Efficient Enrichment Of Phosphopeptides Research Articles

Complementary multiple hydrogen-bond-based magnetic composite microspheres for high coverage and efficient phosphopeptide enrichment in bio-samples.

Due to the number of phosphorylation sites, mono- and multiple-phosphopeptides exhibit significantly different biological effects. Therefore, comprehensive profiles of mono- and multiple-phosphopeptides are vital for the analysis of these biological and pathological processes. However, the most commonly used affinity materials based on metal oxide affinity chromatography (MOAC) show stronger selectivity toward mono-phosphopeptides, thus losing most information on multiple-phosphopeptides. Herein, we report polymer functionalized magnetic nanocomposite microspheres as an ideal platform to efficiently enrich both mono- and multiple-phosphopeptides from complex biological samples. Driven by complementary multiple hydrogen bonding interactions, the composite microspheres exhibited remarkable performance for phosphopeptide enrichment from model proteins and real bio-samples. Excellent selectivity (the molar ratio of nonphosphopeptides/phosphopeptides was 5000 : 1), high enrichment sensitivity (2 fmol) and coverage, as well as high capture rates of multiple-phosphopeptides revealed their great potential in comprehensive phosphoproteomics studies. More importantly, we successfully captured the cancer related phosphopeptides (from the phosphoprotein Stathmin-1) and identified their relevant phosphorylation sites from oral carcinoma patients' saliva and tissue lysate, demonstrating the potential of this material for phosphorylated disease marker detection and discovery.

A sensitive and selective phosphopeptide enrichment strategy by combining polyoxometalates and cysteamine hydrochloride-modified chitosan through layer-by-layer assembly

Highly efficient enrichment of phosphopeptides in low abundance prior to mass spectrometry analysis is essential in phosphopeptidomics analysis. Herein, an affinity probe possessing both interactions of polyoxometalates (POMs) and hydrophilic interaction chromatography (HILIC) was facilely prepared. POMs and cysteamine hydrochloride-modified chitosan (CYECS) were assembled onto magnetic nanoparticles (MNPs) via a layer-by-layer approach. The developed MNPs-(POM/CYECS) material owned merits of large metal oxide surfaces, positive charge, and hydrophilicity as well as superparamagnetism. By combining with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), the prepared material was utilized for effective enrichment of phosphopeptides. High selectivity (mass ratio of β-casein and bovine serum albumin digests of 1:5000) and low detection limit (0.02 fmol) toward phosphopeptides were achieved. The recovery of phosphopeptides was measured to be 92.6%. The specific enrichment of phosphopeptides from complex samples (nonfat milk, human saliva, serum, and A549 cell lysate) with MNPs-(POM/CYECS) material further confirmed its bright prospects for isolation of phosphopeptides from biological samples.

Boronic Acid-Functionalized Magnetic Metal–Organic Frameworks via a Dual-Ligand Strategy for Highly Efficient Enrichment of Phosphopeptides and Glycopeptides

To date, abundant strategies and affinity materials are proposed to enrich phosphopeptides and glycopeptides from biological systems. However, few affinity materials can meet the demand for enrichi...

ZrO2 doped magnetic mesoporous polyimide for the efficient enrichment of phosphopeptides

FexOy and ZrO2 nanoparticles co-doped layered porous polyimide, polyimide-FexOy-ZrO2 is prepared with a one-step strategy, shortly termed as PI-FexOy-ZrO2. The layered and porous structure of the polymer offers a supported platform for metallic oxide anchoring, exhibiting a mesopore size of 3.93 nm and providing a surface area of 198.47 m2 g−1. The metallic oxides were uniformly and highly dispersed in the PI-FexOy-ZrO2 nanocomposite with percentages of 15.81 and 20.53 wt% for Fe and Zr, respectively. The magnetic FexOy provides driving force for rapid separation. The high doping of ZrO2 facilitates effective enrichment of phosphopeptides, even at a very low mass ratio of 1:1000 for tryptic digest of phosphopeptides/non-phosphopeptides, e.g., β-casein/BSA in this particular case. In addition, the PI-FexOy-ZrO2 nanocomposite exhibits better adsorption performance to phosphopeptides with respect to commercial titanium dioxide nanoparticles. The effectiveness of low-abundant phosphopeptides isolation and enrichment from human serum is further identified and demonstrated by means of MALDI-TOF MS and LC-ESI-MS/MS.

