-cell receptor stimulation (8, 9). In mice, TSLP can also activ...

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“…14 There are a number of metal oxidebased phosphopeptide enrichment approaches. [15][16][17][18][19][20][21][22][23][24] Ikeguchi and Nakamura reported phosphopeptide enrichment using titania. 15 Other metal oxides, such as zirconia 23 and aluminium hydroxide, 22 have also been employed.…”

Section: Introductionmentioning

confidence: 99%

“…17,19,24,28,29 However, no system has yet been applied to real complex samples, such as cell lysates, although recently Cantin et al analyzed a moderately complex sample with a titania column-trap column system. 29 The reason for the failure to achieve practical application is mainly that the dynamic range between phosphorylated and non-phosphorylated peptides is much wider in real samples, and this results in poor identification of phosphopeptides.…”

Section: Introductionmentioning

confidence: 99%

Automated Phosphoproteome Analysis for Cultured Cancer Cells by Two-Dimensional NanoLC-MS Using a Calcined Titania/C18 Biphasic Column

et al. 2008

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We have developed an on-line automated system for phosphoproteome analysis using titania-based phosphopeptide enrichment followed by nanoLC-MS/MS. Titania beads were prepared by calcination of commercial chromatographic titania beads at 800˚C to convert the crystalline structure. The obtained rutile-form titania exhibited higher selectivity in phosphopeptide enrichment than commercial titania, even in the absence of a competitive chelating reagent for nonphosphopeptides. For phosphoproteome analysis of human cervical cancer HeLa cells, tryptic digests of the cell extracts were directly injected into this on-line system, and 696 non-redundant phosphopeptides with 671 unambiguously determined phosphorylation sites, derived from 512 phosphoproteins, were successfully identified. This is the first successful application of an on-line automated phosphoproteome analysis system to complex biological samples.

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Phosphopeptide-selective Column-switching RP-HPLC with a Titania Precolumn

Cited by 99 publications

References 16 publications

Modulation of Protein Phosphorylation, N-Glycosylation and Lys-Acetylation in Grape (Vitis vinifera) Mesocarp and Exocarp Owing to Lobesia botrana Infection

Modulation of Protein Phosphorylation, N-Glycosylation and Lys-Acetylation in Grape (Vitis vinifera) Mesocarp and Exocarp Owing to Lobesia botrana Infection

TSLP Signaling Network Revealed by SILAC-Based Phosphoproteomics

Automated Phosphoproteome Analysis for Cultured Cancer Cells by Two-Dimensional NanoLC-MS Using a Calcined Titania/C18 Biphasic Column

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