Low-abundance Phosphopeptides Research Articles

Development of a high-throughput kinase activity platform using nanoLC-MS/MS with DIA approach for studying the anti-cancer mechanism of Taxol in ovarian cancer

BackgroundProtein phosphorylation by protein kinases plays a pivotal role in increasing protein diversity, thereby influencing various cellular functions. However, due to the relatively low abundance of phosphopeptides in a mixture of peptides and the ion-suppression effect of non-phosphorylated peptides, the detection of phosphopeptides is not straightforward. ResultsHerein, a quantitative high-throughput platform was developed for assessing multikinase activity using nano-LC-MS/MS with a data-independent acquisition (DIA) approach. This platform was evaluated by studying the kinase activity in Taxol-treated SKOV3 cells. A library containing 38 peptide substrates was designed and analyzed to determine the activities of major kinases involved in cancer development. Twenty-three synthetic peptide substrates showed significant phosphorylation changes in triplicate biological experiments, as further verified by western blotting. Our findings reveal that Taxol suppressed SKOV3 cell survival by activating AMPK and suppressing the PI3K-Akt-dependent pathway, ultimately leading to mTOR inhibition. Furthermore, in combination with ERK, Akt, SGK, CK1, and ErbB2 inhibitors, Taxol enhanced the inhibitory effect on ovarian cancer. SignificanceThis platform can be an attractive approach for large-scale kinase activity studies to comprehensively uncover the mechanisms of drug-disease treatment and to investigate a more effective therapy strategy.

Polyoxometalate functionalized magnetic metal-organic framework with multi-affinity sites for efficient enrichment of phosphopeptides.

The reasonable design of metal-organic framework (MOF)-derived nanomaterial has important meaning in increasing the enrichment efficiency in the study of protein phosphorylation. In this work, a polyoxometalate (POM) functionalized magnetic MOF nanomaterial (Fe3O4@MIL-125-POM) was designed and fabricated. The nanomaterial with multi-affinity sites (unsaturated metal sites and metal oxide clusters) was used for the enrichment of phosphopeptides. Fe3O4@MIL-125-POM had high-efficient enrichment performance towards phosphopeptides (selectivity, a mass ratio of bovine serum albumin/α-casein/β-casein at 5000:1:1; sensitivity, 0.1 fmol; satisfactory repeatability, ten times). Furthermore, Fe3O4@MIL-125-POM was employed to enrich phosphopeptides from non-fat milk digests, saliva, serum, and A549 cell lysate. The enrichment results illustrated the great potential of Fe3O4@MIL-125-POM for efficient identification of low-abundance phosphopeptides.

Cobalt Phthalocyanine-Modified Magnetic Metal-Organic Frameworks for Specific Enrichment of Phosphopeptides.

For mass spectrometry (MS)-based phosphoproteomics studies, sample pretreatment is an essential step for efficient identification of low-abundance phosphopeptides. Herein, a cobalt phthalocyanine-modified magnetic metal-organic framework (MOF) (Fe3O4@MIL-101-CoPc) was prepared and applied to enrich phosphopeptides before MS analysis. Fe3O4@MIL-101-CoPc exhibited an excellent magnetic response (74.98 emu g-1) and good hydrophilicity (7.75°), which were favorable for the enrichment. Fe3O4@MIL-101-CoPc showed good enrichment performance with high selectivity (1:1:5000), sensitivity (0.1 fmol), reusability (10 circles), and recovery (91.3%). Additionally, the Fe3O4@MIL-101-CoPc-based MS method was able to successfully detect 827 phosphopeptides from the A549 cell lysate, demonstrating a high enrichment efficiency (89.3%). This study promotes the application of postfunctionalized MOFs for phosphoproteomics analysis.

An amino-rich polymer-coated magnetic nanomaterial for ultra-rapid separation of phosphorylated peptides in the serum of Parkinson's disease patients.