Preparation of quaternized cellulose/chitosan microspheres for selective enrichment of phosphopeptides.

As one of the most important posttranslational modifications, protein phosphorylation plays an important role in vital movement. However, an efficiency enrichment treatment prior to MS detection is still a crucial step to protein phosphorylation analysis. In this work, a novel hybrid microsphere for efficient phosphopeptide enrichment was prepared by reverse-phase suspension polymerization of cellulose derivative and chitosan. The microspheres bore different kinds of amine groups and the main enrichment mechanism was based on anion exchange. This approach exhibited high selectivity for phosphopeptides from β-casein, α-casein, and non-fat milk. Three phosphopeptides could still be detected when the amount of β-casein was as low as 10fmol. This study demonstrated a new attractive solid-phase support for phosphopeptide enrichment to meet the increasing need of phosphoproteomics analysis.

Highly efficient enrichment of phosphopeptides by a magnetic lanthanide metal-organic framework

Highly efficient enrichment of phosphopeptides from complex biological samples is crucial prior to mass spectrometry analysis due to the low abundance and ion suppression effects. In this study, a facile route was designed for preparation of a magnetic erbium(Er)-based metal-organic framework (denoted as Fe3O4@PDA@Er(btc)), which was synthesized with 1,3,5-benzenetricarboxylic acid(H3btc) as ligand and grafted on the polydopamine (PDA) – coated Fe3O4. The as-prepared material exhibited ultra-high sensitivity (detection limit of 20amol/μL) and selectivity at a low mass ratio of β-Casein/BSA (1:500). Moreover, it was also investigated for enrichment of phosphopeptides from human serum, which provided a promising technique for highly efficient enrichment of low-abundance phosphorylated peptides in the practical application.

Phosphopeptide enrichment with inorganic nanofibers prepared by forcespinning technology

Efficient selective sample enrichment is often a key procedure in protocols for analyses of complex samples. This applies not only to trace sample components but also to species with weak detection response. For example enrichment of phosphopeptides using selective affinity techniques prior to mass spectrometry analysis is necessary to increase detection sensitivity of phosphopeptides from highly complex peptide mixtures. In this work we have developed inorganic nanofibrous materials based on titanium or zirconium dioxides for selective and efficient enrichment of phosphopeptides for MALDI/MS detection. In comparison to the common bead based materials the presented nanofibrous materials exhibit much higher permeability allowing their use not only for batch mode or packed in the column operation, but also in the pipette tip format without the need for high pressure. Both the methods of preparation and characterization of the resulting materials are presented.

Polydopamine-coated eppendorf tubes for Ti4+ immobilization for selective enrichment of phosphopeptides

Mass spectrometric technique has emerged as a preferred technique in the analysis of protein phosphorylation. Owing to the low stoichiometry of phosphopeptides and the signal suppression effect by non-phosphopeptides, there is a demand for efficient enrichment of phosphopeptides. The selective enrichment of phosphopeptides in modified eppendorf tubes prior to mass spectrometry analysis, which can minimize sample loss as well as nonspecific interferences effectively, has become a hot topic in current proteomics field. In our work, an easy-to-use phosphopeptide-selective eppendorf tube was initially prepared, with its inner surface being modified with a Ti4+-immobilized polydopamine (PDA) layer. The unique Ti4+-immobilized PDA-modified eppendorf tubes (EP tube@PDA-Ti4+) are investigated for its application in selective enrichment of phosphopeptides from complex biological samples. Due to the high Ti4+ loading amount on the surface of PDA, the EP tube@PDA-Ti4+ exhibits remarkable phosphopeptide enrichment ability in protein digests and human serum, which presents a powerful evidence for its high selectivity in detecting the low-abundance phosphopeptides from complex biological samples.

