Enrichment of phosphopeptides by arginine-functionalized magnetic chitosan nanoparticles.

One of the most crucial and prevalent post-translational modifications is the phosphorylation of proteins. The study and examination of protein phosphorylation hold immense importance in comprehending disease mechanisms and discovering novel biomarkers. However, the inherent low abundance, low ionization efficiency, and coexistence with non phosphopeptides seriously affect the direct analysis of phosphopeptides by mass spectrometry. In order to tackle these problems, it is necessary to carry out selective enrichment of phosphopeptides prior to conducting mass spectrometry analysis. Herein, magnetic chitosan nanoparticles were developed by incorporating arginine, and were then utilized for phosphopeptide enrichment. A tryptic digest of ß-casein was chosen as the standard substance. After enrichment, combined with matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), the detection limit of the method was 0.4 fmol. The synthesized magnetic material demonstrated great potential in the detection of phosphopeptides in complex samples, as proven by its successful application in detecting phosphopeptides in skim milk and human saliva samples.

Facile synthesis of Fe3+ immobilized magnetic polydopamine-polyethyleneimine composites for phosphopeptide enrichment.

As one of the most common post-translational modification of proteins, protein phosphorylation plays a vital role in many physiological processes. The enrichment of phosphopeptides is highly important before the mass spectrometry detection since phosphopeptides are susceptible to interferences from high-abundance non-phosphopeptides. In this study, we designed a novel magnetic composite (Fe3O4@PDA-PEI-Fe3+) for phosphopeptide enrichment with a facile protocol. The developed Fe3O4@PDA-PEI-Fe3+ is a marvelous material with multiple functional groups, and can effectively enrich phosphopeptides through the synergistic effect of three mechanisms, i.e., immobilized metal ion affinity chromatography raised form Fe3+, electrostatic interaction between amine and phosphate groups, and hydrogen bond between the hydrogen atoms of amine groups and oxygen atoms of phosphate groups. Combined with mass spectrometry, the material shows excellent enrichment performance, high sensitivity (0.4 fmol), good selectivity (ß-casein:BSA= 1:500, w:w), and stable reusability (at least 5 cycles). In addition, the material was successfully applied to enrich phosphopeptides from skim milk and human saliva samples, implying that it is an ideal adsorbent for the phosphopeptide enrichment in complex biological samples and provides valuable insights into the field of phosphopeptide analysis.

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/ß-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.

Simultaneous Profiling of Palmitoylomics and Glycomics with Photo/pH Dual-Responsive Magnetic Nanocomposites.

Following an in-depth examination of a single type of protein posttranslational modification, the synergistic analysis of two or more modification types has gradually emerged as a focal point in proteomic research. Palmitoylation and glycosylation are both critical for protein, implicated in carcinogenesis and inflammation. In this study, novel dual-responsive magnetic nanocomposites that serve as an ideal platform for the sequential or simultaneous enrichment of palmitoyl and glycopeptides are reported. The nanocomposites denoted as magDVS-VBA are constructed by modifying magnetic nanoparticles with azobenzene and divinyl sulfone (DVS), and self-assembled with 4-vinylbenzeneboronic acid (VBA)-immobilized ß-cyclodextrin, which responds to light. The incorporated DVS component possesses the ability to recognize palmitoyl or glycopeptides under different pH conditions, whereas the introduction of VBA enhances the affinity of the nanocomposite for glycopeptides. Notably, magDVS-VBA exhibits flexible photo-, pH-, and magnetic-responsive capabilities, enabling the simultaneous recognition of hydrophobic palmitoyl peptides and hydrophilic glycopeptides for the first time. The developed platform demonstrates high specificity for sensitive palmitoylomics and glycomics analysis of mouse liver tissue, providing an effective method for studying of their crosstalk, and potential implications in clinical applications.

Carnosine functionalized magnetic metal-organic framework nanocomposites for synergistic enrichment of phosphopeptides.

