Efficient Enrichment Of Phosphopeptides Research Articles

Synthesis of Ti4+ modified COF-based polymer for efficient enrichment of phosphopeptides in colorectal cancer serum

In this study, we propose a Ti4+ modified COF-based polymers (COF-PA-Ti4+) with a facile synthesis method for efficient enrichment of phosphopeptides in colorectal cancer serum. Thanks to the abundant modification sites provided by radical polymerization on COF, as well as the excellent hydrophilicity of vinyl phosphate and the strong chelating ability of Ti4+, COF-PA-Ti4+ have superior performance on phosphopeptides enrichment. Employing COF-PA-Ti4+ as the enrichment material, the detection method exhibits high selectivity (1:1000), low detection limit (low as 0.005 fmol μL−1), high size-exclusion effect (1:10,000), excellent reusability (10 cycles), good recovery (98.7 ± 0.6 %), and loading capacity (50 mg g−1), with analysis performed via MALDI-TOF MS. Most importantly, after enriching phosphopeptides from the serum of colorectal cancer (CRC) patients and healthy individuals by COF-PA-Ti4+, we discovered significant differences in the phosphopeptides between the two groups, validating the feasibility of this approach. Taken together, these results confirm that COF-PA-Ti4+ has an outstanding contribution to the precise capture of phosphopeptides and provides an innovative approach for rapid diagnosis of early CRC.

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.

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.

Packed in-tube solid-phase microextraction with mesoporous zirconium titanium oxides for highly efficient phosphopeptide enrichment and analysis

Phosphorylated peptides are crucial for understanding biological regulation processes. However, the detection of phosphorylated peptides remains challenging in mass spectrometry (MS) due to their high hydration, low abundance, and the significant signal suppression caused by non-phosphorylated peptides. It is essential to enhance the efficiency of phosphopeptide enrichment from complex biological samples to achieve comprehensive analysis and identification of phosphopeptides. Herein, a packed in-tube solid-phase microextraction (IT-SPME) method was developed based on mesoporous zirconium titanium oxides for the selective enrichment and analysis of phosphopeptides. The IT-SPME system achieved the efficient capture of phosphopeptides from complex biological samples followed by high-sensitivity detection (5 fmol). This exceptional performance can be attributed to the high surface area (289.77 m2/g) of mesoporous zirconium titanium oxides, which provided abundant active sites for a high loading capacity. The zirconium titanium oxides with a mesoporous structure (∼4.5 nm) was prepared through a sol–gel and solvothermal process. Importantly, this method proved effective in enriching phosphopeptides from practical samples such as non-fat milk and egg white. It is the first attempt to employ a mesoporous packed IT-SPME approach for phosphopeptide enrichment. These results demonstrate the significant application potential of mesoporous zirconium titanium oxides in phosphoproteomics.

Dielectric barrier discharge induced Oxid-Ti3C2Tx/UIO-66-NH2 composites for efficient phosphopeptides enrichment

Enrichment of phosphopeptides is a crucial step in phosphoproteomics study. At present, combination of multiple affinity chromatography is regard as an efficient phosphopeptide enrichment strategy. Therefore, designing and preparing composites with multiple affinity sites is of great significance. Herein, the Oxid-Ti3C2Tx/UIO-66-NH2 composites with multiple affinity sites are rapidly synthesized by dielectric barrier discharge (DBD) technology at low temperature and atmospheric pressure. The in-situ nucleation of UIO-66-NH2 and TiO2 can be flexibly regulated through the feed ratio of MOF precursors and the DBD duration individually. The UIO-66-NH2 has large specific surface area, high porosity and plentiful Zr-O clusters. Oxid-Ti3C2Tx provides abundant exposed Ti sites and Ti-O clusters. Based on these, Oxid-Ti3C2Tx/UIO-66-NH2 composites exhibited high sensitivity (0.1 fmol μL−1), satisfactory selectivity (BSA: α-casein = 100: 1, molar ratio) and superior repeatability for phosphopeptide enrichment. In addition, the composites performed well in biological samples like nonfat milk (24 phosphopeptides), human saliva (15 endogenous phosphopeptides) and human serum (all 4 endogenous phosphopeptides), demonstrating a huge application potential in phosphoproteomics study. More importantly, this study opens a new path to synthesize MXene/MOF composites with controllable oxidation degree and flexible compositions regulation efficiently, revealing its broad application prospects in various fields.