Newly Developed Poly(Allyl Glycidyl Ether/Divinyl Benzene) Polymer for Phosphopeptides Enrichment and Desalting of Biofluids

The polymeric materials have contributed significantly in the area of bioanalytical science. The functionalization of polymeric backbone after its development brings unique selectivity towards the target biomolecules. In present work, the functionalities of choice have been introduced through the ring-opening of allyl glycidyl ether. The utility of polymer is widened through derivatizations to immobilized metal ion affinity chromatographic (IMAC) material for the phosphopeptides enrichment and Reversed Phase (C-18) for the desalting prior to MALDI-MS analysis. The polymer-IMAC in addition to Fe(3+) is also immobilized with lanthanide ions like La(3+), Eu(3+), and Er(3+). The amount of Fe(3+) immobilized is determined as 0.7928 mg/g. Spherical morphology with narrow particle size dispersion is revealed by scanning electron microscopy (SEM). The surface area, pore volume and size distribution is determined by nitrogen adsorption porosimetery. The elemental composition and purity level is confirmed by energy dispersive X-ray spectroscopy (EDX) data. The derivatization to IMAC and RP is evaluated by Fourier transform infrared (FT-IR) spectroscopy. The polymer enables the efficient phosphopeptide enrichment to equal degree from casein variants, non-fat milk, egg yolk, human serum, and HeLa cell extract. The identification of phosphorylation sites can lead to the phosphorylation pathways to understand the post-translational modifications. The identification with their sequence coverage is made using Mascot and Phosphosite Plus. It is sensitive to enrich the phosphopeptides down to 2 femtomoles with very high selectivity of 1:2000 with BSA background. These attributes are linked to the higher surface area (173.1554 m(2)/g) of the designed polymer. The non-specific bindings, particularly the Fe(3+) linked acidic residues are also avoided. Four characteristic phosphopeptides (fibrinopeptide A and their hydrolytic products) from fibrinogen α-chain are identified from the human serum after the enrichment, which have link to the hepatocellular carcinoma (HCC). The proportions of fibrinogen and their phosphorylation products enriched by poly(AGE/DVB)-IMAC open new horizons in the biomarker discovery.

Preparation of magnetic hydroxyapatite clusters and their application in the enrichment of phosphopeptides

A novel strategy for the effective enrichment of phosphopeptides based on magnetic hydro-xyapatite (HAp) clusters was developed in the current study. The structure of HAp ensures its probable separation capability, including cation exchange with P-sites (negatively charged pairs of crystal phosphates), calcium coordination, anion exchange with C-sites (positively charged pairs of crystal calcium ions). The prepared magnetic HAp clusters showed good performance on the efficient enrichment of phosphopeptides from the digestion mixture of β-casein and BSA. Compared to commercial HAp particles, the magnetic HAp clusters exhibited better selectivity toward phosphopeptides. In addition, the use of magnetic material greatly simplified the enrichment procedure, which avoided the tedious centrifugation steps in a typical phosphopeptides enrichment protocol. Finally, the material was successfully applied in the enrichment of phosphopeptides from human serum. Taken together, the efficient enrichment of the phosphopeptides by the easily prepared magnetic HAp clusters demonstrated a rapid and convenient strategy for the purification of phosphopeptides from complex samples, which may facilitate protein phosphorylation studies.

Ti4+-immobilized multilayer polysaccharide coated magnetic nanoparticles for highly selective enrichment of phosphopeptides.

Highly selective and efficient enrichment of trace phosphorylated proteins or peptides from complex biological samples is of profound significance for the discovery of disease biomarkers in biological systems. In this study, a novel immobilized metal affinity chromatography (IMAC) material has been synthesized to improve the enrichment specificity and sensitivity for phosphopeptides by introducing a titanium phosphate moiety on a multilayer polysaccharide (hyaluronate (HA) and chitosan (CS)) coated Fe3O4@SiO2 nanoparticle (denoted as Fe3O4@SiO2@(HA/CS)10-Ti4+ IMAC). The thicker multilayer polysaccharide endows excellent hydrophilic properties and a higher binding capacity of the titanium ion to the IMAC material. Due to the combination of uniform magnetic properties, highly hydrophilic properties and enhanced binding capacity of the titanium ion, the Fe3O4@SiO2@(HA/CS)10-Ti4+ nanoparticle possesses many merits, such as high selectivity for phosphopeptides (phosphopeptides/non-phosphopeptides at a molar ratio of 1 : 2000), extreme detection sensitivity (0.5 fmol), large binding capacity (100 mg g-1), high enrichment recovery (85.45%) and rapid magnetic separation (within 10 s). Moreover, the as-prepared IMAC nanoparticle provides effective enrichment of phosphopeptides from real samples (human serum and nonfat milk), showing great potential as a tool for the detection and identification of low-abundance phosphopeptides in biological samples.

