Isolation Of Phosphopeptides Research Articles

Electrostatic Repulsion Hydrophilic Interaction Chromatography for Isocratic Separation of Charged Solutes and Selective Isolation of Phosphopeptides

If an ion-exchange column is eluted with a predominantly organic mobile phase, then solutes can be retained through hydrophilic interaction even if they have the same charge as the stationary phase. This combination is termed electrostatic repulsion-hydrophilic interaction chromatography (ERLIC). With mixtures of solutes that differ greatly in charge, repulsion effects can be exploited to selectively antagonize the retention of the solutes that normally would be the best retained. This permits the isocratic resolution of mixtures that normally require gradients, including peptides, amino acids, and nucleotides. ERLIC affords convenient separations of highly charged peptides that cannot readily be resolved by other means. In addition, phosphopeptides can be isolated selectively from a tryptic digest.

Mesoporous Fe 2O 3 microspheres: Rapid and effective enrichment of phosphopeptides for MALDI-TOF MS analysis

Mesoporous Fe 2O 3 microspheres have been successfully synthesized by the polymerization (urea and formaldehyde)-induced ferric hydroxide colloid aggregation. The urea-formaldehyde resin was removed by calcination in air. The obtained mesoporous Fe 2O 3 materials have spherical morphology with uniform particle size of ∼ 3.0 μm and porous surface with large inter-particle pores of ∼ 48.0 nm . The surface area is as large as ∼ 33.3 m 2 / g and the pore volume is 0.31 cm 3/g. The mesoporous Fe 2O 3 microspheres were used for the enrichment of phosphopeptides for the first time, in which high sensitivity, selectivity and capacity of specifically enriched phosphopeptides were achieved under a mild condition in a relative short time. After enriched from tryptic digest products of β-casein by the novel mesoporous Fe 2O 3 microspheres, phosphopeptides can be selectively detected with high intensity in MALDI-TOF mass spectrometry. Elimination of “shadow effect” was observed by using mesoporous Fe 2O 3 microspheres, and the detectable limitation is 5 × 10 −10 M . This material is also effective for enrichment of phosphopeptides from the complex tryptic digests of commercial phosphoprotein casein, with much more phosphorylated sites (26 in 27 of total) and higher signal/noise ratio in the MALDI-TOF mass spectrometry, compared to commercial Fe 2O 3 nanoparticles. It shows a great potential application in the field of rapid and effective isolation of phosphopeptides.

Highly specific enrichment of phosphopeptides by zirconium dioxide nanoparticles for phosphoproteome analysis

Large-scale characterization of phosphoproteins requires highly specific methods for the purification of phosphopeptides because of the low abundance of phosphoproteins and substoichiometry of phosphorylation. A phosphopeptide enrichment method using ZrO2 nanoparticles is presented. The high specificity of this approach was demonstrated by the isolation of phosphopeptides from the digests of model phosphoproteins. The strong affinity of ZrO2 nanoparticles to phosphopeptides enables the specific enrichment of phosphopeptides from a complex peptide mixture in which the abundance of phosphopeptides is two orders of magnitude lower than that of nonphosphopeptides. Superior selectivity of ZrO2 nanoparticles for the enrichment of phosphorylated peptides than that of conventional immobilized metal affinity chromatography was observed. Femtomole phosphopeptides from digestion products could be enriched by ZrO2 nanoparticles and can be well detected by MALDI mass spectrometric analysis. ZrO2 nanoparticles were further applied to selectively isolate phosphopeptides from the tryptic digestion of mouse liver lysate for phosphoproteome analysis by nanoliter LC MS/MS (nano-LC-MS/MS) and MS/MS/MS. A total of 248 defining phosphorylation sites and 140 phosphorylated peptides were identified by manual validation using a series of rigid criteria.