Metal-phenolic networks modified two-dimensional magnetic MXene-based composites for highly efficient enrichment of phosphopeptides

Efficient and specific enrichment of phosphopeptides is a precondition for the research of phosphoproteomics-related processes, while the development of more superior affinity materials remains a long-term challenge. In this work, Zr-tannic acid metal-phenolic networks (MPNs) modified magnetic composites based on two-dimensional MXene substrates, denoted as Ti3C2Tx@Fe3O4@MPNs-TA/ZrIV, were facilely prepared and innovatively applied for phosphopeptides enrichment. By virtue of large specific surface, excellent magnetic responsiveness, and massive affinity sites provided by TA/ZrIV-based MPNs, the Ti3C2Tx@Fe3O4@MPNs-TA/ZrIV composites exhibited outstanding enrichment performance with high sensitivity (1 fmol μL−1), excellent selectivity (β-casein: bovine serum albumin = 1:5000, molar ratio), and satisfactory repeatability (5 times). Encouraged by the remarkable enrichment results, we further employed the Ti3C2Tx@Fe3O4@MPNs-TA/ZrIV composites to detect phosphopeptides from non-fat milk and human saliva, which confirmed the great potential of Ti3C2Tx@Fe3O4@MPNs-TA/ZrIV composites in phosphopeptide enrichment from intricate biological specimens. The enrichment results demonstrate that Ti3C2Tx@Fe3O4@MPNs-TA/ZrIV composites are ideal candidates for the research of phosphoproteomics and also promote the application of surface-functionalized MXene-based materials in the biomedical field.

Profiling Phosphoproteome Landscape in Circulating Extracellular Vesicles from Microliters of Biofluids through Functionally Tunable Paramagnetic Separation.

Many biological processes are regulated through dynamic protein phosphorylation. Monitoring disease-relevant phosphorylation events in circulating biofluids is highly appealing but also technically challenging. We introduce here a functionally tunable material and a strategy, extracellular vesicles to phosphoproteins (EVTOP), which achieves one-pot extracellular vesicles (EVs) isolation, extraction, and digestion of EV proteins, and enrichment of phosphopeptides, with only a trace amount of starting biofluids. EVs are efficiently isolated by magnetic beads functionalized with TiIV ions and a membrane-penetrating peptide, octa-arginine R8 + , which also provides the hydrophilic surface to retain EV proteins during lysis. Subsequent on-bead digestion concurrently converts EVTOP to TiIV ion-only surface for efficient enrichment of phosphopeptides for phosphoproteomic analyses. The streamlined, ultra-sensitive platform enabled us to quantify 500 unique EV phosphopeptides with only a few μL of plasma and over 1200 phosphopeptides with 100 μL of cerebrospinal fluid (CSF). We explored its clinical application of monitoring the outcome of chemotherapy of primary central nervous system lymphoma (PCNSL) patients with a small volume of CSF, presenting a powerful tool for broad clinical applications.

Profiling Phosphoproteome Landscape in Circulating Extracellular Vesicles from Microliters of Biofluids through Functionally Tunable Paramagnetic Separation

AbstractMany biological processes are regulated through dynamic protein phosphorylation. Monitoring disease‐relevant phosphorylation events in circulating biofluids is highly appealing but also technically challenging. We introduce here a functionally tunable material and a strategy, extracellular vesicles to phosphoproteins (EVTOP), which achieves one‐pot extracellular vesicles (EVs) isolation, extraction, and digestion of EV proteins, and enrichment of phosphopeptides, with only a trace amount of starting biofluids. EVs are efficiently isolated by magnetic beads functionalized with TiIV ions and a membrane‐penetrating peptide, octa‐arginine R8+, which also provides the hydrophilic surface to retain EV proteins during lysis. Subsequent on‐bead digestion concurrently converts EVTOP to TiIV ion‐only surface for efficient enrichment of phosphopeptides for phosphoproteomic analyses. The streamlined, ultra‐sensitive platform enabled us to quantify 500 unique EV phosphopeptides with only a few μL of plasma and over 1200 phosphopeptides with 100 μL of cerebrospinal fluid (CSF). We explored its clinical application of monitoring the outcome of chemotherapy of primary central nervous system lymphoma (PCNSL) patients with a small volume of CSF, presenting a powerful tool for broad clinical applications.