The elucidation of disease pathogenesis can be achieved by analyzing the low-abundance phosphopeptides in organisms. Herein, we developed a novel and easy-to-prepare polymer-coated nanomaterial. By improving the hydrophilicity and spatial conformation of the material, we effectively enhanced the adsorption of phosphopeptides and demonstrated excellent enrichment properties. The material was able to successfully enrich the phosphopeptides in only 1min. Meanwhile, the material has high selectivity (1:2000), good loading capacity (100μg/mg), excellent sensitivity (0.5 fmol), and great acid and alkali resistance. In addition, the material was applied to real samples, and 70 phosphopeptides were enriched from the serum of Parkinson's disease (PD) patients and 67 phosphopeptides were enriched from the serum of normal controls. Sequences Logo showed that PD is probably associated with threonine, glutamate, serine, and glutamine. Finally, gene ontology (GO) analysis was performed on phosphopeptides enriched in PD patients' serum. The results showed that PD patients expressed abnormal expression of thecholesterol metabolic process and cell-matrix adhesion in thebiological process (BP), endoplasmic reticulum and lipoprotein in thecellular component (CC), and heparin-binding, lipid-binding, and receptor-binding in themolecular function (MF) as compared with normal individuals. All the experiments indicate that the nanomaterials have great potential in proteomics studies.

Facile fabrication of Ti4+-immobilized magnetic nanoparticles by phase-transitioned lysozyme nanofilms for enrichment of phosphopeptides.

In this study, titanium (IV)-immobilized magnetic nanoparticles (Ti4+-PTL-MNPs) were firstly synthesized via a one-step aqueous self-assembly of lysozyme nanofilms for efficient phosphopeptide enrichment. Under physiological conditions, lysozymes readily self-organized into phase-transitioned lysozyme (PTL) nanofilms on Fe3O4@SiO2 and Fe3O4@C MNP surfaces with abundant functional groups, including -NH2, -COOH, -OH, and -SH, which can be used as multiple linkers to efficiently chelate Ti4+. The obtained Ti4+-PTL-MNPs possessed high sensitivity of 0.01 fmol μL-1 and remarkable selectivity even at a mass ratio of β-casein to BSA as low as 1:400 for phosphopeptide enrichment. Furthermore, the synthesized Ti4+-PTL-MNPs can also selectively identify low-abundance phosphopeptides from extremely complicated human serum samples and their rapid separation, good reproducibility, and excellent recovery were also proven. This one-step self-assembly of PTL nanofilms facilitated the facile and efficient surface functionalization of various nanoparticles for proteomes/peptidomes.

Cotton Ti-IMAC: Developing Phosphorylated Cotton as a Novel Platform for Phosphopeptide Enrichment.

Protein phosphorylation is an important post-translational modification (PTM), which is involved in many important cellular functions. Understanding protein phosphorylation at the molecular level is critical to deciphering its relevant biological processes and signaling networks. Mass spectrometry (MS) has become a powerful tool for the comprehensive profiling of protein phosphorylation. Yet the low ionization efficiency and low abundance of phosphopeptides among complex biological samples make its MS analysis challenging; an enrichment strategy with high efficiency and selectivity is always necessary prior to MS analysis. In this study, we developed a phosphorylated cotton-fiber-based Ti(IV)-IMAC material (termed as Cotton Ti-IMAC) that can serve as a novel platform for phosphopeptide enrichment. The cotton fiber can be effectively grafted with phosphate groups covalently in a single step, where the titanium ions can then be immobilized to enable capturing phosphopeptides. The material can be prepared using cost-effective reagents within only 4 h. Benefiting from the flexibility and filterability of cotton fibers, the material can be easily packed as a spin-tip and make the enrichment process convenient. Cotton Ti-IMAC successfully enriched phosphopeptides from protein standard digests and exhibited a high selectivity (BSA/β-casein = 1000:1) and excellent sensitivity (0.1 fmol/μL). Moreover, 2354 phosphopeptides were profiled in one LC-MS/MS injection after enriching from only 100 μg of HeLa cell digests with an enrichment specificity of up to 97.51%. Taken together, we believe that Cotton Ti-IMAC can serve as a widely applicable and robust platform for achieving large-scale phosphopeptide enrichment and expanding our knowledge of phosphoproteomics in complex biological systems.

