Preparation and characterization of a biomimetic honeycomb cross-linked chitosan membrane and its application in the serum of gastric cancer patients.

Chitosan, as a biological macromolecule with excellent biocompatibility, has great potential for application in immobilized metal affinity chromatography (IMAC) strategies. In-depth analysis of low-abundance phosphopeptides in organisms can help reveal the pathological mechanisms of diseases. Here, we developed an IMAC material based on a biomimetic honeycomb chitosan membrane. The material demonstrates excellent biocompatibility, good hydrophilicity, and strong metal chelating capacity, which collectively confer outstanding enrichment properties. The material has high sensitivity (0.05 fmol), great selectivity (1:2000), excellent cycling stability (at least 10 cycles) and acid-base stability. In addition, the material was employed in human serum, successfully enriching 129 phosphopeptides from the serum of gastric cancer patients and 146 phosphopeptides from healthy controls. Sequence logo suggests a potential association between gastric cancer and glutamine. Ultimately, an in-depth gene ontology analysis was carried out on the phosphopeptides that were enriched in the serum samples. Compared to normal controls, our results demonstrated dysregulated expression of biological process, cellular component, and molecular function in gastric cancer patients. This suggests that the disease involves, such as blood coagulation pathways, cholesterol metabolism, and heparin binding. All experimental outcomes converge to demonstrate the substantial promise of the material for applications within proteomics research.

One-step fabrication of dipeptide-based bifunctional polymer for individual enrichment of glycopeptides and phosphopeptides from serum.

A dipeptide-based bifunctional material immobilized with Ti4+ (denoted as APE-MBA-VPA-Ti4+) was developed using precipitation polymerization. This polymer combines hydrophilic interaction liquid chromatography (HILIC) and immobilized metal affinity chromatography (IMAC) enrichment strategies, allowing for the individual and simultaneous enrichment of glycopeptides and phosphopeptides. It demonstrated high sensitivity (0.1 fmol µL-1 for glycopeptides, 0.005 fmol µL-1 for phosphopeptides), strong selectivity (molar ratio HRP: BSA = 1:1000, ß-casein: BSA = 1:2500), consistent reusability (10 cycles) and satisfactory recovery rate (93.5 ± 1.8 % for glycopeptides, 91.6 ± 0.6 % for phosphopeptides) in the individual enrichment. Utilizing nano LC-MS/MS technology, the serum of liver cancer patients was analyzed after enrichment individually, resulting in the successful capture of 333 glycopeptides covering 262 glycosylation sites, corresponding to 131 glycoproteins, as well as 67 phosphopeptides covering 57 phosphorylation sites, related to 48 phosphoproteins. In comparison, the serum of normal healthy individuals yielded a total of 283 glycopeptides covering 244 glycosylation sites corresponding to 126 glycoproteins, as well as 66 phosphopeptides covering 56 phosphorylation sites related to 37 phosphoproteins. Label-free quantification identified 10 differentially expressed glycoproteins and 8 differentially expressed phosphoproteins in the serum of liver cancer patients. Among them, glycoproteins (HP, BCHE, AGT, C3, and PROC) and phosphoproteins (ZYX, GOLM1, GP1BB, CLU, and TNXB) showed upregulation and displayed potential as biomarkers for liver cancer.

Exploitation of porphyrin-based titanium-rich porous organic polymers for targeted phosphopeptide enrichment from the serum of colorectal cancer individuals.

A porphyrin-based titanium-rich porous organic polymer (Th-PPOPs@Ti4+) was designed based on immobilized metal ion affinity chromatography technique and successfully applied to phosphopeptide enrichment with 5,10,15,20-tetrakis(4-carboxyphenyl) porphine tetramethyl ester (TCPTE), 2,3-dihydroxyterephthalaldehyde (DHTA), and 2,3,4-trihydroxybenzaldehyde (THBA) as raw materials. Th-PPOPs@Ti4+ exhibited remarkable sensitivity (0.5 fmol), high selectivity (ß-casein: BSA = 1:2000, molar ratio), outstanding recovery (95.0 ± 1.9%), reusability (10 times), and superior loading capacity (143 mg·g-1). In addition, Th-PPOPs@Ti4+ exhibited excellent ability to specifically capture phosphopeptides from the serum of colorectal cancer (CRC) individuals and normal subjects. Sixty phosphopeptides assigned to 35 phosphoproteins were obtained from the serum of CRC individuals, and 43 phosphopeptides allocated to 28 phosphoproteins were extracted in the serum of healthy individuals via nano-LC-MS/MS. Gene ontology assays revealed that the detected phosphoproteins may be inextricably tied to CRC-associated events, including response to estrogen, inflammatory response, and heparin binding, suggesting that it is possible that these correlative pathways may be implicated in the pathogenesis of CRC.

Ti4+ functionalized zirconium metal-organic frameworks with polymer brushes for specific identification of phosphopeptides in human serum and skimmed milk.