Robust phosphoproteome enrichment using monodisperse microsphere–based immobilized titanium (IV) ion affinity chromatography

Mass spectrometry (MS)-based proteomics has become the preferred tool for the analysis of protein phosphorylation. To be successful at such an endeavor, there is a requirement for an efficient enrichment of phosphopeptides. This is necessary because of the substoichiometric nature of phosphorylation at a given site and the complexity of the cell. Recently, new alternative materials have emerged that allow excellent and robust enrichment of phosphopeptides. These monodisperse microsphere-based immobilized metal ion affinity chromatography (IMAC) resins incorporate a flexible linker terminated with phosphonate groups that chelate either zirconium or titanium ions. The chelated zirconium or titanium ions bind specifically to phosphopeptides, with an affinity that is similar to that of other widely used metal oxide affinity chromatography materials (typically TiO(2)). Here we present a detailed protocol for the preparation of monodisperse microsphere-based Ti(4+)-IMAC adsorbents and the subsequent enrichment process. Furthermore, we discuss general pitfalls and crucial steps in the preparation of phosphoproteomics samples before enrichment and, just as importantly, in the subsequent mass spectrometric analysis. Key points such as lysis, preparation of the chromatographic system for analysis and the most appropriate methods for sequencing phosphopeptides are discussed. Bioinformatics analysis specifically relating to site localization is also addressed. Finally, we demonstrate how the protocols provided are appropriate for both single-protein analysis and the screening of entire phosphoproteomes. It takes ∼2 weeks to complete the protocol: 1 week to prepare the Ti(4+)-IMAC material, 2 d for sample preparation, 3 d for MS analysis of the enriched sample and 2 d for data analysis.

Tuning of Ti-doped mesoporous silica for highly efficient enrichment of phosphopeptides in human placenta mitochondria

Extraction of phosphopeptides from rather complex biological samples has been a tough issue for deep and comprehensive investigation into phosphoproteomes. In this paper, we present a series of Ti-doped mesoporous silica (Ti-MPS) materials with tunable composition and controllable morphology for highly efficient enrichment of phosphopeptides. By altering the molar ratio of silicon to titanium (Si/Ti) in the precursor, the external morphology, Ti content, internal long-rang order, and surface area of Ti-MPS were all modulated accordingly with certain regularity. Tryptic digests of standard phosphoprotein α- and β-casein were employed to assess the phosphopeptide enrichment capability of Ti-MPS series. At the Si/Ti molar ratio of 8:1, the optimum enrichment performance with admirable sensitivity and capacity was achieved. The detection limit for β-casein could reach 10 fmol, and 15 phosphopeptides from the digest of α-casein were resolved in the spectrum after enrichment, both superior to the behavior of commercial TiO(2) materials. More significantly, for the digest of human placenta mitochondria, 396 phosphopeptides and 298 phosphoproteins were definitely detected and identified after enrichment with optimized Ti-MPS material, demonstrating its remarkable applicability for untouched phosphoproteomes. In addition, this research also opened up a universal pathway to construct a composition-tunable functional material in pursuit of the maximum performance in applications.

Nanoparticle-modified monolithic pipette tips for phosphopeptide enrichment

We have developed nanoparticle-modified monoliths in pipette tips for selective and efficient enrichment of phosphopeptides. The 5 μL monolithic beds were prepared by UV-initiated polymerization in 200 μL polypropylene pipette tips and either iron oxide or hydroxyapatite nanoparticles were used for monolith modification. Iron oxide nanoparticles were prepared by a co-precipitation method and stabilized by citrate ions. A stable coating of iron oxide nanoparticles on the pore surface of the monolith was obtained via multivalent electrostatic interactions of citrate ions on the surface of nanoparticles with a quaternary amine functionalized poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) monolith. Hydroxyapatite nanoparticles were incorporated into the poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) monolith by simply admixing them in the polymerization mixture followed by in situ polymerization. The nanoparticle-modified monoliths were compared with commercially available titanium dioxide pipette tips. Performance of the developed and commercially available sorbents was demonstrated with the efficient and selective enrichment of phosphopeptides from peptide mixtures of α-casein and β-casein digests followed by off-line MALDI/MS analysis.