Specific capture of phosphopeptides on matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry targets modified by magnetic affinity nanoparticles

Specific capture of phosphopeptides from protein digests is a critical step for identification of phosphoproteins by mass spectrometry. In this study, we report a novel phosphopeptide-capture approach based on the specific interaction of phosphopeptides with a stainless steel target modified with magnetic affinity nanoparticles. The modification which was carried out by loading the suspension of nanoparticles into sample wells of the target did not require any pretreatment procedure to the target and did not involve chemical binding reactions. To isolate phosphopeptides, digests were loaded into the wells of the modified target for 10 min incubation, followed by rinsing with washing buffer to remove unbound species; matrix was then added to the captured phosphopeptides prior to analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Capturing the phosphopeptides on the modified target simplified significantly analytical operations and reduced sample loss. This approach has been applied to solution digests of alpha-casein, beta-casein, and a mixture of five proteins; a number of phosphopeptides were confidently detected. Phosphopeptides from digests of 10 fmol beta-casein could be isolated and detected by MALDI-TOFMS with this method. In addition, this approach has been applied successfully to the isolation of phosphopeptides from in-gel digestive products of sub-pmol phosphoproteins after separation by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE).

Interorganelle Trafficking of Ceramide Is Regulated by Phosphorylation-dependent Cooperativity between the PH and START Domains of CERT

The synthesis and transport of lipids are essential events for membrane biogenesis. However, little is known about how intracellular trafficking of lipids is regulated. Ceramide is synthesized at the endoplasmic reticulum (ER) and transported by the ceramide transfer protein CERT to the Golgi apparatus, where it is converted to sphingomyelin. CERT has a phosphoinositide-binding pleckstrin homology (PH) domain for Golgi-targeting and a lipid transfer START domain for intermembrane transfer of ceramide. We here show that CERT receives multiple phosphorylations at a serine-repeat motif, a possibe site for casein kinase I, and that the phosphorylation down-regulates the ER-to-Golgi transport of ceramide. In vitro assays show that the phosphorylation induces an autoinhibitory interaction between the PH and START domains and consequently inactivates both the phosphoinositide binding and ceramide transfer activities of CERT. Loss of sphingomyelin and cholesterol from cells causes dephosphorylation of CERT to activate it. The cooperative control of functionally distinct domains of CERT is a novel molecular event to regulate the intracellular trafficking of ceramide.

Analytical Currents: Light-emitting nanofibers for lab-on-a-chip applications | Microfluidic system for dynamic microarrays | COINs for Raman-based tissue analysis | Scanning outside the NMR tube | Assessing phosphopeptide isolation methods | Breath analysis without sample pretreatment | Apoptosis probe with a quantifiable readout | Patterned paper for cheap, portable bioassays | Proteotypic peptides in the spotlight

Analytical Currents: Light-emitting nanofibers for lab-on-a-chip applications | Microfluidic system for dynamic microarrays | COINs for Raman-based tissue analysis | Scanning outside the NMR tube | Assessing phosphopeptide isolation methods | Breath analysis without sample pretreatment | Apoptosis probe with a quantifiable readout | Patterned paper for cheap, portable bioassays | Proteotypic peptides in the spotlight

TOOLbox: Automatic validation of phosphopeptide identifications | Assessing human protein interaction maps | Faster database searches | ByOnic | Assessing phosphopeptide isolation methods | Proteomics of Huntington?s disease | Large-scale phosphoproteomics studies with ETD | A photoactive semiconductor to digest proteins | Host proteins copurify with blood flukes

ADVERTISEMENT RETURN TO ISSUEPREVCURRENTSNEXTTOOLbox: Automatic validation of phosphopeptide identifications | Assessing human protein interaction maps | Faster database searches | ByOnic | Assessing phosphopeptide isolation methods | Proteomics of Huntington?s disease | Large-scale phosphoproteomics studies with ETD | A photoactive semiconductor to digest proteins | Host proteins copurify with blood flukesCite this: J. Proteome Res. 2007, 6, 4, 1237–1239Publication Date (Web):April 9, 2007Publication History Published online9 April 2007Published inissue 1 April 2007https://doi.org/10.1021/pr070731zRIGHTS & PERMISSIONSArticle Views89Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (180 KB) Get e-AlertsSUBJECTS:Anatomy,Protein identification,Isolation,Proteomics,Semiconductors Get e-Alerts