Light, pH, and Temperature Triple-Responsive Magnetic Composites for Highly Efficient Phosphopeptide Enrichment.

Smart materials can dynamically and reversibly change their structures and functions in response to external stimuli. In this study, we designed a smart magnetic composite (MNP-pSPA-b-pNIPAm) with a triple response to ultraviolet (UV) light, pH, and temperature. Relying on the response of spiropyranyl acrylate (SPA) and N-isopropylacrylamide (NIPAm) to external stimuli (light, pH, and temperature), MNP-pSPA-b-pNIPAm was used for the controlled capture and release of phosphopeptides. The established phosphopeptide enrichment platform exhibits high sensitivity (detection limit of 0.04 fmol), high selectivity (BSA/β-casein, 1000:1), and good reusability (6 cycles). In addition, the method was also applied to the enrichment of phosphopeptides in real samples (skim milk, human saliva, and serum), demonstrating the feasibility of this method for phosphoproteomic analysis. After enriching from human nonsmall cell lung cancer cell (A549) lysates with MNP-pSPA-b-pNIPAm, 2595 phosphopeptides corresponding to 2281 phosphoproteins were identified. The novel responsive enrichment probe is highly specific for phosphoproteomic analysis and provides an effective method for studying the significance of protein phosphorylation in complex biological samples.

Post-synthesis of a titanium-rich magnetic COF nanocomposite with flexible branched polymers for efficient enrichment of phosphopeptides from human saliva and serum.

A Ti4+-functionalized magnetic covalent organic framework material with flexible branched polymers (mCOF@ε-PL@THBA-Ti4+) built via an immobilized metal ion affinity chromatography (IMAC) enrichment strategy was proposed through post-synthesis modification. Hydrophilic ε-poly-L-lysine (ε-PL) rich in amino active groups was first introduced in the fabrication of the phosphopeptide enrichment material to increase the hydrophilicity while providing more functional modification pathways of the material. 2,3,4-Trihydroxy-benzaldehyde (THBA) provides abundant binding sites for the immobilization of numerous Ti4+, which is advantageous for the subsequent efficient phosphopeptide enrichment. The magnetic nanocomposite exhibited outstanding performance of phosphopeptide enrichment with good selectivity (1 : 5000), a low detection limit (2 fmol), and relatively high loading capacity (66.7 mg g-1). What's more, after treatment with mCOF@ε-PL@THBA-Ti4+, 16 endogenous phosphopeptides from fresh saliva of healthy people were recognized by MALDI-TOF MS, and 50 phosphopeptides belonging to 35 phosphoproteins from the serum of uremia patients were detected by nano-LC-MS/MS. Proteomics data analysis for the differential protein selection between uremia and normal controls was conducted using R software, and four down-regulated and three up-regulated proteins were obtained. The results suggested that the prepared material has potential applications in biomarker discovery.

A novel molybdenum disulfide nanosheet loaded Titanium/Zirconium bimetal oxide affinity probe for efficient enrichment of phosphopeptides in A549 cells

In this paper, we developed a facile route for the preparation of a novel bimetal oxide affinity chromatography (MOAC) material. The TiO2/ZrO2@MoS2 was constructed by the electrostatic interaction between titanium oxide/zirconia (w:w, 10:1) and molybdenum disulfide nanosheet. The nanocomposite has the large specific surface area (186.30 m2⋅g−1) and pore volume (0.37 cm3⋅g−1). Compared with single-metal probes, the combination of bimetallic oxides probe (TiO2/ZrO2) and hydrophilicity MoS2 support offered multitudinous affinity sites for phosphopeptides capturing from tryptic digests of protein samples under 50% acetonitrile-1% trifluoroacetate conditions. Singnificant feasibility of the TiO2/ZrO2@MoS2 nanomaterial for the enrichment of phosphopeptides under optimal conditions was proved via the bovine serum albumin (BSA) and the mixtures of β-casein. The phosphopeptide expression was identified using ultra-performance liquid chromatography (uHPLC) separation and-linear ion trap mass spectrometry (MSn). With these affinity characters of TiO2/ZrO2@MoS2, it exhibited higher binding capacity (25 mg⋅g−1), better selectivity for phosphopeptides from β-casein/BSA (1:2000) tryptic digests, high sensitivity (1 fmol⋅µL−1) towards phosphopeptides from β-casein tryptic digests, and great reusability of 8 cycles test for capturing phosphopeptides. In addition, the TiO2/ZrO2@MoS2 with high sensitivity and selectivity was successfully applied to enriching phosphopeptides from nonfat milk and human serum samples. More importantly, the TiO2/ZrO2@MoS2 was further successfully applied to multi-phosphopeptides enrichment, 1779 serine, threonine and tyrosine phosphosites can be identified in A549 cell protein tryptic digest. Compared with commercial TiO2 from enrichening 416 phosphopeptide from A549 cell lysates, the successful locating of 44 phosphosites were overlapped.