In the present study, click chemistry and Schiff base reactions were simultaneously applied to prepare polymer brush (PEG)-functionalized MOF materials (UiO-66-NH2) and immobilized with Ti4+ (MOF-Brush-THBA-Ti4+) for phosphopeptide analysis. The material has a detection limit of 0.5 fmol, a selectivity of 2000:1, and a loading capacity of 133 mg/g for phosphopeptides. It also demonstrated great repeatability (10 cycles) and recovery rate (96.7 ± 1.4%). During the analysis of bio-samples, 4 specific phosphopeptides were identified in endogenous breast cancer serum, while 11 phosphopeptides were identified in skimmed milk. Moreover, 47 phosphopeptides correlated with 29 phosphorylated proteins were selectively identified from normal control serum, and 66 phosphopeptides correlated with 26 phosphorylated proteins were identified from breast cancer serum. Further analysis of gene ontology (GO) revealed that the detected phosphorylated proteins associated with breast cancer included positive regulation of receptor-mediated endocytosis, proteolysis, extracellular exosome, heparin binding, and chaperone binding. These findings suggest that these associated pathways might contribute to the etiology of breast cancer. Overall, this application exhibits enormous potential in the identification of phosphorylated peptides within bio-samples.

In-Depth Endogenous Phosphopeptidomics of Serum with Zirconium(IV)-Grafted Mesoporous Silica Enrichment.

Detection of endogenous peptides, especially those with modifications (such as phosphorylation) in biofluids, can serve as an indicator of intracellular pathophysiology. Although great progress has been made in phosphoproteomics in recent years, endogenous phosphopeptidomics has largely lagged behind. One main hurdle in endogenous phosphopeptidomics analysis is the coexistence of proteins and highly abundant nonmodified peptides in complex matrices. In this study, we developed an approach using zirconium(IV)-grafted mesoporous beads to enrich phosphopeptides, followed by analysis with a high resolution nanoRPLC-MS/MS system. The bifunctional material was first tested with digests of standard phosphoproteins and HeLa cell lysates, with excellent enrichment performance achieved. Given the size exclusion nature, the beads were directly applied for endogenous phosphopeptidomic analysis of serum samples from pancreatic ductal adenocarcinoma (PDAC) patients and controls. In total, 329 endogenous phosphopeptides (containing 113 high confidence sites) were identified across samples, by far the largest endogenous phosphopeptide data set cataloged to date. In addition, the method was readily applied for phosphoproteomics of the same set of samples, with 172 phosphopeptides identified and significant changes in dozens of phosphopeptides observed. Given the simplicity and robustness of the proposed method, we envision that it can be readily used for comprehensive phosphorylation studies of serum and other biofluid samples.

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 1 min. Meanwhile, the material has high selectivity (1:2000), good loading capacity (100 µg/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 the cholesterol metabolic process and cell-matrix adhesion in the biological process (BP), endoplasmic reticulum and lipoprotein in the cellular component (CC), and heparin-binding, lipid-binding, and receptor-binding in the molecular function (MF) as compared with normal individuals. All the experiments indicate that the nanomaterials have great potential in proteomics studies.

Bimetallic Ordered Large-Pore MesoMOFs for Simultaneous Enrichment and Dephosphorylation of Phosphopeptides.

Despite the fact that bimetallic metal-organic frameworks (MOFs) could afford multiple functionalities by a synergistic effect of individual metallic centers, their intrinsic microporous structure frequently restricts their wide applications with bulky molecules involved. An urgent need is consequently triggered to design bimetallic hierarchical mesoporous MOFs (mesoMOFs). Herein, Zr/Ce mesoMOFs with a uniform pore size of up to 8 nm was successfully synthesized by a copolymer template strategy with the aid of a Hoffmeister ion. The obtained Zr/Ce mesoMOFs feature high porosity, good chemical and thermal stabilities, and tunable element components, and up to 70% Zr could be incorporated into the mesoporous Ce-based framework without deteriorating its crystallinity. Thanks to the synergistic effect of inherent Ce and Zr as well as the large and open pore channels, a broad range of phosphopeptides with different molecule sizes could be effectively checked out, thanks to their simultaneous enrichment and dephosphorylation capabilities. Such an ability to efficiently concentrate phosphopeptides remained intact even in the presence of abundant non-phosphorylated species. The practical detection of phosphopeptides from human serum was also verified, prefiguring the great potentials of bimetallic large-pore mesoMOFs for the proteome applications.

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 ß-cyclodextrin (ß-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 ß-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.

Serum levels of bone formation and resorption markers in relation to vitamin D status in professional gymnastics and physically active men during upper and lower body high-intensity exercise.