Synapsin Phosphorylation by Src Tyrosine Kinase Enhances Src Activity in Synaptic Vesicles

Synapsins are synaptic vesicle-associated phosphoproteins implicated in the regulation of neurotransmitter release. Synapsin I is the major binding protein for the SH3 domain of the kinase c-Src in synaptic vesicles. Its binding leads to stimulation of synaptic vesicle-associated c-Src activity. We investigated the mechanism and role of Src activation by synapsins on synaptic vesicles. We found that synapsin is tyrosine phosphorylated by c-Src in vitro and on intact synaptic vesicles independently of its phosphorylation state on serine. Mass spectrometry revealed a single major phosphorylation site at Tyr(301), which is highly conserved in all synapsin isoforms and orthologues. Synapsin tyrosine phosphorylation triggered its binding to the SH2 domains of Src or Fyn. However, synapsin selectively activated and was phosphorylated by Src, consistent with the specific enrichment of c-Src in synaptic vesicles over Fyn or n-Src. The activity of Src on synaptic vesicles was controlled by the amount of vesicle-associated synapsin, which is in turn dependent on synapsin serine phosphorylation. Synaptic vesicles depleted of synapsin in vitro or derived from synapsin null mice exhibited greatly reduced Src activity and tyrosine phosphorylation of other synaptic vesicle proteins. Disruption of the Src-synapsin interaction by internalization of either the Src SH3 or SH2 domains into synaptosomes decreased synapsin tyrosine phosphorylation and concomitantly increased neurotransmitter release in response to Ca(2+)-ionophores. We conclude that synapsin is an endogenous substrate and activator of synaptic vesicle-associated c-Src and that regulation of Src activity on synaptic vesicles participates in the regulation of neurotransmitter release by synapsin.

Reproducible isolation of distinct, overlapping segments of the phosphoproteome

The ability to routinely analyze and quantitatively measure changes in protein phosphorylation on a proteome-wide scale is essential for biological and clinical research. We assessed the ability of three common phosphopeptide isolation methods (phosphoramidate chemistry (PAC), immobilized metal affinity chromatography (IMAC) and titanium dioxide) to reproducibly, specifically and comprehensively isolate phosphopeptides from complex mixtures. Phosphopeptides were isolated from aliquots of a tryptic digest of the cytosolic fraction of Drosophila melanogaster Kc167 cells and analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry. Each method reproducibly isolated phosphopeptides. The methods, however, differed in their specificity of isolation and, notably, in the set of phosphopeptides isolated. The results suggest that the three methods detect different, partially overlapping segments of the phosphoproteome and that, at present, no single method is sufficient for a comprehensive phosphoproteome analysis.

Isolation of Phosphopeptides by pI-Difference-Based Electrophoresis

Effective proteomics studies of protein phosphorylation require an efficient enrichment method for phosphopeptides, which remains a challenge. Here, we describe the discovery of pI differences between methylated phosphopeptides (typically <7.4) and methylated nonphosphorylated peptides (typically >9.0). This pI difference allows isolation of methylated phosphopeptides from the methylated nonphosphopeptides by in-solution isoelectric focusing. We proved the principle of such a novel approach by isolating a phosphorylated peptide from nonphosphorylated tryptic digest of myoglobin. While the principle for pI-based, in-solution electrophoresis is proven, it requires further development for practical application. The method described here provides a stepping stone toward more reliable, convenient method for efficient isolation of phosphopeptides.