Layer-by-layer assembly of multilayereddouble hydroxides/polyoxometalate-coated magnetic nanoparticles for highly efficient phosphopeptide enrichment.

Alayer-by-layer (LbL) assembly strategy was developed to prepare multilayered double hydroxide/polyoxometalate shell-coated magnetic nanoparticles. The introduction of functional shells not only offered abundant affinity sites of metal oxide and metal ions but also increased the surface area for thecontact with targets. By combining the enrichment strategies of immobilized metal ion affinity chromatography and metal oxide affinity chromatography, the nanomaterial can capture phosphopeptides via a synergistic effect. The method presented a low detection limit of 0.1 fmol in combination withmatrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. The nanomaterial showed satisfactory selectivity (1:1:5000M ratio of α-/β-casein/bovine serum albumin), goodrecovery (92.07%), highadsorption capacity (117.6mgg-1), and ten timesreusability for capturing phosphopeptides.

Two-dimensional magnetic bimetallic organic framework nanosheets for highly efficient enrichment of phosphopeptides.

Highly selective enrichment and sensitive detection of phosphopeptides is pivotal for comprehensive phosphoproteomics analysis; however, it also poses a long-standing challenge. Here, a novel two-dimensional (2D) magnetic bimetallic organic framework (MOF) nanosheet with Zr-O clusters and Ti-O clusters (denoted as the Fe3O4@Zr-Ti BPDC nanosheet) is prepared via a solvothermal method and in situ deposition of Fe3O4 nanoparticles for the first time. Taking advantage of the abundant dual affinities of Zr-O and Ti-O clusters for phosphopeptides, large surface area and high chemical stability, the Fe3O4@Zr-Ti BPDC nanosheets exhibit excellent enrichment performance for phosphopeptides. Within the framework of density functional theory, the interaction between Zr-O clusters, Ti-O clusters and phosphorylated molecules was studied to find the possible reason behind the superior adsorption performance of the bimetallic MOF nanosheets. We found that electrons would migrate from Ti to Zr spontaneously after doping Ti element and enhance the electrostatic traction between Zr species and phosphorylated molecules, demonstrating that the synergistic effect of Zr-Ti was helpful to improve the enrichment efficiency for phosphopeptides. Furthermore, the Fe3O4@Zr-Ti BPDC nanosheets showed good enrichment performance in complex bio-samples, including nonfat milk, human saliva, and a breast cancer cell lysate, indicating their tremendous potential in the analysis of trace phosphorylated biomolecules in complex bio-samples.

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.

Ti4+-immobilized hierarchically porous zirconium-organic frameworks for highly efficient enrichment of phosphopeptides

Ti4+-immobilized hierarchically porous zirconium-organic frameworks for highly efficient enrichment of phosphopeptides

In-Depth Characterization of the Staphylococcus aureus Phosphoproteome Reveals New Targets of Stk1

AbstractStaphylococcus aureus is a major cause of infections worldwide, and infection results in a variety of diseases. As of no surprise, protein phosphorylation is an important game player in signaling cascades and has been shown to be involved in S. aureus virulence. Albeit long neglected, eukaryotic-type serine/threonine kinases in S. aureus have been implicated in this complex signaling cascades. Due to the substoichiometric nature of protein phosphorylation and a lack of suitable analysis tools, the knowledge of these cascades is, however, to date, still limited. Here, were apply an optimized protocol for efficient phosphopeptide enrichment via Fe3+-IMAC followed by LC-MS/MS to get a better understanding of the impact of protein phosphorylation on the complex signaling networks involved in pathogenicity. By profiling a serine/threonine kinase and phosphatase mutant from a methicillin-resistant S. aureus mutant library, we generated the most comprehensive phosphoproteome data set of S. aureus to date, aiding a better understanding of signaling in bacteria. With the identification of 3800 class I p-sites, we were able to increase the number of identifications by more than 21 times compared with recent literature. In addition, we were able to identify 74 downstream targets of the only reported eukaryotic-type Ser/Thr kinase of the S. aureus strain USA300, Stk1. This work allowed an extensive analysis of the bacterial phosphoproteome and indicates that Ser/Thr kinase signaling is far more abundant than previously anticipated in S. aureus.

