Selective Enrichment Of Phosphopeptides Research Articles

Combination of click chemistry and Schiff base reaction: Post-synthesis of covalent organic frameworks as an immobilized metal ion affinity chromatography platform for efficient capture of global phosphopeptides in serum with chronic obstructive pulmonary disease.

Reasonable design and construction of functionalized materials are of great importance for the enrichment of global phosphopeptides. In this work, Ti4+ functionalized hydrophilic covalent organic frameworks by introducing glutathione (GSH) and 2,3,4-trihydroxy benzaldehyde (THBA) via click chemistry and Schiff base reaction (COF-V@GSH-THBA-Ti4+ ) was constructed and applied for selective enrichment of phosphopeptides in serum. Benefit from the high surface area, excellent hydrophilicity as well as regular mesoporous structure, COF-V@GSH-THBA-Ti4+ displayed high selectivity (molar ratio of 2000:1), low limit of detection (0.5 fmol), high load capacity (100.0mg/g) and excellent size-exclusion effect (1:10000) for enrichment of phosphopeptides. For actual bio-sample analysis, 15 phosphopeptides assigned to 10 phosphoproteins with 16 phosphorylated sites and 33 phosphopeptides assigned to 25 phosphoproteins with 34 phosphorylated sites were detected from the serum of patients with chronic obstructive pulmonary disease (COPD), and normal controls. Biological processes and molecular functions analysis further disclosed the difference of serums with phosphoproteomics between COPD and normal controls.

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.

A nitrogen-doped graphene tube composite based on immobilized metal affinity chromatography for the capture of phosphopeptides

A novel immobilized metal affinity chromatography (IMAC) functional composite, mNi@N-GrT@PDA@Ti4+, was fabricated based on ultrathin magnetic nitrogen-doped graphene tube (mNi@N-GrT) after chelated Ti4+ with polydopamine, following as a magnetic solid-phase extraction sorbent for rapidly selective enrichment and mass spectrometry identification of phosphorylated peptides. After optimized, the composite exhibited high specificity in the enrichment of phosphopeptides from the digest mixture of β-casein and bovine serum albumin (BSA). The robust method presented the low detection limits (1 fmol, 200 μL) and excellent selectivity (1:100) in the molar ration mixture of β-casein and BSA digests. Furthermore, the selective enrichment of phosphopeptides in the complex bio-samples, was successfully carried out. The results showed that 28 phosphopeptides were finally detected in mouse brain, and 2087 phosphorylated peptides were identified in the HeLa cells extracts with specific selectivity of 95.6%. The enrichment performance of mNi@N-GrT@PDA@Ti4+ was satisfactory, suggesting that the functional composite provided a potential application in the enrichment of trace phosphorylated peptides from the complex biological matrix.

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.

Preparation of Core‐Shell Structured Magnetic Superhydrophilic Extractant for Enrichment of Phosphopeptides

A core-shell structured magnetic superhydrophilic Fe3O4@C@TiO2 microspheres are successfully prepared. The as-prepared Fe3O4@C@TiO2 magnetic microspheres possess fast magnetic response, superhydrophilic and high affinity. The magnetic superhydrophilic Fe3O4@C@TiO2 microspheres are employed for selective enrichment phosphopeptides and exhibit excellent enrichment performance such as high selectivity, low detection and excellent reusability. Sixteen phosphopeptides and four endogenous phosphopeptides can be identified from non-fat milk and human serum by Fe3O4@C@TiO2 magnetic microspheres, indicating Fe3O4@C@TiO2 will be a powerful and helpful IMAC material in understanding and analyzing essential biological phosphorylation processes in phosphorylated proteomics research.

Design and fabrication of reusable core-shell composite microspheres based on nanodiamond for selective enrichment of phosphopeptides.