A novel carbon-based material with titanium and zirconium ions etched on hollow mesoporous carbon tubes for specific capture of phosphopeptides and exosomes

The analysis of low abundance phosphopeptides in organisms and specific capture exosomes are crucial for unraveling the pathogenesis of diseases. For this reason, titanium-zirconium ions and highly biocompatible dopamine and polyimide tubes (PITs) were introduced, and a novel carbon-based material with titanium and zirconium ions etched on hollow mesoporous carbon tubes (HMCT), denoted as G@C@Ti–Zr-HMCT, comes into being after high-temperature calcination. Attributing to the tightly bound titanium and zirconium ions to HMCT and the high carbon content of the polydopamine carbonaceous layer, G@C@Ti–Zr-HMCT displays satisfactory capability of enriching phosphopeptides with satisfactory detection limit (0.2 fmol), extraordinary selectivity (1:2000), and good loading capacity (100 μg/mg). In addition, 25 phosphopeptides related to 25 phosphoproteins from the serum of Parkinson's disease (PD) patients and 30 phosphopeptides attributed to 26 phosphoproteins from the serum of healthy individuals were enriched by G@C@Ti–Zr-HMCT, respectively. In addition, bioinformatics analysis of the above results revealed that PD were associated with serine, threonine, and leucine of high frequency, blood coagulation in BP, Golgi apparatus and mitochondrial outer membrane in CC, and heparin binding in MF. Moreover, the phospholipid bilayer of exosomes and metallic titanium and zirconium ions interact to produce the following results: this highly biocompatible carbon-based material was successfully applied to capture exosomes, which offers a promising platform for isolating exosomes. To sum up, these delightful results confirmed without doubt that G@C@Ti–Zr-HMCT has enjoyed a splendiferous future in the specific capture of phosphopeptides and exosomes isolation.

Ti(IV) immobilized bisphosphate fructose-modified magnetic Zr metal organic framework (MOF) for specific enrichment of phosphopeptides

Highly selective enrichment of low abundance phosphopeptides from intricate biological samples before mass spectrometry detection is a vital prerequisite for in-depth phosphoproteomics. Herein, a magnetic material mMOF-FBP-Ti4+ is fabricated by grafting UiO-66-NH2 metal–organic framework (Zr-MOF) onto the surface of Fe3O4, and subsequently immobilizing Ti(IV) on the mMOF with fructose 1,6-bisphosphate as chelating ligands. The obtained mMOF-FBP-Ti4+ showed large specific surface area (202.4 m2 g−1) and superparamagnetism (47.5 emu/g). The intrinsic Zr − O clusters of mMOF and the immobilized Ti(IV) ions on the surface of mMOF could interact with phosphopeptides as metal oxide affinity chromatography (MOAC) and immobilized metal affinity chromatography (IMAC), respectively. The introduction of fructose 1,6-bisphosphate also improved the hydrophilicity of the material, which enhanced the hydrophilic chromatography interaction (HILIC) between mMOF-FBP-Ti4+ and phosphopeptides. The above strong affinities provided low limit of detection (0.2 fmol μL−1), high selectivity (α-/β-casein/BSA tryptic digests with a mass ratio of 1:1:500), great reusability (three times circles) and good postenrichment recovery (90.2 %) towards phosphopeptides. Furthermore, the mMOF-FBP-Ti4+ was successfully applied to capture phosphopeptides from non-fat milk tryptic digest and human saliva, indicating its great potential for highly efficient enrichment of low-abundance phosphopeptides in complex biological samples.

Epitaxial Growth of Guanidyl-Functionalized Magnetic Metal-Organic Frameworks with Multiaffinity Sites for Selective Capture of Global Phosphopeptides.