Protein phosphorylation regulates the conformations and function of proteins, which plays an important part in organisms. However, systematic and in-depth analysis of phosphorylation often hinders on account of the low abundance and suppressed ionization of phosphopeptides. Various materials based on single enrichment mechanism show potential in phosphopeptides enrichment, but the enrichment performance is typically not satisfactory. Herein, we developed a carnosine (Car) functionalized magnetic metal organic framework designed as Fe3O4@NH2@ZIF-90@Car. Benefiting from the multiple recognition groups of Car and massive metal ions site of ZIF-90, the as-fabricated Fe3O4@NH2@ZIF-90@Car was utilized as a multifunctional material with synergistic effect for phosphopeptides enrichment. On the basis of combined immobilized metal ion affinity chromatography (IMAC) and amine-based affinity enrichment mechanism, Fe3O4@NH2@ZIF-90@Car exhibited higher enrichment performance of phosphopeptides compared with Fe3O4@NH2@ZIF-90 (single IMAC mechanism). Besides, the feasibility of Fe3O4@NH2@ZIF-90@Car nanocomposites in complicated samples was further verified by enriching phosphopeptides from nonfat milk, human fluids such as serum and saliva, demonstrating its bright application prospects in phosphoproteomics analysis.

Engineering Magnetic Guanidyl-Functionalized Supramolecular Organic Framework for Efficient Enrichment of Global Phosphopeptides.

Comprehensive mass spectrometry-based proteomics analysis is currently available but remains challenging, especially for post-translational modifications of phosphorylated proteins. Herein, multifunctional magnetic pillar[5]arene supramolecular organic frameworks were fabricated and immobilized with arginine (mP5SOF-Arg) for highly effective enrichment of global phosphopeptides. The specific phosphate-P5/phosphate-guanidine affinities and large surface area with regular porosity contribute to the high enrichment capacity. By coupling with mass spectrometry, high detection sensitivity (0.1 fmol), excellent selectivity (1:5000 molar ratios of ß-casein/cytochrome c), and high recyclability (seven cycles) were achieved for phosphopeptide analysis. mP5SOF-Arg can efficiently enrich phosphopeptides from practical samples, including defatted milk, egg yolk, and human saliva. Notably, a total of 450 phosphopeptides were explored for highly selective identification from A594 cells and 1445 phosphopeptides were identified from mouse liver tissue samples. mP5SOF-Arg exhibited great potential to serve as the basis for peptidomic research to identify phosphopeptides and provided insight for biomarker discovery.

Bifunctional Magnetic Supramolecular-Organic Framework: A Nanoprobe for Simultaneous Enrichment of Glycosylated and Phosphorylated Peptides.

Protein glycosylation and phosphorylation are two important protein post-translational modifications. Mass spectrometry (MS) has been proved to be a powerful technique in comprehensive characterization of protein glycosylation and phosphorylation; however, the complexity of biological matrices and weak ionization efficiency bring a big challenge. Capturing glycopeptides and phosphopeptides from complicated biological samples is indispensable before MS determinations. In this study, a bifunctional gallium ion immobilized magnetic pertriflated pillar[5]arene supramolecular-organic framework (magOTfP5SOF-Ga3+) was designed for the one-step simultaneous enrichment of glycopeptides and phosphopeptides. Thanks to the abundant sulfonic acid groups, the material owns strong hydrophilicity and leads to hydrophilic interaction chromatography for glycopeptides enrichment. Simultaneously, the high loading amount of gallium ion provides immobilized metal ion affinity for phosphopeptides enrichment. The established platform possesses quick magnetic response performance, high sensitivity (detection limits as low as 0.1 fmol and 0.05 fmol for glycopeptides and phosphopeptides, respectively), and good reusability. In addition, the method was applied to the determination of glycopeptides and phosphopeptides in clinical specimens, cell lysates, and mouse liver tissue samples, demonstrating its highly sensitive and specific glycoproteomics and phosphorproteomics analysis in complex biosamples.

A polymer monolith composed of a perovskite and cucurbit[6]uril hybrid for highly selective enrichment of phosphopeptides prior to mass spectrometric analysis.

A hybrid monolith was prepared from perovskite and cucurbit[6]uril [poly(hydroxyethyl methacrylate-pentaerythritol triacrylate) monolith] for the enrichment of phosphopeptides. By coupling with mass spectrometry, three goals were simultaneously realized, viz. (a) selective enrichment of phosphopeptides from non-phosphopeptides, (b) identification of mono- and multi-phosphopeptides, and (c) recognition of tyrosine phosphopeptides. The perovskite introduced into the monolith warrants high selectivity for phosphopeptides even at a high (10,000:1) ratio of non-phosphopeptides to phosphopeptides, and and enables identification of eight mono- and multi-phosphopeptides from standard ß-casein tryptic digests. Tyrosine phosphopeptides were specifically detected via the recognition capability of cucurbit[6]uril integrated into the monolith. The method has remarkably specific enrichment capacity for phosphopeptides from samples including human serum, nonfat milk, and human acute myelocytic leukemia cell lysate. Graphical abstractSchematic representation of a monolith integrated with perovskite and cucurbit[6]uril. The monolithic column was coupled with mass spectrometry and applied to the enrichment of phosphopeptides. The method has remarkably specific enrichment capacity for phosphopeptides from complex biological samples.

