Evidence for phosphorylation of serine 753 in CFTR using a novel metal‐ion affinity resin and matrix‐assisted laser desorption mass spectrometry

Neville, David C. A.; Rozanas, Christine R.; Price, Elmer M.; Gruis, Darren B.; Townsend, R. Reid

doi:10.1002/pro.5560061117

Cited by 237 publications

(119 citation statements)

References 47 publications

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“…However, the binding of phosphoproteins and phosphoamino acids to metal ions, first demonstrated by Andersson and Porath in 1986 [39], gave the technique new potential. IMAC was further extended by Neville et al to the enrichment of phosphopeptides obtained from proteolytically digested proteins [40], and the IMAC technique has since been used extensively for enrichment of phosphopeptides prior to MS analysis [41][42][43][44][45][46][47].…”

Section: Immobilized Metal Ion Affinity Chromatographymentioning

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: Immobilized Metal Ion Affinity Chromatographymentioning

confidence: 99%

Analytical strategies for phosphoproteomics

2009

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“…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.…”

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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“…Furthermore, because we sought to carry out relative quantitative measurements it was essential to preserve the ratios of modified to unmodified peptides that would be representative of any phosphorylation within the original protein. A phospho-enrichment procedure would negate this and would likely result in differential enrichment of the various phosphorylated species; therefore we avoided using enrichment procedures such as IMAC (immobilized metal-ion affinity chromatography) [32][33][34][35], strong cation exchange [36,37], or phospho-specific immunoprecipitation [38]. Using an optimized MS approach developed by monitoring known phosphorylation sites, Ser-118 and Ser-167, we demonstrated that Ser-154 in ER␣-NTD, long suspected but unconfirmed as being phosphorylated under endogenous intracellular conditions [27], is indeed partially phosphorylated under both ligand-dependent and ligand-independent human breast cancer growth conditions.…”

mentioning

confidence: 99%

A novel serine phosphorylation site detected in the n-terminal domain of estrogen receptor isolated from human breast cancer cells

Britton

Scott

Schilling

et al. 2008

J. Am. Soc. Mass Spectrom.

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Activated estrogen receptor (ER␣) plays a critical role in breast cancer development and is a major target for drug treatment. Serine phosphorylation within the N-terminal domain (NTD) contributes to ER␣ activation and may also cause drug resistance. Previous biochemical identification of phosphorylated ER␣ residues was limited to protein artificially overexpressed in transfected cell lines. We report mass spectrometric methods that have allowed the identification of a new site within the NTD of ER␣ isolated from cultured human breast cancer cells. Immunoprecipitation, trypsin digestion, and analysis by nano-LC-ESI-MS/MS (Q-STAR, MDS Sciex) and vMALDI-MS n (Finnigan™ LTQ™, Thermo-Electron) identified peptides containing 8 of 14 serine residues within the NTD, one being partially phosphorylated Ser-167, known but not previously reported by MS. Chymotrypsin digestion revealed other known sites at Ser-102/104/106 and 118. Tandem methods developed for the peptide containing Ser-118 and the use of hypothesis-driven experiments-i.e., the assumption that an intact phosphopeptide showing no molecular ion might yield fragment ions including loss of phosphoric acid in vMALDI-MS/MS-allowed the identification of a novel site at Ser-154. Quantitation by selected reaction monitoring demonstrated 6-fold and 2.5-fold increases in Ser-154 phosphorylation in estradiol-and EGF-treated cells, respectively, compared to controls, confirmed by immunoblotting with a novel rabbit polyclonal antibody. Thus, the protein isolation and MS strategies described here can facilitate discovery of novel phosphorylation sites within low abundance, clinically important cancer targets like ER␣, and may thereby contribute to our understanding of the role of phosphorylation in the development of breast cancer. (J Am

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Evidence for phosphorylation of serine 753 in CFTR using a novel metal‐ion affinity resin and matrix‐assisted laser desorption mass spectrometry

Cited by 237 publications

References 47 publications

Analytical strategies for phosphoproteomics

Analytical strategies for phosphoproteomics

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

A novel serine phosphorylation site detected in the n-terminal domain of estrogen receptor isolated from human breast cancer cells

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