Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome

Oda, Yoshiya; Nagasu, Takeshi; Chait, Brian T.

doi:10.1038/86783

Cited by 796 publications

(633 citation statements)

References 19 publications

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“…Under alkaline conditions, b-elimination of phosphate groups present on serine and threonine residues produces dehydroalanine and b-methyldehydroalanine (dehydroaminobutyric acid), respectively. By subsequently adding a chemical compound containing a free sulfhydryl group to dehydroalanine or b-methyldehydroalanine acid by Michael addition, a di-thiol is created in the peptide, which can serve as a cross-linker to a biotin-tag [98] or for direct affinity purification [99]. Knight and co-workers used the combination of b-elimination, Michael addition reaction and proteolysis to aid the assignment of phosphorylation sites using tandem MS analysis [100].…”

Section: Chemical Derivatization Strategiesmentioning

confidence: 99%

Analytical strategies for phosphoproteomics

2009

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Protein phosphorylation is a key regulator of cellular signaling pathways. It is involved in most cellular events in which the complex interplay between protein kinases and protein phosphatases strictly controls biological processes such as proliferation, differentiation, and apoptosis. Defective or altered signaling pathways often result in abnormalities leading to various diseases, emphasizing the importance of understanding protein phosphorylation. Phosphorylation is a transient modification, and phosphoproteins are often very low abundant. Consequently, phosphoproteome analysis requires highly sensitive and specific strategies. Today, most phosphoproteomic studies are conducted by mass spectrometric strategies in combination with phosphospecific enrichment methods. This review presents an overview of different analytical strategies for the characterization of phosphoproteins. Emphasis will be on the affinity methods utilized specifically for phosphoprotein and phosphopeptide enrichment prior to MS analysis, and on recent applications of these methods in cell biological applications.

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Section: Chemical Derivatization Strategiesmentioning

confidence: 99%

Analytical strategies for phosphoproteomics

2009

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“…Average intensity of the 2L unassigned peaks; (5). Number of fragment ion neutral losses (FNL); (6). Average intensity of fragment ion neutral losses; (7).…”

Section: Supplementary Materialsmentioning

confidence: 99%

Automatic Validation of Phosphopeptide Identifications from Tandem Mass Spectra

Ruse

et al. 2007

Anal. Chem.

128

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We developed and compared two approaches for automated validation of phosphopeptides tandem mass spectra identified using database searching algorithms. Phosphopeptide identifications were obtained through SEQUEST searches of a protein database appended with its decoy (reversed sequences). Statistical evaluation and iterative searches were employed to create a high quality dataset of phosphopeptides. Automation of post-search validation was approached by two different strategies. By using statistical multiple testing, we calculate a p-value for each tentative peptide phosphorylation. In a second method, we use a support vector machine (a machine learning algorithm) binary classifier to predict whether a tentative peptide phosphorylation is true or not. We show good agreement (85%) between post-search validation of phosphopeptide/spectrum matches by multiple testing and that from support vector machines. Automatic methods confirm very well with manual expert validation in a blinded test. Additionally, the algorithms were tested on the identification of synthetic phosphopeptides. We show that phosphate neutral losses in tandem mass spectra can be used to assess the correctness of phosphopeptide/spectrum matches. An SVM classifier with a radial basis function provided classification accuracy from 95.7% to 96.8% of the positive dataset, depending on search algorithm used. Establishing the efficacy of an identification is a necessary step for further post-search interrogation of the spectra for complete localization of phosphorylation sites. Our current implementation performs validation of phosphoserine/ phosphothreonine containing peptides having 1 or 2 phosphorylation sites from data gathered on an ion trap mass spectrometer. The SVM-based algorithm has been implemented in a software package DeBunker. We illustrate the application of the SVM-based software DeBunker on a large phosphorylation dataset.

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“…Antibody-based methods are used mainly for the selective isolation of phosphotyrosinecontaining proteins or peptides (5)(6)(7)(8). Chemical derivatization methods begin with the ␤-elimination of phosphates from phosphoserine and phosphothreonine (9) or the formation of phosphoramidates by reactions with amines (10) to selectively immobilize phosphopeptides. Metal ion-based affinity capture techniques use immobilized metal affinity chromatography (IMAC) with Fe(III) (11,12) or Ga(III) (13) and, for the past a few years, more successful metal oxide approaches (i.e.…”

mentioning

confidence: 99%

In-depth Analyses of Kinase-dependent Tyrosine Phosphoproteomes Based on Metal Ion-functionalized Soluble Nanopolymers

Iliuk

Martin

Alicie

et al. 2010

Molecular & Cellular Proteomics

148

180

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The ability to obtain in-depth understanding of signaling networks in cells is a key objective of systems biology research. Such ability depends largely on unbiased and reproducible analysis of phosphoproteomes. We present here a novel proteomics tool, polymer-based metal ion affinity capture (PolyMAC), for the highly efficient isolation of phosphopeptides to facilitate comprehensive phosphoproteome analyses. This approach uses polyamidoamine dendrimers multifunctionalized with titanium ions and aldehyde groups to allow the chelation and subsequent isolation of phosphopeptides in a homogeneous environment. Compared with current strategies based on solid phase micro-and nanoparticles, PolyMAC demonstrated outstanding reproducibility, exceptional selectivity, fast chelation times, and high phosphopeptide recovery from complex mixtures. Using the PolyMAC method combined with antibody enrichment, we identified 794 unique sites of tyrosine phosphorylation in malignant breast cancer cells, 514 of which are dependent on the expression of Syk, a protein-tyrosine kinase with unusual properties of a tumor suppressor. The superior sensitivity of PolyMAC allowed us to identify novel components in a variety of major signaling networks, including cell migration and apoptosis. PolyMAC offers a powerful and widely applicable tool for phosphoproteomics and molecular signaling. Molecular & Cellular Proteomics 9:2162-2172, 2010.Reversible phosphorylation of proteins is a major mechanism for the regulation of multiple cellular processes (1, 2). Mass spectrometry-based phosphoproteomics provides a method for the global analysis of protein phosphorylation and molecular signaling in cells (3,4). Despite the great progress that has been made over the past few years, the isolation of phosphopeptides and their analysis by mass spectrometry are still a considerable challenge because of the typically low stoichiometry of protein phosphorylation and the resulting low abundance of phosphopeptides. An early step in any phosphoproteome analysis is the isolation of phosphopeptides, preferably with high efficiency, selectivity, sensitivity, and reproducibility. Currently, there are three major strategies for the isolation of phosphopeptides: antibody-based affinity capture, chemical derivatization of phosphoamino acids, and metal ion-based affinity capture. Antibody-based methods are used mainly for the selective isolation of phosphotyrosinecontaining proteins or peptides (5-8). Chemical derivatization methods begin with the ␤-elimination of phosphates from phosphoserine and phosphothreonine (9) or the formation of phosphoramidates by reactions with amines (10) to selectively immobilize phosphopeptides. Metal ion-based affinity capture techniques use immobilized metal affinity chromatography (IMAC) with Fe(III) (11,12) or Ga(III) (13) and, for the past a few years, more successful metal oxide approaches (i.e. TiO 2 (14, 15) and ZrO 2 (16, 17)) for the selective binding of phosphorylated peptides. Almost all of the current isolation methods are b...

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Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome

Cited by 796 publications

References 19 publications

Analytical strategies for phosphoproteomics

Analytical strategies for phosphoproteomics

Automatic Validation of Phosphopeptide Identifications from Tandem Mass Spectra

In-depth Analyses of Kinase-dependent Tyrosine Phosphoproteomes Based on Metal Ion-functionalized Soluble Nanopolymers

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