Cobalt Phthalocyanine Tetracarboxylic Acid Functionalized Polymer Monolith for Selective Enrichment of Glycopeptides and Glycans.

In this study, poly(glycidyl methacrylate-ethyleneglycol dimethacrylate) monolith functionalized with cobalt phthalocyanine tetracarboxylic acid is prepared. The polymer monolithic material is used for glycopeptides enrichment coupled with MALDI-TOF MS. By taking advantage of cobalt phthalocyanine including hydrogen bonds between isoindole subunits of phthalocyanine and glycans, coordination interaction between cobalt and glycopeptides, the monolithic material is successfully applied to the enrichment of glycopeptides efficiently and selectively. With IgG and horse radish peroxidase as the model glycoproteins, 28 and 17 glycopeptides could be identified respectively after enrichment with the monolith, only four and three glycopeptides could be obtained for direct analysis. The monolith is also employed to the digests mixture of BSA and IgG (50:1, m/m), indicating the high enrichment selectivity of glycopeptides even in the presence of a large interference ratio. The detection limit is determined to be 6.7 fmol, implying that the present method had great potential for trace sample analysis. In addition, the monolith was successfully applied to the enrichment of N-linked glycans in human serum samples, demonstrating its great potential for the analysis of glycoproteins.

Aptamer-Functionalized Magnetic Conjugated Organic Framework for Selective Extraction of Traces of Hydroxylated Polychlorinated Biphenyls in Human Serum.

A solid-phase extraction adsorbent based on an aptamer-functionalized magnetic conjugated organic framework (COF) was developed for selective extraction of traces of hydroxylated polychlorinated biphenyls. This material has advantages such as superparamagnetism of the magnetic core, high surface area and porous structure of the COF, and high specific affinity of the aptamer. In combination with HPLC-MS, the aptamer-functionalized magnetic COF was used for the capture of hydroxy-2',3',4',5,5'-pentachlorobiphenyl in human serum. The method provided a linear range of 0.01-40 ng mL-1 with a good correlation coefficient (R2 =0.9973). The limit of detection was as low as 2.1 pg mL-1 . Furthermore, the material showed good reusability and could be applied in at least ten extraction cycles with recoveries greater than 90 %.

Self-Assembling Glutamate-Functionalized Cyclodextrin Molecular Tube for Specific Enrichment of N-Linked Glycopeptides.

Cyclodextrin molecular tube (CDMT), a new comer of cyclodextrin family, possesses large and hydrophilic outer area and stable structure. Its development and applications remain highly desired, especially in the field of separation and enrichment. Herein, we developed a CDMT-based enrichment platform focusing on the specific capture of glycopeptides. To enhance the hydrophilicity of CDMT, it was functionalized with glutamate (glu). The prepared gluCDMT exhibited large hydrophilic surface, high stability, and good acidic/alkalic resistance. A solid monolithic support was employed to immobilize gluCDMT by a host-guest self-assembly synthetic strategy, which did not occupy the surface hydrophilic sites. The gluCDMT-based monolith exhibited high binding capacity (∼50 mg g-1), good ability to capture glycopeptides (23 HRP glycopeptides and 28 IgG glycopeptides), and high selectivity (horseradish peroxidase/bovine serum albumin = 1:10 000). Moreover, the developed platform was successfully applied to analyze glycopetides in acute myelogenous leukemia cell lysate and human serum samples.

Design of a calix[4]arene-functionalized metal-organic framework probe for highly sensitive and selective monitor of hippuric acid for indexing toluene exposure.

In the present work, a novel metal-organic framework (MOF) fluorescent probe was prepared by post-synthetic modification of MIL-53-NH2(Al) with carboxylatocalix[4]arene (CC[4]A). The introduced CC[4]A could not only enhance the fluorescence performance and the recognition ability of the probe, but also sustain the high stability under UV light and moisture conditions. A method based on the as-synthesized CC[4]A@MIL-53-NH2(Al) probe was established for sensing hippuric acid. The detection limit was determined to be as low as 3.7 µg mL-1. Over the concentration range of 0.005-3 mg mL-1, the calibration curve was obtained with a satisfactory linearity (R2 = 0.993). The method was successfully used for rapid and highly selective direct monitor of hippuric acid in human urine. The sensor had great potential to be used as a simple diagnostic tool for hippuric acid in human urine which is regarded as a biological index of toluene exposure.

