State-of-the-art in phosphoproteomics

Reinders, Joerg; Sickmann, Albert

doi:10.1002/pmic.200401289

Cited by 319 publications

(256 citation statements)

References 97 publications

(95 reference statements)

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“…Phosphorylation is a key regulator of intracellular biological processes and at present, it is the most studied and best understood PTM [1,2]. Protein phosphorylation is known to be involved in the regulation of diverse processes including metabolism, transcriptional, and translational regulation, degradation of proteins, homeostasis, cellular signaling and communication, proliferation, differentiation, and cell survival [3,4].…”

Section: Introductionmentioning

confidence: 99%

“…Genomic sequencing has revealed that 2-3% of all eukaryotic genes are likely to code for protein kinases [6] and more than 100 human protein phosphatases have been predicted by genome annotation [7], emphasizing the ubiquitous role of protein phosphorylation. At present, no studies have been able to elucidate how many proteins are in fact regulated by phosphorylation, but it has been estimated that more than 50 percent of all proteins are phosphorylated during their lifetime [2], and that more than 100 000 phosphorylation sites may exist in the human proteome [8]. Despite its ubiquitous role, phosphoproteins are generally present at relatively low abundance within the cell and phosphorylated forms of individual proteins also tend to be present at much lower ratios than their native counterparts.…”

Section: Introductionmentioning

confidence: 99%

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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: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Analytical strategies for phosphoproteomics

2009

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“…We also report that such neutral losses may be reduced by fragmenting peptide-alkali metal adducts, such as sodiated or potassiated peptides. (J Am Soc Mass Spectrom 2007, 18, 1617-1624) © 2007 American Society for Mass Spectrometry P hosphopeptides are the most frequently targeted post-translational modifications in proteomics studies because of their biological significance [1][2][3][4][5]. Sulfopeptides also display an 80 Da mass increase as well as similar chromatographic and mass spectrometric behavior [6].…”

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confidence: 99%

Sulfopeptide fragmentation in electron-capture and electron-transfer dissociation

Medzihradszky

Guan

Maltby

et al. 2007

J. Am. Soc. Mass Spectrom.

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Sulfopeptides can be misassigned as phosphopeptides because of the isobaric nature of the sulfo-and the phosphomoieties. Instruments having the ability to measure mass with high accuracy may be employed to distinguish these moieties based on their mass defect (the sulfo-group is 9 mmu lighter than the phosphomoiety). However, the assignment of the exact site(s) of post-translational modification is required to probe biological function. We have reported earlier that peptides with identical sequences containing either O-sulfo-or Ophospho-modifications display different fragmentation behavior (K. F. Medzihradszky et al., Mol. Cell. Proteom. 2004, 3, 429 -440). We have also established that O-sulfo moieties are susceptible to side-chain fragmentation during collision-induced dissociation. Our present study provides evidence that neutral SO 3 losses can also occur in electron capture dissociation and electron-transfer dissociation experiments. We also report that such neutral losses may be reduced by fragmenting peptide-alkali metal adducts, such as sodiated or potassiated peptides. ( -5]. Sulfopeptides also display an 80 Da mass increase as well as similar chromatographic and mass spectrometric behavior [6]. The sulfopeptides are 9 mmu lighter than their phosphorylated counterparts. They can easily be misidentified as phosphopeptides. However, the most significant difference between phosphoand sulfopeptides is revealed during MS/MS analysis. In CID analyses phosphorylated Tyr residues retain the modification, and even their modified immonium ion is sometimes observed, while phosphorylated Ser and Thr residues undergo ␤-elimination of H 3 PO 4 . Thus, the fragments detected in the CID spectra of phosphopeptides either display a ϩ80 Da or a Ϫ18 Da mass shift in comparison to the fragments of an unmodified peptide. Under identical conditions, collisional activation of Osulfopeptides leads to a gas-phase rearrangement reaction that completely eliminates the sulfate from the molecular ion, and the CID fragment spectrum is practically identical to that observed for the unmodified molecule [6]. We have analyzed numerous O-sulfopeptides and have not detected any CID fragments that still contained the sulfate group.While the different CID behavior proved to be a reliable tool for the differentiation of these two isobaric modifications, the gas-phase elimination of the sulfomoiety also prevented the assignment of the modification site. Therefore, it seemed essential to evaluate electron-capture dissociation based on reports that most of the labile side chains of peptides and proteins studied remain intact [7]. Electron-transfer dissociation [8,9] was also employed to analyze synthetic phospho-and sulfopeptides. Here we report our observations on the ECD and ETD fragmentation of sulfopeptides. Experimental MaterialsPhosphopeptides and sulfopeptides were synthesized as described earlier [6]. Their sequences are as follows: RIEVALsTK; RIEVALpTK; LAGLQDEIGsSLR; LAGLQDEIGpSLR; Ac-LAGLQDEIGsSLR-amide. Caerulein, ϽQQDsYTGWMDF-ami...