A kind of core-shell composite microsphere (CM) with nano-on-micro structure was synthesized via grafting amine-modified nanodiamonds onto the surface of monodisperse nonporous polymeric microsphere. In this way, the agglomeration of nanodiamond particles in the solutionwas avoided. After modification with pyrogallol groups, CM could chelate titanium ions (Ti4+) and thus be utilized as immobilized metal affinity chromatography (IMAC) sorbent to enrich phosphopeptides from biological samples. The resulting Ti4+-CM exhibited high enrichment efficiency and specificity to phosphopeptides. A total of 106 of unique phosphopeptides mapped to 29 phosphoproteins were clearly identified from 5 µL ofa milk digest after enrichment. Owing to the strong chelation between Ti4+ and pyrogallol ligands, the Ti4+is not releasedfrom thesorbent after completion of theenrichment process. As a result, the Ti4+-CM sorbent could be reused, and no significant loss of enrichment efficiency occurred even on the fourth run employing a β-casein digest as the sample. The strategy adopted presents a new way to prepare ahigh-performance reusable IMAC sorbent.

Synthesis of a metal oxide affinity chromatography magnetic mesoporous nanomaterial and development of a one-step selective phosphopeptide enrichment strategy for analysis of phosphorylated proteins

Protein phosphorylation is one of the most vital protein post-translational modifications, which regulates numerous biological processes including protein structure transformation, molecular interaction, cell proliferation and apoptosis, as well as various malignant diseases. Though mass spectrometry (MS)-based technologies provide a feasible strategy to analyze phosphorylated proteins, label-free quantitative analysis for phosphopeptides in biological samples is still a challenging task due to sample losses from complicated preparation and necessary phosphopeptide enrichment. In this study, magnetic mesoporous TiO2 microspheres, Fe3O4@nSiO2@mSiO2@TiO2, a metal oxide affinity chromatography material, were synthesized with uniform particle size of 500 nm. The synthesized material exhibited good selectivity (molar ratio of β-casein:BSA = 1:2000, great sensitivity (0.5 fmol μL−1β-casein), and good adsorption capacity (120 mg g−1). Moreover, the proper mesopores on the surface of the enrichment material displayed the unique property of size-exclusion, which could ‘capture’ the trypsin and form a temporary immobilized enzyme reactor, reducing the tryptic digestion procedure from at least 16 h to 2 h. Based on the synthesized material, a facile one-step selective phosphopeptide enrichment method was successfully developed and consisted with protein digestion and phosphopeptide enrichment, which was further applied to selectively enrich phosphopeptides from the complex biological samples of nonfat milk and HepG2 cells, indicating its great feasibility and potential of applications in real sample analysis. More importantly, combining with MS, a label-free phosphoproteomic quantitative strategy was established and performed for determination of the mixture samples containing BSA and various concentrations of β-casein. The results showed that the obtained calibration curve was linear with R = 0.9987 by plotting the intensities of unique phosphopeptide of β-casein (m/z 2061.77) versus the concentrations of β-casein. Our study will provide a novel analytical strategy for selective enrichment of phosphopeptides and facile label-free relative quantitative analysis of phosphorylated proteins.

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.

Magnetic mesoporous silica nanoparticles modified by phosphonate functionalized ionic liquid for selective enrichment of phosphopeptides

In this study, new magnetic nanoparticles (denotated as Fe3O4@mSiO2-PFIL-Ti4+) have been prepared by immobilizing titanium ions with phosphonate functionalized ionic liquid (PFIL) on the wall of core-shell structured mesoporous nanomaterials. The resulting nanoparticles possess large specific surface area, strong hydrophilicity and fast magnetic response. The composites can capture traces of phosphopeptides from the tryptic β-casein digest (0.08 fmol), a digest mixture of β-casein and BSA (1 : 10 000, molar ratio) as well as a blend of β-casein digest and a great quantity of phosphorylated protein (β-casein) and non-phosphorylated protein (BSA) (1 : 2000 : 2000, mass ratio), respectively, showing excellent sensitivity, selectivity and size exclusion ability. Additionally, Fe3O4@mSiO2-PFIL-Ti4+ shows excellent steadiness and can be reused at least 12 times. Moreover, this material was successfully applied to enrich endogenous phosphopeptides from complex bio-samples, including human saliva and serum.