Design of pH-Responsive Polymer Monolith Based on Cyclodextrin Vesicle for Capture and Release of Myoglobin.

ß-Cyclodextrin vesicles (CDVs) were first introduced into the polymer monolith to prepare a pH-responsive adsorption material and used for capture and release of a cardiac biomarker, myoglobin (Myo). SH-CDV was decorated with adamantane-modified SH-octapeptide to enhance the encapsulation and release rates of Myo. Afterward, SH-CDV was introduced into the polymer monolith via click reaction to produce a pH-responsive monolith. Combining with the mass spectrometry detection, the CDV-based pH-responsive monolith was used for the enrichment of Myo glycopeptides from the mixture of glycopeptides and nonglycoprotein (bovine serum albumin) tryptsin digests reach up to 1:10 000. A limit of detection of 0.1 fmol was obtained for Myo glycopeptides in the blood sample, indicating the high sensitivity of the method. The prepared CDV-based hybrid monolith demonstrated itself to be a promising material for capture of glycoproteins in complex samples, which provides an efficient strategy for the identification and discovery of biomarkers of acute myocardial infarction.

Anchoring ß-cyclodextrin modified lysine to polymer monolith with biotin: specific capture of plasminogen.

Plasminogen (Plg) is a kind of glycoprotein which plays an important role in cell migration. The determination of Plg content can directly reflect the abnormal manifestation of fibrinolytic system dysfunctions. In the present work, lysine (Lys)-based adsorbents were prepared for the specific capture of Plg through the covalent binding of Lys with a polymer monolithic substrate. Lys was modified with ß-cyclodextrin (ß-CD) via a click reaction and anchored to the substrate with biotin by host-guest interaction. The biotin-Lys-CD based monolithic material was employed for the specific capture of Plg. Combining with mass spectrometry (MS) determinations, the method exhibited a low detection limit of 1.0 fmol with relative standard deviations below 10.0% for Plg. Considering the advantages of simplicity, sensitivity, and high specificity, the developed approach was successfully applied to the determination of Plg in human plasma samples and opened a gallery for testing Plg as a biomarker for the diagnosis of fibrinolytic system dysfunctions.

Immobilization of ß-Cyclodextrin-Conjugated Lactoferrin onto Polymer Monolith for Enrichment of Ga in Metabolic Residues of Ga-Based Anticancer Drugs.

Biological-material-functionalized porous monoliths were prepared with lactoferrin and ß-cyclodextrin via a click reaction. With the monolith as an extraction medium, a method combined with ICP-MS was developed for the determination of total gallium originating from metabolic residues of orally bioavailable gallium complexes with tris(8-quinolinolato)gallium (GaQ3) as a representative. The method exhibited favorable adsorption behaviors for gallium with high selectivity, low detection limit (2 ng L-1), and an enrichment factor of 29-fold with the sample throughput of 30 min-1. The developed approach was validated by the analysis of gallium from GaQ3 metabolic residues in a human cell line. Additionally, the practical applicability of this method was evaluated by the determination of gallium in human blood and urine samples from cancer patients. Results illustrated that the prepared monolith had potential in Ga-based anticancer drug analysis in complex biological samples.

Peanut agglutinin and ß-cyclodextrin functionalized polymer monolith: Microextraction of IgG galactosylation coupled with online MS detection.

A facile online method coupling polymer monolithic microextraction (PMME) with mass spectrometry (MS) was developed for the detection of Immunoglobulin G (IgG) galactosylation glycopeptides. A peanut agglutinin-ß-cyclodextrin (PNA-ß-CD) functionalized poly(hydroxyethyl methylacrylate-ethyleneglycol dimethacrylate) monolith was designed via a click reaction. Thanking to the specificity of PNA-ß-CD for the targets, the material exhibited enhanced enrichment selectivity and extraction efficiency for IgG galactosylation glycopeptides. Under optimal conditions, the developed method gave a linear range of 0.005-5 pmol for IgG glycopeptides with the regression coefficient greater than 0.9990, and the detection limit of IgG galactosylation glycopeptides as low as 0.5 fmol was achieved. The PMME-MS method was applied to IgG galactosylation glycopeptides in real samples including human serum and acute myelogenous leukemia (AML) cell lysate. A series of unique IgG galactosylation glycopeptides were captured by the monolith in the complex samples, indicating satisfactory enrichment ability for IgG galactosylation glycopeptides. The quick and integrated online PMME-MS method exhibited high selectivity for IgG galactosylation, demonstrating its perspectives on the development and broad applications of MS in studying galactosylation proteins regulated biological processes.