The flexible and controlled synthesis of metal-organic framework (MOF)-derived hybrid nanostructures is of great significance in fine tuning of their enrichment performance in large-scale and in-depth phosphoproteome analysis. Herein, a magnetic guanidyl-functionalized MOF hybrid coating with multiaffinity sites, denoted as Fe3O4@G-ZIF-8, was fast fabricated via a one-pot epitaxial growth strategy for the first time and applied for selective and highly efficient enrichment of global phosphopeptides. The intrinsic unsaturated metal sites of ZIF-8 endow the surface-mounted MOF coatings with immobilized metal ion affinity chromatography interaction with multiphosphorylated peptides. The oriented anchoring of bifunctional guanidineacetic acid on the magnetic MOF nanospheres provides additional affinity sites (guanidyl groups) for specific recognition of phosphopeptides by "salt bridge" interaction, as well as active site carboxyl groups for the coordination with the metal ions. The as-prepared Fe3O4@G-ZIF-8 exhibits large surface area (382.5 m2 g-1), good superparamagnetic property (41.6 emu g-1) and stability, and size-exclusion effect (1.73 nm), which can serve as a specific adsorbent for global phosphopeptide analysis with satisfactory selectivity, great detection sensitivity (1 fmol), and rapid magnetic separation. Moreover, the successful application of Fe3O4@G-ZIF-8 for selective capture of both multi- and mono-phosphopeptides from human saliva and serum demonstrated the great potential of magnetic surface-mounted MOF coatings in effective identification of low-abundance phosphopeptides by matrix-assisted laser desorption ionization time-of-flight mass spectrometry from complicated biological matrices.

Design of a Spiropyran-Based Smart Adsorbent with Dual Response: Focusing on Highly Efficient Enrichment of Phosphopeptides.

Smart responsive materials have attractive application prospects due to their tunable behaviors. In this work, we design novel spiropyran (SP)-based magnetic nanoparticles (MNP-SP) with dual response to ultraviolet light and pH and apply them to the enrichment of phosphopeptides. SP is modified on the surface of magnetic nanoparticles through a simple esterification reaction, based on which an MNP-SP-MS phosphopeptide identification platform is established. The capture and release of phosphopeptides are facilely adjusted by changing external light and the pH of the solution. The smart responsive MNP-SP has fast magnetic response performance, high sensitivity (detection limit of 0.4 fmol), and good reusability (6 cycles). In addition, MNP-SP is used for the enrichment of phosphopeptides in skimmed milk, human saliva, and human serum samples, indicating that it is an ideal adsorbent for enriching low-abundance phosphopeptides in complex biological environments.

In Situ Controllable Fabrication of Two-Dimensional Magnetic Fe3O4/TiO2@Ti3C2Tx Composites for Highly Efficient Phosphopeptides Enrichment.

Highly efficient enrichment of phosphopeptides is of great significance for phosphoproteomics-related biological and pathological processes research, but it remains challenging due to the lack of affinity materials which hold high enrichment efficiency and capacity. Ti3C2Tx MXene, a novel two-dimensional material with outstanding physicochemical properties, has attracted wide research interests for application in various fields. However, there are few reports on the use of MXene-derived materials for phosphopeptides separation in the biomedical field. In this work, we proposed a facile one-pot method that in situ oxidation and modification of Ti3C2Tx MXene, to prepare two-dimensional (2D) magnetic Fe3O4/TiO2@Ti3C2Tx composites for potential application in phosphopeptides enrichment. Benefiting from the outstanding magnetic responsiveness and multiaffinity sites (Ti-O, Fe-O, and NH2 groups), the Fe3O4/TiO2@Ti3C2Tx composites possessed excellent enrichment performance with high sensitivity (0.1 fmol μL-1), excellent selectivity (β-casein: bovine serum albumin = 1:5000, molar ratio), good repeatability (5 times), and high enrichment capacity (200 mg g-1). Moreover, the results of selective enrichment of phosphopeptides from nonfat milk, human saliva, human serum, and rat brain lysates indicated the great potential of Fe3O4/TiO2@Ti3C2Tx composites in low-abundance phosphopeptides enrichment from complex biological samples. This work has put forward a versatile method to prepare magnetic MXene composites and promoted the use of MXene composites in phosphoproteome in biomedicine.

