New Approach for Analysis of the Phosphotyrosine Proteome and Its Application to the Chicken B Cell Line, DT40

Zolodz, Melissa D.; Wood, Karl V.; Regnier, Fred E.; Geahlen, Robert L.

doi:10.1021/pr049967i

Cited by 23 publications

(10 citation statements)

References 45 publications

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“…In conclusion, we demonstrate that Syk positively affects the function of adherens junctions. Zolodz et al (2004) carried out a phosphoproteomic analysis on a Syk-negative chicken B cell line in which Syk expression was restored by transfection with murine Syk and activated with PV. Not using the quantitative SILAC approach, a significantly lesser amount of potential Syk signaling effector proteins were identified, excluding proteins expressed uniquely in nonhematopoietic cells such as the cell-cell adhesion and cell polarization components described above.…”

Section: Discussionmentioning

confidence: 99%

Phosphoproteomic analysis of Syk kinase signaling in human cancer cells reveals its role in cell–cell adhesion

et al. 2009

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The spleen tyrosine kinase Syk has predominantly been studied in hematopoietic cells in which it is involved in immunoreceptor-mediated signaling. Recently, Syk expression was evidenced in numerous nonhematopoietic cells and shown to be involved in tumor formation and progression. The Syk downstream signaling effectors in nonhematopoietic cells remain, however, to be uncovered, and were investigated using MS-based quantitative phosphoproteomics. Two strategies, based on the inhibition of the Syk catalytic activity and on the loss of Syk expression were employed to identify phosphotyrosine-dependent complexes. Quantitative measurements were obtained on 350 proteins purified with phosphotyrosine affinity columns using the SILAC method. Forty-one proteins are dependent on both Syk expression and catalytic activity and were selected as signaling effectors. They are involved in a variety of biological processes such as signal transduction, cell-cell adhesion and cell polarization. We investigated the functional involvement of Syk in cell-cell adhesion and demonstrated the phosphorylation of E-cadherin and acatenin. In addition, Syk is localized at cell-cell contacts, and Syk-mediated phosphorylation of E-cadherin seems to be important for the proper localization of p120-catenin at adherens junctions. Identification of the biochemical pathways regulated by Syk in human cancer cells will help to uncover its role in tumor formation and progression.

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Section: Discussionmentioning

confidence: 99%

Phosphoproteomic analysis of Syk kinase signaling in human cancer cells reveals its role in cell–cell adhesion

et al. 2009

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“…Furthermore, phophotyrosine is not sensitive to the belimination reaction. Zolodz et al [38] used this aspect as a tool for reduction in the complexity of a mixture of phosphopeptides prior to IMAC when searching for pTyrcontaining peptides.…”

Section: Chemical Modificationsmentioning

confidence: 99%

Phospho‐proteomic analysis of cellular signaling

Graauw¹,

Hensbergen²,

Water³

2006

Electrophoresis

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Reversible protein phosphorylation plays an important role in the regulation of many different processes, such as cell growth, differentiation, migration, metabolism, and apoptosis. Identification of differentially phosphorylated proteins by means of phospho-proteomic analysis provides insight into signal transduction pathways that are activated in response to, for example, growth factor stimulation or toxicant-induced apoptosis. This review summarizes recent advances made in the field of phospho-proteomics and provides examples of how phospho-proteomic techniques can be combined to quantitatively investigate the dynamic changes in protein phosphorylation in time. By linking experimental data to clinical data (e.g., disease progression or response to therapy) new disease markers could be identified, which could then be validated for applications in disease diagnosis and progression or prediction of a response to drugs.

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“…pTyr‐containing proteins can be first purified from whole cell lysate by IP using anti‐pTyr monoclonal antibodies. After tryptic digestion, phosphopeptides were further enriched by IMAC and analysed by LC‐MS (Zolodz et al, 2004; Zheng, Hu, Quinn, & Wang, 2005). Lombardi combined IP (with a mixture of pY100, 4G10, and PT66) and TiO 2 enrichment, and demonstrated that this IP‐TiO 2 strategy was more comprehensive for enriching phosphopeptides and allowed the identification of a large set of proteins known to be phosphorylated (715 protein groups) (Lombardi et al, 2015).…”

Section: Enrichment Strategies For Phosphoproteome Analysismentioning

confidence: 99%

Widening the Bottleneck of Phosphoproteomics: Evolving Strategies for Phosphopeptide Enrichment

et al. 2020

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Phosphorylation is a form of protein posttranslational modification (PTM) that regulates many biological processes. Whereas phosphoproteomics is a scientific discipline that identifies and quantifies the phosphorylated proteome using mass spectrometry (MS). This task is extremely challenging as ~30% of the human proteome is phosphorylated; and each phosphoprotein may exist as multiple phospho‐isoforms that are present in low abundance and stoichiometry. Hence, phosphopeptide enrichment techniques are indispensable to (phospho)proteomics laboratories. These enrichment methods encompass widely‐adopted techniques such as (i) affinity‐based chromatography; (ii) ion exchange and mixed‐mode chromatography (iii) enrichment with phospho‐specific antibodies and protein domains, and (iv) functionalized polymers and other less common but emerging technologies such as hydroxyapatite chromatography and precipitation with inorganic ions. Here, we review these techniques, their history, continuous development and evaluation. Besides, we outline associating challenges of phosphoproteomics that are linked to experimental design, sample preparation, and proteolytic digestion. In addition, we also discuss about the future outlooks in phosphoproteomics, focusing on elucidating the noncanonical phosphoproteome and deciphering the “dark phosphoproteome”. © 2020 John Wiley & Sons Ltd.

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New Approach for Analysis of the Phosphotyrosine Proteome and Its Application to the Chicken B Cell Line, DT40

Cited by 23 publications

References 45 publications

Phosphoproteomic analysis of Syk kinase signaling in human cancer cells reveals its role in cell–cell adhesion

Phosphoproteomic analysis of Syk kinase signaling in human cancer cells reveals its role in cell–cell adhesion

Phospho‐proteomic analysis of cellular signaling

Widening the Bottleneck of Phosphoproteomics: Evolving Strategies for Phosphopeptide Enrichment

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