Novel Two-Dimensional MoS2-Ti4+ Nanomaterial for Efficient Enrichment of Phosphopeptides and Large-Scale Identification of Histidine Phosphorylation by Mass Spectrometry.

Due to its key roles in regulating the occurrence and development of cancer, protein histidine phosphorylation has been increasingly recognized as an important form of post-translational modification in recent years. However, large-scale analysis of histidine phosphorylation is much more challenging than that of serine/threonine or tyrosine phosphorylation, mainly because of its acid lability. In this study, MoS2-Ti4+ nanomaterials were synthesized using a solvothermal method and taking advantage of the electrostatic adsorption between MoS2 nanosheets and Ti4+. The MoS2-Ti4+ nanomaterials have the advantage of the combined affinity of Ti4+ and Mo toward phosphorylation under medium acidic conditions (pH = 3), which is crucial for preventing hydrolysis and loss of histidine phosphorylation during enrichment. The feasibility of using the MoS2-Ti4+ nanomaterial for phosphopeptide enrichment was demonstrated using mixtures of β-casein and bovine serum albumin (BSA). Further evaluation revealed that the MoS2-Ti4+ nanomaterial is capable of enriching synthetic histidine phosphopeptides from 1000 times excess tryptic-digested HeLa cell lysate. Application of the MoS2-Ti4+ nanomaterials for large-scale phosphopeptide enrichment results in the identification of 10 345 serine, threonine, and tyrosine phosphosites and the successful mapping of 159 histidine phosphosites in HeLa cell lysates, therefore indicating great potential for deciphering the vital biological roles of protein (histidine) phosphorylation.

Glycocyamine functionalized magnetic layered double hydroxides with multiple affinity sites for trace phosphopeptides enrichment

Efficient enrichment and identification of phosphopeptides are of great significance in biological applications. Glycocyamine functionalized magnetic layered double hydroxides (Fe3O4@LDH@NH2-GAA) was fabricated through an easy process. The magnetic composite possessed high surface area, good biocompatibility, and fast magnetic response. Fe3O4@LDH@NH2-GAA, combining not only metal ions (Cu2+ and Ga3+) in LDH but also functional guanidyl groups in glycocyamine, offered multiple affinity sites for phosphopeptides enrichment. With these favorable characters, it exhibited high selectivity (β-casein: bovine serum albumin = 1:5000), low detection limit (0.1 fmol), satisfactory enrichment recovery (94.5%), high adsorption capacity (82.4 mg g−1), and good repetitiveness. Moreover, the efficient enrichment of phosphopeptides by Fe3O4@LDH@NH2-GAA from nonfat milk, human saliva, serum, and A549 cell lysates further confirmed its great potential for trace biological detection and proteomic analysis.

Highly sensitive detection of phosphopeptides with superparamagnetic Fe3O4@mZrO2 core-shell microspheres-assisted mass spectrometry

Protein phosphorylation is one of the most important post-translational modifications. It is an active research area to study phosphoproteomics for discovery of disease biomarkers and druggable targets. Here we report the development of superparamagnetic Fe3O4@mZrO2 core-shell microspheres with mesoporous structures for highly efficient enrichment of phosphopeptides. We have demonstrated that the mesoporous ZrO2 layer dramatically improves the selective enrichment of phosphopeptides. Our approach allows for in-situ elution and sensitive identification of both mono-phosphorylated and multi-phosphorylated peptides in MALDI-TOF mass spectrometry, with the detection limit of down to the femtomole range. The target phosphopeptides can reliably be enriched for MS analysis from various complex samples including the spiked protein digests and tumor cell lysates. The Fe3O4@mZrO2 core-shell microspheres promise a useful tool for phosphoproteomics by allowing for highly efficient and selective enrichment of the crucial signaling regulators in a low abundance.