One-step preparation of carbonaceous spheres rich in phosphate groups via hydrothermal carbonization for effective phosphopeptides enrichment

A green strategy was developed to prepare carbonaceous spheres rich in phosphoric acid groups on the surface with D-Glucose 6-phosphate sodium salt (called G6PNa2) as a sole carbon source through one-step hydrothermal carbonization method. The method is simple and facile and meets the standards of green chemistry as water is the sole solvent employed. Following the hydrothermal carbonization synthesis, the carbonaceous spheres were further functionalized with Ti4+. The main factors including reaction temperature, reaction time, and concentration of G6PNa2 were systematically studied in order to obtain the desirable morphology and the optimum phosphopeptides enrichment, for the resulting Ti4+ functionalized carbonaceous spheres (CS-Ti4+). The performance evaluation of the CS-Ti4+ prepared under the optimum conditions demonstrated excellent selectivity (1:1000), low detection limit (1 fmol) and high recovery rate (85%) towards phosphopeptides. Furthermore, 24 low-abundance phosphopeptides were captured from human saliva using CS-Ti4+, indicating its great potential in mass spectrometry-based phosphoproteome studies.

Application of SILAC Labeling in Phosphoproteomics Analysis.

The analysis of disease-related changes in the phosphorylation status of cellular signal transduction networks is of major interest to biomedical researchers. Mass spectrometry-based proteomics allows the analysis of phosphorylation in a global manner. However, several technical challenges need to be addressed when the phosphorylation of proteins is analyzed. Low-abundant phosphopeptides need to be enriched before analysis, thereby introducing additional steps in sample preparation. Consequently, the applied quantification strategies should be robust towards elaborate sampling handling, rendering label-based quantification strategies the methods of choice in many experiments. Here, we present a protocol for SILAC labeling and the subsequent isolation of phosphopeptides using TiO2 affinity chromatography. We outline the corresponding LC-MS/MS analysis and the essential steps of data processing.

Glass Fiber-Supported Hybrid Monolithic Spin Tip for Enrichment of Phosphopeptides from Urinary Extracellular Vesicles.

Extracellular vesicles (EVs) are attracting increasing interest with their intriguing role in intercellular communications. Protein phosphorylation in EVs is of great importance for understanding intercellular signaling processes. However, the study of EV phosphoproteomics is impeded by their relatively low amount in limited clinical sample volumes, and it is necessary to have a sensitive and efficient enrichment method for EV phosphopeptides. Herein, a novel Ti(IV)-functionalized and glass fiber-supported hybrid monolithic spin tip, termed PhosTip, was prepared for enriching phosphopeptides from urinary EVs. Glass fiber as the stationary phase positions the hybrid monolith in a standard pipet tip and prevents the monolith from distortion during experiments. The preparation procedure for the new PhosTip is simple and time-saving. The hybrid monolithic PhosTip provides excellent enrichment efficiency of low-abundance phosphopeptides from cell digests and urinary EVs with minimum contamination and sample loss. Using the PhosTip, we demonstrate that 5373 and 336 unique phosphopeptides were identified from 100 and 1 μg of cell lysates, while 3919 and 217 unique phosphopeptides were successfully identified from 10 and 1 mL of urinary samples, respectively. The PhosTip was finally applied to enrich phosphopeptides in urine EVs from prostate cancer patients and healthy controls and quantify 118 up-regulated proteins with phosphosites in prostate cancer samples. These results demonstrated that the PhosTip could be a simple and convenient tool for enriching phosphopeptides from clinical samples and for broader applications in biomarker discovery.

Targeted immobilization of titanium (IV) on magnetic mesoporous nanomaterials derived from metal-organic frameworks for high-efficiency phosphopeptide enrichment in biological samples.

A selectively modified porous metal/carbon nanocomposite was fabricated to enhance the enrichment of low-abundance phosphopeptides from biological samples. The carbon matrix derived from the metal-organic framework provides a suitable pore size to allow the diffusion of peptides, while the deliberately modified metal nanoparticles within the pores enhance their interaction with the phosphopeptides. This nanocomposite shows extremely high enrichment selectivity for phosphopeptides in the MALDI-TOF MS detection, even when the molar ratio of α-casein digests versus bovine serum albumin digests was up to about 1:20,000. By combining such nanocomposite with nano-LC-MS/MS, 4556 unique phosphopeptides were identified with high selectivity (95.2%) from HeLa cell extracts. Furthermore, phosphopeptides from prostate tissue digests were also determined. A total of 277 and 1242 phosphopeptides were identified from normal and tumor tissues of a patient with prostate cancer, respectively. This indicates that phosphorylation and prostate cancer can be related to each other.