PhosphoSiteAnalyzer: A Bioinformatic Platform for Deciphering Phospho Proteomes Using Kinase Predictions Retrieved from NetworKIN

Phosphoproteomic experiments are routinely conducted in laboratories worldwide, and because of the fast development of mass spectrometric techniques and efficient phosphopeptide enrichment methods, researchers frequently end up having lists with tens of thousands of phosphorylation sites for further interrogation. To answer biologically relevant questions from these complex data sets, it becomes essential to apply computational, statistical, and predictive analytical methods. Here we provide an advanced bioinformatic platform termed "PhosphoSiteAnalyzer" to explore large phosphoproteomic data sets that have been subjected to kinase prediction using the previously published NetworKIN algorithm. NetworKIN applies sophisticated linear motif analysis and contextual network modeling to obtain kinase-substrate associations with high accuracy and sensitivity. PhosphoSiteAnalyzer provides an algorithm to retrieve kinase predictions from the public NetworKIN webpage in a semiautomated way and applies hereafter advanced statistics to facilitate a user-tailored in-depth analysis of the phosphoproteomic data sets. The interface of the software provides a high degree of analytical flexibility and is designed to be intuitive for most users. PhosphoSiteAnalyzer is a freeware program available at http://phosphosite.sourceforge.net .

Efficient enrichment of phosphopeptides by magnetic TiO2 -coated carbon-encapsulated iron nanoparticles

Titanium dioxide (TiO₂) has been widely used for phosphopeptide enrichment. Several approaches have been reported to produce magnetic TiO₂ affinity probes. In this report, we present a facile approach to immobilize TiO₂ onto poly(acrylic acid)-functionalized magnetic carbon-encapsulated iron nanoparticles as affinity probes for efficient enrichment of phosphopeptides. By using the new magnetic TiO₂ affinity probes, denoted as TiO₂-coated Fe@CNPs, rapid and effective MALDI-TOF MS profiling of phosphopeptides was demonstrated in different model systems such as tryptic digests of β-casein, and complex β-casein/BSA mixture. The TiO₂-coated Fe@CNPs out-performed the commercial TiO₂-coated magnetic beads for detection of phosphopeptides from tryptic digests of β-casein/BSA mixture with a molar ratio of 1:100. The new TiO₂-coated magnetic probes were also proven to be applicable for real life samples. The magnetic TiO₂-coated Fe@CNPs were employed to selectively isolate phosphopeptides from tryptic digests of HeLa cell lysates and out-performed the commercial magnetic TiO₂ beads in the number of identified phosphopeptides and phosphorylation sites. In a 200-μg equivalent of HeLa cell lysates, we identified 1415 unique phosphopeptides and 1093 phosphorylation sites, indicating the good performance of the new approach.

Iron oxide nanoparticle coating of organic polymer‐based monolithic columns for phosphopeptide enrichment

A new monolithic capillary column with an iron oxide nanoparticle coating has been developed for selective and efficient enrichment of phosphopeptides. Iron oxide nanoparticles were prepared by a co-precipitation method and stabilized by citrate ions. A stable coating of nanoparticles was obtained via multivalent electrostatic interactions of citrate ions on the surface of iron oxide nanoparticles with a quaternary amine functionalized poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith. A high dynamic binding capacity of 86 μmol/mL column volume was measured with an adenosine-5'-triphosphate. Performance of the monolithic column was demonstrated with the efficient and selective enrichment of phosphopeptides from peptide mixtures of α-casein and β-casein digests and their MALDI/MS characterization in off-line mode.