Facile Preparation of Chitosan‐modified Mesoporous Titanium Dioxide Film on Fused‐silica Capillary for Selective Enrichment of Phosphopeptides

AbstractChitosan (Cts), a positively charged natural polysaccharide, is employed to functionalize mesoporous titanium dioxide (mTiO2) nanoparticles. The prepared Cts/mTiO2 composite was used as a stationary phase to coat the inner wall of fused‐silica capillary and obtain uniform Cts/mTiO2 film. The Cts/mTiO2 film is applied to detect and enrich phosphopeptides in complex biological samples based on typical in‐tube solid phase microextraction (IT‐SPME) system. This unique Cts/mTiO2 film exhibits excellent enrichment performances of high selectivity (β‐casein and BSA in molar ratio of 1 : 1000), sensitivity (detection limit as low as 40 fmol of β‐casein tryptic digest solution) and high adsorption capacity (124.5 μg mg−1). Meanwhile, the Cts/mTiO2 film possesses preferable reusability and can be reused more than 10 times. These results confirm that the Cts/mTiO2 film possesses potential and satisfactory prospects in phosphoproteomics analysis.

Facile “one-pot” preparation of phosphonate functional polythiophene based microsphere via Friedel-Crafts reaction for selective enrichment of phosphopeptides from milk

A novel kind of phosphonate functionalized polythiophene microsphere was designed and fabricated via Friedel-Crafts reaction. Diethyl (thiophen-2-ylmethyl) phosphonate (DTYP) and thiophene were co-polymerized by Fe (III) catalysis, without any surfactant, stabilizer and initiator. Functional phosphonate group was directly introduced into the microsphere without redundant modification steps. The adsorption amount of the as-synthesized microsphere, Ti-poly(Th-co-DTYP), was as high as 66.7 mg/g, which was higher than that of commercial Ti4+-IMAC microsphere (49.7 mg/g). The microsphere was explored on the specific capture of phosphopeptides from either tryptic digests of milk or HeLa cell protein. As a result, 88 of unique phosphopeptides mapping to 21 phosphoproteins were identified from 150 μg of milk tryptic digest after enrichment, and a total of 2534 unique phosphopeptides mapping to 1087 phosphoproteins was identified from HeLa cell. It is expected that such a robust and facile approach will be explored in other functional microspheres to be commercialized in the future.

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.

Development of dual-ligand titanium (IV) hydrophilic network sorbent for highly selective enrichment of phosphopeptides

A hydrophilic metal-organic network based on Ti4+ and dual natural ligand, tannic acid (TA) and phytic acid (PA), has been developed to enrich phosphopeptides from complex bio-samples prior to liquid chromatography-mass spectrometric analysis. Due to the strong chelation ability of TA and PA, abundant Ti4+ can be immobilized in the material, forming hydrophilic network by one-step coordination-driven self-assembly approach. The sorbent, TA-Ti-PA@Fe3O4, exhibited satisfactory selectivity for the phosphopeptides in the tryptic digest of β-casein, and can eliminate the interference components in 1000-fold excess of bovine serum albumin. The adsorption capacity of the sorbents for phosphopeptides was up to 35.2 mg g−1 and the adsorbing equilibrium can be reached in 5 min. The adsorbing mechanism has been investigated and the results indicated that the Ti4+ in forms of [Ti(f-TA)(H2O)4]2+, [Ti(f-PA)(H2O)4]2+ and Ti(f-PA)2(H2O)2 may play an important role in the adsorption process. The sorbent of the TA-Ti-PA@Fe3O4 has been applied to enrichment of the phosphopeptides in tryptic digest of rat liver lysate, and 3408 phosphopeptides have been identified, while the numbers of the identified phosphopeptides were 2730 and 1217 when the sample was enriched by the commercial TiO2 and Fe3+-IMAC kit, respectively. This work provides a strategy to enrich phosphopeptides from complex samples and shows great potential application in phosphoproteome research.

Design of Gd3+-immobilized two-dimensional magnetic magadiite nanosheets for highly selective enrichment of phosphopeptides.