In situ synthesis of a novel metal oxide affinity chromatography affinity probe for the selective enrichment of low-abundance phosphopeptides.

Due to the dynamic nature of phosphorylation states and the low stoichiometry of phosphopeptides, it is still a challenge to efficiently capture phosphopeptides from complex biological samples before mass spectrometry analysis. Among the enrichment strategies, metal oxide affinity chromatography (MOAC) is one of the most widely used and the one with the most potential. It is based on reversible Lewis acid-base interactions between the metal oxides and the negatively charged phosphate groups to achieve the specific selection of phosphopeptides. A novel MOAC affinity probe, denoted as G@PDA@ZrO2 , was successfully synthesized by in situ grafting ZrO2 onto the surface of graphene (G) modified with polydopamine (PDA). The novel MOAC probe thus obtained was used for phosphopeptide enrichment. This novel MOAC affinity probe when used to selectively enrich phosphopeptides from standard protein digest solutions exhibited a high selectivity (β-casein:bovine serum albumin = 1:1000), a low detection limit (4 fmol), and a high loading capacity (400 mg/g). At the same time, the experimental results proved that G@PDA@ZrO2 had great recyclability (five cycles), stability, and reproducibility. Subsequently, G@PDA@ZrO2 was applied to enrich phosphopeptides from human saliva and human serum, in which 25 and 4 phosphopeptide peaks, respectively, were detected. This novel MOAC affinity probe (G@PDA@ZrO2 ) showed good performance in enriching phosphopeptides. Thus, G@PDA@ZrO2 has good potential in phosphopeptidomics analysis.

Facile synthesis of guanidyl-based magnetic ionic covalent organic framework composites for selective enrichment of phosphopeptides

Protein phosphorylation plays vital roles in the regulation of various biological processes involving in protein folding, molecular recognition, cell growth, and metabolism. It is prerequisite to develop effective enrichment methods of trace phosphopeptides before mass spectrometry (MS) analysis. In this study, we proposed a facile strategy to synthesize magnetic ionic covalent organic frameworks (Fe3O4@iCOFs) for the capture of phosphopeptides with guanidyl as the ionic ligand instead of the post-synthetic functionalization strategy. The developed Fe3O4@iCOFs contain a large amount of amino groups, positive charge, as well as owned superparamagnetism. The enrichment of phosphopeptides is based on the electrostatic interaction and hydrogen bonds formed between phosphate groups and guanidyl groups. By combing with MS determinations, high sensitivity of phosphopeptides (the lowest detection amount being 0.4 fmol) was achieved. The obtained material provided selective enrichment capacity of phosphopeptides from non-fat milk digest and HeLa cells, showing great potential in the detection of low-abundance phosphopeptides in complex real samples.

High-Throughput Characterization of Histidine Phosphorylation Sites Using UPAX and Tandem Mass Spectrometry.

Liquid chromatography (LC)-tandem mass spectrometry (MS/MS) is key for the characterization of phosphorylation sites in a high-throughput manner, and its application has proven essential to elucidate the phosphoproteome of many biological systems. Following proteolytic digestion of proteins extracted from tissues or cells, phosphopeptides are typically enriched by affinity chromatography using TiO2 or metal-ions (e.g., Fe3+) coupled to solid-phase materials, prior to LC-MS/MS analysis. Separation of relatively low abundance phosphopeptides from nonphosphorylated peptides in these types of extremely complex mixtures is essential to maximize coverage of the phosphoproteome. Maintaining acidic conditions during these IMAC or TiO2-based enrichment minimizes the concurrent unwanted binding of highly acidic peptides. However, while peptides containing phosphomonoesters, namely, phosphoserine (pSer), phosphothreonine (pThr), and phosphotyrosine (pTyr), are stable under these acidic binding conditions, phosphopeptides containing acid-labile phosphate group such as phosphohistidine (pHis), are not. Consequently, hydrolysis of these types of phosphopeptides occurs during standard phosphopeptide enrichment, and subsequent phosphosite identification by LC-MS/MS is severely compromised. Here we describe UPAX, unbiased phosphopeptide enrichment using strong anion exchange, for the separation of both acid-stable (pSer, pThr, pTyr) and acid-labile phosphopeptides (including those containing pHis) from nonphosphorylated peptides. We outline how implementation of UPAX prior to a minimally modified standard proteomics workflow can be used to identify sites of pHis as well as other acid-labile, as well as acid-stable phosphosites.