Isolation of Phosphopeptides by Immobilized Metal Ion Affinity Chromatography

The identification of protein phosphorylation sites from cell-derived proteins is crucial to the understanding of signal transduction pathways. While determining the modified sites on individual proteins can present a significant challenge, recent progress in the rapid, large-scale identification of phosphopeptides from complex protein mixtures by combinations of affinity chromatography and mass spectrometry provides a powerful tool to decipher the phosphoproteome. A set of protocols is described for sample preparation, fractionation, immobilized metal ion affinity chromatography (IMAC), and mass spectrometric analysis of phosphorylation sites. Parts of the protocols can be combined in different ways to adapt to sample amounts, complexity, and available equipment. Up to thousands of unique phosphopeptides can be sequenced from a single sample in a day, revealing a unique snapshot of global cellular phosphorylation sites on proteins and facilitating in-depth study of the identified phosphoproteins.

PhosphoPep—a phosphoproteome resource for systems biology research in Drosophila Kc167 cells

The ability to analyze and understand the mechanisms by which cells process information is a key question of systems biology research. Such mechanisms critically depend on reversible phosphorylation of cellular proteins, a process that is catalyzed by protein kinases and phosphatases. Here, we present PhosphoPep, a database containing more than 10 000 unique high-confidence phosphorylation sites mapping to nearly 3500 gene models and 4600 distinct phosphoproteins of the Drosophila melanogaster Kc167 cell line. This constitutes the most comprehensive phosphorylation map of any single source to date. To enhance the utility of PhosphoPep, we also provide an array of software tools that allow users to browse through phosphorylation sites on single proteins or pathways, to easily integrate the data with other, external data types such as protein–protein interactions and to search the database via spectral matching. Finally, all data can be readily exported, for example, for targeted proteomics approaches and the data thus generated can be again validated using PhosphoPep, supporting iterative cycles of experimentation and analysis that are typical for systems biology research.

Selective sampling of multiply phosphorylated peptides by capillary electrophoresis for electrospray ionization mass spectrometry analysis

The ionization of phosphorylated peptides in positive ion mode mass spectrometry is generally less efficient compared with the ionization of their non-phosphorylated counterparts. This can make phosphopeptides much more difficult to detect. One way to enhance the detection of phosphorylated proteins and peptides is by selectively isolating these species. Current approaches of phosphopeptide isolation are based on the favorable interactions of phosphate groups with immobilized metals. While these methods can be effective in the extraction, they can lead to incomplete sample recovery, particularly for the most strongly bound multiply phosphorylated components. A non-sorptive method of phosphopeptide isolation using capillary electrophoresis (CE) was recently reported [Zhang et al., Anal. Chem. 77 (2005) 6078]. The relatively low isoelectric points of phosphopeptides cause them to remain anionic at acidic sample pH. Hence, they can be selectively injected into the capillary by an applied field after the electroosmotic flow (EOF) is suppressed. The technique was previously coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). In this work, the exploitation of selective sampling in conjugation with electrospray ionization mass spectrometry (ESI-MS) is presented. The transition was not immediately straightforward. A number of major alterations were necessary for ESI interfacing. These adaptations include the choice of a suitable capillary coating for EOF control and the incorporation of organic solvent for efficient ESI. As expected, selective injection of phosphopeptides greatly enhanced the sensitivity of their detection in ESI-MS, particularly for the multiply phosphorylated species that were traditionally most problematic. Furthermore, an electrophoretic separation subsequent to the selective injection of the phosphopeptides was performed prior to analysis by ESI-MS. This allowed us to resolve the multiply phosphorylated peptides present in the samples, predominantly based on the number of phosphorylation sites on the peptides.

A Clean, More Efficient Method for In-Solution Digestion of Protein Mixtures without Detergent or Urea

Proteolytic digestion of a complicated protein mixture from an organelle or whole-cell lysate is usually carried out in a dilute solution of a denaturing buffer, such as 1-2 M urea. Urea must be subsequently removed by C18 beads before downstream analysis such as HPLC/MS/MS or complete methylation followed by IMAC isolation of phosphopeptides. Here we describe a procedure for digesting a complicated protein mixture in the absence of denaturants. Proteins in the mixture are precipitated with trichloroacetic acid/acetone for denaturation and salt removal and resuspended in NH4HCO3 buffer. After trypsinolysis, the resulting peptides are not contaminated by urea or other nonvolatile salts and can be dried in a SpeedVac to remove NH4HCO3. When this protocol was applied to an extract of A431 cells, 96.8% of the tryptic peptides were completely digested (i.e., had no missed cleavage sites), in contrast to 87.3% of those produced by digestion in urea buffer. We successfully applied this digestion method to analysis of the phosphoproteome of adiposomes from HeLa cells, identifying 33 phosphorylation sites in 28 different proteins. Our digestion method avoids the need to remove urea before HPLC/MS/MS analysis or methylation and IMAC, increasing throughput while reducing sample loss and contamination from sample handling. We believe that this method should be valuable for proteomics studies.