Exfoliated magadiite nanosheets embedded with Fe3O4 were constructed. Advantage was taken of the strong coordination between the silanol groups in magadiite nanosheets and the Gd3+ ion to prepare the final adsorbent, Gd3+-immobilized magnetic magadiite nanosheets. The adsorbent with two-dimensional (2D) morphology offered high surface area and abundant Gd3+ contents for phosphopeptides enrichment, on which Fe3O4 with positive electricity incorporated the magnetic properties. Combining with matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF-MS), the method showed low detection limit (0.05 fmol). The feasibility of using the 2D nanocomposite for phosphopeptides enrichment was demonstrated using mixtures of β-casein and bovine serum albumin (1:5000). The standard deviation of captured phosphopeptides in three repeated experiments were in the range 0.15-0.42 (< 0.5% RSD). Further evaluation revealed that the nanocomposite was capable of enriching phosphopeptides from non-fat milk, human saliva, and serum. A novel Gd3+-immobilized two-dimensional magnetic magadiite nanosheets-based enrichment platform was designed. The developed material was employed as the adsorbent for the selective enrichment of phosphopeptides by coupling with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The material was successfully applied to enrich phosphopetides from standard peptide mixtures, nonfat milk, human saliva, and serum.

Removal of adsorption sites on the external surface of mesoporous adsorbent for eliminating the interference of proteins in enrichment of phosphopeptides/nucleotides

Various mesoporous adsorbents are of great promise for enriching small molecules from biological samples based on the size-exclusion effect. At present, the mesoporous adsorbents have adsorption sites distributed uniformly on the internal and external surfaces of mesopores. However, the adsorption sites on the external surface can adsorb proteins, interfering with the enrichment of small molecules. Herein, a novel immobilized-Ti4+ magnetic mesoporous adsorbent removing the adsorption sites on the external surface was facile prepared via the coupling chemistry of isocyanate with amine and consequent hydrolysis of urea linkage by urease. The adsorbent enables fast and selective enrichment of phosphopeptides and nucleotides from biological samples. In addition, sensitive detection methods for phosphopeptides and nucleotides in human serum are developed by coupling the magnetic solid-phase extraction with matrix-assisted laser desorption/ionization time of flight mass spectrometry and liquid chromatography-mass spectrometer, respectively. Under optimal conditions, response is linear (R2 ≥ 0.9923), limits of detection are low (0.41–9.48 ng mL−1), and reproducibility is acceptable (inter- and intra-day assay RSDs of≤15.0%) for six nucleotides. The developed strategy offers an effective method to eliminate the interference of proteins in the enrichment of small molecules from real biological samples.

A new Ti-based IMAC nanohybrid with high hydrophilicity and enhanced absorption capacity for the selective enrichment of phosphopeptides

Ti-based immobilized metal affinity chromatography (IMAC) nanomaterial has shown high potential in phosphoproteome mass-spectrometric (MS) analysis. However, the limited surface area and poor solubility will greatly restrict its use in phosphoproteome research. To overcome these two key drawbacks, a novel Ti-based IMAC nanomaterial was prepared by Ti-bonded β-cyclodextrin (β-CD) anchored on the surface of carbon nanotubes (CNTs) (denoted as COOH-CNTs-CD-Ti) and successfully applied as a biofunctional adsorbent for selectively enriching trace phosphopeptides. In the selective enrichment process, CNTs provided greater surface area for the absorption of phosphopeptides, while β-CD also offered a greater opportunity for the interaction between phosphopeptides and Ti4+. COOH-CNTs-CD-Ti with the aforementioned properities exhibited higher selectivity for phosphopeptides from the standard protein digests, the tryptic digests of nonfat milk and human serum, showing a great selective enrichment capability towards complex biological samples.

Rapid Shotgun Phosphoproteomics Analysis.

In this chapter, we describe a rapid workflow for the shotgun global phosphoproteomics analysis. The strategy is based on the use of accelerated in-solution trypsin digestion under an ultrasonic field by high-intensity focused ultrasound (HIFU) coupled to titanium dioxide (TiO2) selective phosphopeptide enrichment, fractionation by strong cation exchange chromatography (SCX), and analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a high-resolution mass spectrometer (LTQ-Orbitrap XL). The strategy was optimized for the global phosphoproteome analysis of Jurkat T-cells. Using this accelerated workflow, HIFU-TiO2-SCX-LC-MS/MS, 15,367 phosphorylation sites from 13,029 different phosphopeptides belonging to 3,163 different phosphoproteins can be efficiently identified in less than 15h.