Design of a ruthenium(III) immobilized affinity material based on a ß-cyclodextrin-functionalized poly(glycidyl methacrylate-ethylene dimethacrylate) monolith for the enrichment of hippuric acid.

Immobilized metal affinity chromatography has drawn great attention as a widespread separation and purification approach. In this work, ruthenium was firstly introduced into the preparation of immobilized metal affinity chromatography considering its affinity to N,O-donor ligands. A ß-cyclodextrin-functionalized poly(glycidyl methacrylate-ethylene dimethacrylate) monolith was designed and employed as the supporting material in immobilized metal affinity chromatography. Thiosemicarbazide was introduced into the synthesis process, which not only acted as a bridge between ß-cyclodextrin and glycidyl methacrylate, but also chelated with ruthenium because of its mixed hard-soft donor characteristics. The developed monolithic ruthenium(III)-immobilized metal affinity chromatography column was utilized for the adsorption and separation of hippuric acid, a biological indicator of toluene exposure. To achieve high extraction capacity, the parameters affecting the extraction efficiency were investigated with an orthogonal experiment design, L9 (34 ). Under the optimized conditions, the enrichment factor of hippuric acid was 16.7-fold. The method reproducibility was investigated in terms of intra- and interday precisions with relative standard deviations lower than 8.7 and 9.5%, respectively. In addition, ruthenium(III)-immobilized metal affinity chromatography material could be used for up to 80 extractions without an apparent change in extraction recovery.

Specific enrichment of glycoproteins with polymer monolith functionalized with glycocluster grafted ß-cyclodextrin.

The low abundance of glycoproteins in complex samples results in the prerequisite role of efficient and selective enrichment of them. In the present work, we designed a new kind of glycosylation poly(hydroxyethyl methacrylate-pentaerythritol triacrylate) monolith functionalized with glycocluster grafted ß-cyclodextrin for the enrichment of glycoproteins. The introduced modifiers endowed the monolithic material with enhanced hydrophilicity and surface area, which benefitted to improve the enrichment selectivity and extraction efficiency for glycopeptides. By combining with MALDI-MS detections, 22 glycopeptides from horseradish peroxidase digest were captured with the developed monolith while 4 glycopeptides were enriched by commercially available agarose matrix column. LOD of 6.6pmol was attained. When applied to the enrichment of glycopeptides from complex protein samples and human lymphoma (U937) cell line, the prepared monolith exhibited high selectivity for glycopeptides.

The role of liquid-liquid transition in glass formation of CuZr alloys.

Some glass-forming liquids have different liquid phases that have the same composition but different structure, density and entropy. Based on experimental and molecular dynamics simulation, we here report thermodynamic, dynamic, and structural evidence of the liquid-liquid transition (LLT) in ten Cu-Zr glass-forming liquids well above the liquidus temperature. We find that for Cu-Zr alloys, the LLT is beneficial to glass formation, and there is a close relationship between the relative transition strength (RTS) of the LLT and the critical thickness representing the glass forming ability (GFA): the bigger the value of the RTS parameters, the stronger the GFA of Cu-Zr alloys. This work not only uncovers the role of the LLT of melts in the glass formation of solids, but also sheds light on the inheritance of properties of glassy solids from the aspect of the detectable dynamics of high-temperature melts.

Preparation of a polymer monolith modified with delaminated layered double hydroxides for the microextraction of ß-agonists.

In the present study, a novel poly(acrylamide-co-N-isopropylacrylamide-co-ethylene dimethacrylate) monolithic column modified with saline-modified delaminated layered double hydroxides was developed and exploited as the stationary phase in the polymer monolith microextraction of ß-agonists. Transmission electron microscopy, Fourier transform infrared spectroscopy, scanning electron microscopy, X-ray photoelectron spectroscopy, and thermal gravimetric analysis with derivative thermogravimetric determination were employed to characterize the monoliths. By coupling with high-performance liquid chromatography determinations, extraction parameters affecting the extraction efficiency, including sample volume, flow rate, sample pH, eluent volume, and eluent flow rate were investigated with an orthogonal experiment design, L16 (45 ). Results demonstrated that under the optimal experimental conditions, low limits of detection in the range of 0.025-0.280 ng/mL were obtained with relative standard deviations in the range of 4.3-7.6% (intraday) and 6.7-8.8% (interday). When the developed method was applied to the analysis of ß-agonists in pork samples, recovery values were obtained in the range of 80.5-108.6%.