Nonretentive Solid-Phase Extraction of Phosphorylated Peptides from Complex Peptide Mixtures for Detection by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Widespread interest in protein phosphorylation has led to the development of a variety of methods for the analysis of phosphoproteomes of different types of organisms. Many applications involve pretreatment of the sample before mass spectrometric measurement and can crucially improve the detection efficiency of individual phosphopeptides. Despite intense research efforts, separation and extraction of phosphorylated peptides, especially multiphosphorylated ones, remain challenging tasks and need to be further explored and expanded with unconventional approaches. In this study, we describe the application of nonretentive solid-phase extraction (SPE) to the analysis of phosphopeptides using the highly cross-linked polystyrene-divinylbenzene material Strata-X. This study indicates that the procedure allows for the preferential extraction of phosphopeptides regardless of their extent of phosphorylation. The Strata-X material primarily retains nonphosphorylated peptides by hydrophobic interaction, whereas the inherent hydrophilicity of phosphorylated peptides leads to their partitioning into the aqueous phase. Phosphopeptides that were rapidly segregated out of tryptic digest mixtures and collected in the early aqueous fractions generated intense signals in mass spectra. The method was developed using SPE Strata-X columns, then suited for detection and sequencing of phosphopeptides by miniaturizing the system to the scale of custom-made microcolumns. This provided fast isolation of phosphopeptides from protein digests along with direct MALDI on-target deposition. The possibility of on-target washing during sample preparation is also presented.

Selective Zirconium Dioxide-Based Enrichment of Phosphorylated Peptides for Mass Spectrometric Analysis

Due to the dynamic nature and low stoichiometry of protein phosphorylation, enrichment of phosphorylated peptides from proteolytic mixtures is often necessary prior to their characterization by mass spectrometry. Several phosphopeptide isolation strategies have been presented in the literature, including immobilized metal ion affinity chromatography. However, that technique suffers from poor selectivity and reproducibility. Recently, titanium dioxide-based columns have been successfully employed for phosphopeptide enrichment by several research groups. Here, we present, to our knowledge, the first demonstration of the utility of zirconium dioxide microtips for phosphopeptide isolation prior to mass spectrometric analysis. These microtips display similar overall performance as TiO2 microtips. However, more selective isolation of singly phosphorylated peptides was observed with ZrO2 compared to TiO2 whereas TiO2 preferentially enriched multiply phosphorylated peptides. Thus, these two chromatographic materials possess complementary properties. For alpha- and beta-casein, Glu-C digestion provided no evident advantage compared to trypsin digestion when combined with TiO2 or ZrO2 phosphopeptide enrichment.

Two-dimensional HPLC on-line analysis of phosphopeptides using titania and monolithic columns

Conventional and comprehensive two-dimensional (2D) HPLC systems using the combination of titania and monolithic columns were established for the on-line analysis of phosphopeptides. Compared with immobilized metal affinity chromatography of a general method for the analysis of phosphopeptides, the use of titania columns in the analysis permits the specific isolation of phosphopeptides in a higher yield. Using the current 2D HPLC systems, phosphopeptides were specifically isolated from nonphosphorylated peptides by the first-dimension titania column, followed by the high-speed separation of the phosphopeptides by the second-dimension monolithic column. Proteolytic digests of β-casein were analyzed within 30 min using the comprehensive 2D HPLC system; all phosphopeptides from β-casein could be efficiently isolated and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The comprehensive 2D HPLC system coupled with mass spectrometry will be useful for high-throughput and on-line phosphoproteome analyses.