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“…However, the amount of phosphorylated proteins in eukaryotic cells often exists in substoichiometry level, which makes the identification of phosphorylation sites challenging [3]. To date, mass spectrometry (MS) analysis has become an effective and commonly used method for characterization of phosphoproteins [4].…”

Section: Introductionmentioning

confidence: 99%

Monoliths with immobilized zirconium ions for selective enrichment of phosphopeptides

Wang

Duan

et al. 2011

J of Separation Science

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To meet the demands of protein phosphorylation study, immobilized zirconium ion affinity chromatography (Zr 41 -IMAC) monolith was prepared by combining UV-initiated polymerization of monolithic support and subsequent photografting in both capillary columns and microchannels. Hydrophilic poly(2-hydroxyethyl methacrylate (HEMA)-coethylene dimethacrylate (EDMA)) monolithic support was prepared under UV irradiation at the wavelength of 365 nm with monomer HEMA, crosslinker EDMA and 2,2-dimethoxy-2-phenylacetophenone as photoinitiator in 1-decanol solution, which provides good biocompatibility and permeability for biomolecule analysis. To introduce chelating ligands, such as phosphate groups, on the pore surface of monolith for metal ion immobilization, photografting of ethylene glycol methacrylate phosphate with benzophenone as the photoinitiator was performed at 254 nm for 300 s. The grafting process and metal ion immobilization can be monitored by measuring the electroosmotic flow produced by the modified monolith, providing a quantitative evaluation of post-modification. This new method for the preparation of Zr 41 -IMAC monolith simplifies the optimization of monolith preparation and avoids the time-consuming chemical modification process. Additionally, advantages include facile preparation in microdevices, easy regenerability and good reproducibility. After optimization, the microchip-based Zr 41 -IMAC monolith was used for phosphopeptide analysis and showed good selectivity in phosphopeptide enrichment with matrix-assisted laser desorption ionization mass spectrometry detection. IntroductionIn recent years, protein phosphorylation has gained much attention because of its significant roles in cellular signal transduction processes [1,2]. However, the amount of phosphorylated proteins in eukaryotic cells often exists in substoichiometry level, which makes the identification of phosphorylation sites challenging [3]. To date, mass spectrometry (MS) analysis has become an effective and commonly used method for characterization of phosphoproteins [4]. To reduce the interference of non-phosphopeptides, selective enrichment strategies, such as immobilized metal ion affinity chromatography (IMAC) [5][6][7], metal oxide affinity chromatography (MOAC) [8,9], and ion exchange chromatography [10][11][12][13], are necessary before MS analysis.Recently, chip-based system has been introduced as a promising platform for phosphopeptide analysis, due to the benefits of low sample amount, high throughput, and easy integration [14]. Several microfluidic devices have been developed by integrating phosphopeptide enrichment with other functions, such as protein alkylation and reduction [15], protein digestion [16], peptide separation and on-line interface for MS instrument [17][18][19][20], which have shown great potentials in the field of phosphoproteome research. However, in these microchip devices, phosphopeptide enrichment components are usually fabricated by packing functionalized particles, such as TiO 2 or IMAC beads, into m...

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State-of-the-art in phosphoproteomics

Cited by 319 publications

References 97 publications

Analytical strategies for phosphoproteomics

Analytical strategies for phosphoproteomics

Sulfopeptide fragmentation in electron-capture and electron-transfer dissociation

Monoliths with immobilized zirconium ions for selective enrichment of phosphopeptides

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