Development of amphiphile 4-armed PEO-based Ti4+ complex for highly selective enrichment of phosphopeptides

Protein phosphorylation is a reversible and important post-translational modification. Identification of phosphopeptides without enrichment is difficult for the low-abundance of phosphopeptides in real complex biological samples. Therefore, the effective and selective concentration of phosphopeptides prior to proteomic identification by mass spectrometer is necessary. In this study, we synthesized a novel titanium-based immobilized metal ion affinity chromatography material for highly selective enrichment of phosphopeptides. To improve material hydrophilia to the maximum extent, titanium ions were immobilized on the 4-armed Poly(ethylene oxide)(4μ-PEO-Ti4+), a totally soluble polymer with large molecular weight (20000 g/mol). The 4μ-PEO-Ti4+ was used to enrich phosphopeptides from tryptic digests of standard proteins and real complex biological samples, followed by MALDI-TOF MS analysis. In enrichment of phosphopeptides from 4 pmol β-casein, the 4μ-PEO-Ti4+ performed the best property with starting material of 99–132 μg, loading buffer of 50% ACN/5% TFA (v/v), elution buffer of 10% NH3·H2O (v/v) and elution time of 30 min. The 4μ-PEO-Ti4+ has a superior detection sensitivity as low as 2 fmol for phosphopeptides. The high selectivity of 4μ-PEO-Ti4+ allows a deep enrichment of phosphopeptides of β-casein from a mixture with BSA of 1000-fold abundant. The 4μ-PEO-Ti4+ shows great stability and endurability and can be recycled up to at least 5 times. In addition, 4μ-PEO-Ti4+ could detect 10 and 15 phosphopeptides from non-fat milk and nonenzymatic human saliva, respectively. In total, 4μ-PEO-Ti4+ is a novel excellent material which shows great sensitive and selective enrichment of low-abundance phosphopeptides in real complex biological samples.

Postsynthetic Functionalization of Zr4+-Immobilized Core-Shell Structured Magnetic Covalent Organic Frameworks for Selective Enrichment of Phosphopeptides.

Chemical modification of covalent organic frameworks (COFs) is indispensable for integrating functionalities of greater complexity and accessing advanced COF materials suitable for more potential applications. Reported here is a novel strategy for fabricating controllable core-shell structured Zr4+-immobilized magnetic COFs (MCNC@COF@Zr4+) composed of a high-magnetic-response magnetic colloid nanocrystal cluster (MCNC) core, Zr4+ ion-functionalized two-dimensional COFs as the shell by sequential postsynthetic functionalization and, for the first time, the application of the MCNC@COF@Zr4+ composites for efficient and selective enrichment of phosphopeptides. The as-prepared MCNC@COF@Zr4+ composites possess regular porosity with large surface areas, high Zr4+ loading amount, strong magnetic responsiveness, and good thermal/chemical stability, which can serve as an ideal adsorbent for selective enrichment of phosphopeptides and simultaneous size exclusion of biomacromolecules, such as proteins. The high detection sensitivity (10 fmol) together with the excellent recovery of phosphopeptides is also obtained. These outstanding features suggest that the MCNC@COF@Zr4+ composites are of great benefit for pretreatment prior to mass spectrometry analysis of phosphopeptides. In addition, the performance of the developed approach in selective enrichment of phosphopeptides from the tryptic digests of defatted milk and directly specific capture of endogenous phosphopeptides from human serum gives powerful proof for its high selectivity and effectiveness in identifying the low-abundance phosphopeptides from complicated biological samples. This study not only provides a strategy for versatile functionalization of magnetic COFs but also opens a new avenue in their use in phosphoproteome analysis.