Phosphoproteomics as a tool to unravel plant regulatory mechanisms

Reversible phosphorylation of proteins plays a key role in many regulatory processes that lie at the basis of life. With plants, much research has focused on protein kinases that are involved in the adaptation to different stress conditions, such as pathogen attack and cold. However, the substrates of these kinases are mostly unknown. With the recent advances in phosphoproteomic techniques, the large‐scale identification of kinase substrates, including their phosphorylation sites, is finally possible. Studies in mainly non‐plant systems have demonstrated the high potential of this method by uncovering numerous novel phosphorylation events. In this minireview, we focus on recent developments in the field of phosphoproteomics that are based on phosphopeptide isolation from complex mixtures by immobilized metal‐affinity chromatography coupled to sequence identification by mass spectrometry. Combination of these methods with labelling techniques now allows quantitative analysis of phosphorylation between different samples. We discuss the potential of this technology to uncover entire phosphoproteomes and signalling pathways in plants in the future.

Identification of a UDP-Glucose-Binding Site of Human UDP-Glucose Dehydrogenase by Photoaffinity Labeling and Cassette Mutagenesis

We have identified a UDP-glucose-binding site within human UDP-glucose dehydrogenase (hUGDH) by photoaffinity labeling with a specific probe, [(32)P]5N(3)UDP-glucose, and cassette mutagenesis using a synthetic hUGDH gene. Photolabel-containing peptides were generated by photolysis followed by tryptic digestion and isolated using the phosphopeptide isolation kit. Photolabeling of these peptides was effectively prevented by the presence of UDP-glucose during photolysis, demonstrating a selectivity of the photoprobe for the UDP-glucose-binding site. Amino acid sequencing and compositional analysis identified the UDP-glucose-binding site of hUGDH as the region containing the sequence, ASVGFGGSXFQK, corresponding to A268-K279 of the amino acid sequence of hUGDH. The unidentified residue, X, can be designated as a photolabeled C276 because the sequences including the cysteine residue in question have a complete identity with those of other UGDH species known. The importance of the C276 residue in the binding of UDP-glucose was further examined with mutant proteins at the C276 site. The mutagenesis at C276 has no effect on the expression of the mutants (C276G, C276K, C276E, C276L, and C276Y). Enzyme activities of the C276 mutants were not measurable under normal assay conditions, suggesting an important role for the C276 residue. No incorporation of [(32)P]5N(3)UDP-glucose was also observed for the mutants. These results indicate that C276 plays an important role for efficient binding of UDP-glucose to hUGDH.

A new maleimide‐bound acid‐cleavable solid‐support reagent for profiling phosphorylation

A new chemical strategy for phosphopeptide profiling is reported in this study. Phosphorylation represents one of the most important classes of posttranslational modifications of proteins. Here we report a generalized strategy that employs solid-phase capture and mass-encoding steps to selectively enrich phosphopeptides from complex mixtures. This method exploits conversion of phosphates into thiols and reactive compounds to selectively isolate products of phosphorylation. Selective isolation of phosphopeptides is achieved with a simple, novel, acid-cleavable, solid-support-bound maleimide reagent. Our chemistry efforts have focused on minimization of linker size and simplification of reagent production with incorporation of common solid-phase peptide synthesis steps. Relative quantitation was demonstrated by modifying phosphopeptides with incorporation of ethanedithiol and propanedithiol. We observed that appropriate normalization is necessary to utilize mass tag strategies for relative quantitation of posttranslational modifications. The utility of solid-phase capture was determined with model phosphopeptides, and the method was demonstrated with enriching phosphopeptides from beta-casein, alpha-casein and ovalbumin. The solid-phase capture and release methods were also demonstrated with unfractionated whole histone protein mixtures to show this compound applicability in real biological samples. The new chemical strategy will ultimately be utilized for high-throughput profiling of phosphorylation and possibly other posttranslational modifications.