Discontinuous pH gradient-mediated separation of TiO 2-enriched phosphopeptides

Global profiling of phosphoproteomes has proven to be a great challenge due to the relatively low stoichiometry of protein phosphorylation and poor ionization efficiency in mass spectrometers. Effective, physiologically relevant, phosphoproteome research relies on the efficient phosphopeptide enrichment from complex samples. Immobilized metal affinity chromatography and titanium dioxide chromatography can greatly assist selective phosphopeptide enrichment. However, the complexity of resultant enriched samples is often still high, suggesting that further separation of enriched phosphopeptides is required. We have developed a pH gradient elution technique for enhanced phosphopeptide identification in conjunction with titanium dioxide chromatography. Using this process, we demonstrated its superiority to the traditional “one-pot” strategies for differential protein identification. Our technique generated a highly specific separation of phosphopeptides by an applied pH gradient between 9.2 and 11.3. The most efficient elution range for high-resolution phosphopeptide separation was between pHs 9.2 and 9.4. High-resolution separation of multiply phosphorylated peptides was primarily achieved using elution ranges greater than pH 9.4. Investigation of phosphopeptide sequences identified in each pH fraction indicated that phosphopeptides with phosphorylated residues proximal to acidic residues, including glutamic acid, aspartic acid, and other phosphorylated residues, were preferentially eluted at higher pH values.

Exploring the Human Leukocyte Phosphoproteome Using a Microfluidic Reversed-Phase−TiO2−Reversed-Phase High-Performance Liquid Chromatography Phosphochip Coupled to a Quadrupole Time-of-Flight Mass Spectrometer

The study of protein phosphorylation events is one of the most important challenges in proteome analysis. Despite the importance of phosphorylation for many regulatory processes in cells and many years of phosphoprotein and phosphopeptide research, the identification and characterization of phosphorylation by mass spectrometry is still a challenging task. Recently, we introduced an approach that facilitates the analysis of phosphopeptides by performing automated, online, TiO(2) enrichment of phosphopeptides prior to mass spectrometry (MS) analysis. The implementation of that method on a "plug-and-play" microfluidic high-performance liquid chromatography (HPLC) chip design will potentially open up efficient phosphopeptide enrichment methods enabling phosphoproteomics analyses by a broader research community. Following our initial proof of principle, whereby the device was coupled to an ion trap, we now show that this so-called phosphochip is capable of the enrichment of large numbers of phosphopeptides from complex cellular lysates, which can be more readily identified when coupled to a higher resolution quadrupole time-of-flight (Q-TOF) mass spectrometer. We use the phosphochip-Q-TOF setup to explore the phosphoproteome of nonstimulated primary human leukocytes where we identify 1012 unique phosphopeptides corresponding to 960 different phosphorylation sites providing for the first time an overview of the phosphoproteome of these important circulating white blood cells.

Highly Specific Capture and Direct MALDI MS Analysis of Phosphopeptides by Zirconium Phosphonate on Self-Assembled Monolayers

The dynamic range and low stoichiometry of protein phosphorylation frequently demands the enrichment of phosphorylated peptides from protein digests prior to mass spectrometry. Several techniques have been reported in literature for phosphopeptide enrichment, including metal oxides such as TiO(2) and ion metal affinity chromatography (IMAC). While the metal oxides have been used with reasonable success, IMAC has suffered from reduced selectivity and poor reproducibility. In this report, we present the first demonstration of the use of immobilized zirconium on a phosphonate-terminated self-assembled monolayer (SAM) for specific phosphopeptide capture and direct analysis by MALDI MS. By using the herein described functionalized-surface-based technology, efficient enrichment of phosphopeptides in different standard test systems such as alpha- or beta-casein digests or synthetic phosphopeptides spiked in nonphosphorylated protein digest has been demonstrated. The limit of detection for a beta-casein phosphopeptide was assessed to be at the low femtomole level. Compared to other state-of the-art technologies, like use of TiO(2) and Fe-IMAC, the presented technique demonstrated a superior performance with respect to specificity and bias with respect to singly or multiply phosphorylated peptides. Additionally, this platform was also successfully applied for ESI sample preparation, providing detailed sequence information of the investigated phosphopeptide. This technology was also proven to be applicable for real life samples such as phosphorylation site analysis of recombinant human MAPK1 and HSP B1 isolated from a 2D-gel spot by phosphopeptide enrichment and direct MALDI MS/MS.