A metal oxide affinity probe derived from MIL-125 for selective enrichment of endogenous phosphopeptides.

Highly effective enrichment of endogenous phosphopeptides from complex biological samples is an essential and crucial theme in the analysis of phosphopeptidomics. Herein, an ordered mesoporous TiO2/C composite (denoted as Ti-MCM) was prepared by the pyrolysis of MIL-125 under a N2 atmosphere. The obtained Ti-MCM possesses a high specific surface area (165 m2 g-1), a uniform pore size (3.75 nm), and a large amount of Ti (46%). By utilizing the selective chelation between Ti-MCM and phosphopeptides, 25 phosphopeptides were detected in α-casein digest after enrichment. The material shows good selectivity even in the presence of 2000-fold excess of interference peptides. It was also used to enrich endogenous phosphopeptides from the complex samples of human serum and saliva and showed a good performance.

Comparative evaluation of MAX-Ti3AlC2 and MXene-Ti3C2 as affinity chromatographic materials for highly selective enrichment of phosphopeptides.

MAX and MXene have received considerable attention owing to their outstanding performance in fields like battery and catalysis. However, their possible biomedical applications have rarely been considered, especially the affinity chromatographic applications in proteomics. In this work, considering the large number of exposed metal sites, small binding potential resistance and fast mass transfer speed, layered ternary carbides MAX-Ti3AlC2 and MXene-Ti3C2 with a two-dimensional nanostructure were successfully explored for the first time as affinity chromatography stationary phases for the specific capture of phosphopeptides from complex biological samples. Helium ion microscopy, transmission electron microscopy, atomic force microscopy, X-ray diffraction spectra, X-ray photoelectron spectroscopy and zeta potential measurement results confirmed that the MXene-Ti3C2 was well exfoliated from the pristine MAX-Ti3AlC2. Ti3AlC2 showed better enrichment specificity than MXene-Ti3C2. The detection limit of Ti3AlC2 was as low as 5 fmol. Even when the molar ratio of BSA to β-casein tryptic digests increased to 1000 : 1, two characteristic phosphopeptides with a relatively clear background could be detected after enrichment. After five cycles of repeated use, the enrichment specificity of Ti3AlC2 still remains. Furthermore, 91 and 830 unique phosphopeptides from 23 and 525 phosphoproteins were identified from milk and BEL7402 cells, respectively. Among them, 27 and 170 phosphopeptides, 12 and 56 phosphoproteins identified from milk and BEL7402 cells were not detected with commercial TiO2 after three independent replicates, which have great potential in providing complementary coverage of phosphoproteome. This work opens up new applications of Ti3AlC2 and MXene-Ti3C2, and will play more important role for phosphorylated proteomics in biomedicine.

Preparation of zirconium arsenate-modified monolithic column for selective enrichment of phosphopeptides.

Protein phosphorylation is a crucial posttranslational modification for the regulation of many different biological functions. Selective enrichment of phosphopeptides from the complex biological samples is an essential step for the mass spectrometry analysis of protein phosphorylation. In this study, an arsenate functionalized monolithic column was first prepared by a single-step copolymerization of p-methacryloylaminophenylarsonic acid and ethylene dimethacrylate. Then the metal ions Zr4+ were attached onto the prepared monolithic column via metal-chelate complex formation by Zr4+ and arsenate groups. The obtained monolithic column was employed as a new sorbent for the phosphopeptide enrichment via immobilized metal affinity chromatography. Phosphopeptides analysis was realized by polymer monolith microextraction using this monolithic column coupled to both matrix-assisted laser desorption/ionization mass spectrometry and liquid chromatography-electrospray ionization tandem mass spectrometry. The proposed method exhibited a high selectivity for phosphopeptide enrichment in complex matrices, and was applied to the analysis of phosphopeptides in human serum and tryptic digests of rat brain proteins. Four phosphopeptides could be selectively captured from human serum and 2608 endogenous phosphopeptides were identified from the tryptic digests of rat brain proteins, indicating a satisfactory performance of this method for the enrichment of phosphopeptides from complex biological samples.