PURPOSE/INTRODUCTION: To compare serum levels of bone turnover markers in athletes and non-athletes, and to evaluate the relationship between serum levels of vitamin D metabolites and exercise-induced changes in biomarker levels. METHODS: Sixteen elite male artistic gymnasts (EG; 21.4 ± 0.8 years-old) and 16 physically active men (the control group, PAM; 20.9 ± 1.2 years-old) performed lower and upper body 30-s Wingate anaerobic tests (LBWT and UBWT, respectively). For biomarker analysis, blood samples were collected before, and 5 and 30 min after exercise. Samples for vitamin D levels were collected before exercise. N-terminal propeptide of type I collagen (PINP) was analysed as a marker of bone formation. C-terminal telopeptide of type I collagen (CTX) was analysed as a marker of bone resorption. RESULTS: UBWT fitness readings were better in the EG group than in the PAM group, with no difference in LBWT readings between the groups. UBWT mean power was 8.8% higher in subjects with 25(OH)D3 levels over 22.50 ng/ml and in those with 24,25(OH)2D3 levels over 1.27 ng/ml. Serum CTX levels increased after both tests in the PAM group, with no change in the EG group. PINP levels did not change in either group; however, in PAM subjects with 25(OH)D3 levels above the median, they were higher than those in EG subjects. CONCLUSION: Vitamin D metabolites affect the anaerobic performance and bone turnover markers at rest and after exercise. Further, adaptation to physical activity modulates the effect of anaerobic exercise on bone metabolism markers.

Elevated Urate Levels Do Not Alter Bone Turnover Markers: Randomized Controlled Trial of Inosine Supplementation in Postmenopausal Women.

OBJECTIVE: Observational studies have consistently demonstrated that serum urate level positively correlates with bone mineral density (BMD). We undertook this study to determine whether moderate hyperuricemia induced by inosine supplements influences bone turnover markers in postmenopausal women over a 6-month period. METHODS: One hundred twenty postmenopausal women were recruited for a 6-month randomized, double-blind, placebo-controlled trial. Key exclusion criteria were osteoporosis, previous fragility fracture, bisphosphonate therapy, gout, kidney stones, and a urine pH level of ≤5.0. Participants were randomized in a 1:1 ratio to receive placebo or inosine. The coprimary end points were change in levels of N-propeptide of type I procollagen (PINP) and change in levels of ß-C-telopeptide of type I collagen (ß-CTX). Change in BMD, as measured by dual x-ray absorptiometry, was an exploratory end point. RESULTS: Administration of inosine led to a significant increase in serum urate concentration over the study period (P < 0.0001 for all follow-up time points). At week 26, the mean change in serum urate concentration was +0.13 mmoles/liter (+2.2 mg/dl) in the inosine group and 0.00 mmoles/liter (0 mg/dl) in the placebo group. There was no difference in PINP or ß-CTX levels between groups over the 6 months. There were no significant changes in bone density between groups over the 6 months. Adverse events and serious adverse events were similar between the 2 groups. CONCLUSION: This clinical trial shows that although inosine supplementation leads to sustained increases in serum urate levels over a 6-month period, it does not alter markers of bone turnover in postmenopausal women. These findings do not support the concept that urate has direct biologic effects on bone turnover.

Efficient separation of phosphopeptides employing a Ti/Nb-functionalized core-shell structure solid-phase extraction nanosphere.

A strategy for effectively enriching global phosphopeptides was successfully developed by using ammonia methyl phosphate (APA) as a novel chelating ligand and Ti4+ and Nb5+ as double functional ions (referred to as Fe3O4@mSiO2@APA@Ti4+/Nb5+). With the advantage of large specific surface area (151.1 m2/g), preeminent immobilized ability for metal ions (about 8% of total atoms), and unbiased enrichment towards phosphopeptides, Fe3O4@mSiO2@APA@Ti4+/Nb5+ displays high selectivity (maximum mass ratio ß-casein to BSA is 1:1500), low limit of detection (LOD, as low as 0.05 fmol), good relative standard deviation (RSD, lower than 7%), recovery rate of 87% (18O isotope labeling method), outstanding phosphopeptide loading capacity (330 µg/mg), and at least five times re-use abilities. In the examination of the actual sample, 24 phosphopeptides were successfully detected in saliva and 4 phosphopeptides were also selectively extracted from human serum. All experiments have shown that Fe3O4@mSiO2@APA@Ti4+/Nb5+ exhibits exciting potential in view of the challenge of low abundance of phosphopeptides. Graphical abstract.

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.

Core-shell magnetic microporous covalent organic framework with functionalized Ti(iv) for selective enrichment of phosphopeptides.

Selective, sensitive and efficient phosphopeptide enrichment is significant in understanding phosphorylation-regulated processes and finding potential biomarkers. Magnetic materials have significant advantages for the separation and enrichment of modified peptides and proteins. In this work, we fabricated a core-shell magnetic Ti4+-functionalized covalent organic framework composite (denoted as Fe3O4@SiO2@TpPa-Ti4+) to selectively capture phosphopeptides in biosamples. The specific porous structure makes high surface area, which provides space and more affinity sites to binding titanium ions. The loading capacity of titanium ions was as high as 14% (wt%). Calculated by the MS results of α-casein tryptic digests, the binding capability could reach 200 mg g-1. Fe3O4@SiO2@TpPa-Ti4+ nanoparticles also demonstrate high sensitivity (above 0.2 fmol µL-1) and selectivity (α-casein : BSA = 1 : 5000). 1083 phosphopeptides were identified within two LC-MS/MS replicates with 91.8% specificity (phosphopeptides/all identified peptides). These MS results demonstrate that with the aid of the magnet, this method based on the magnetic titanium-functionalized covalent organic framework composite is applicable to achieve rapid, selective and efficient phosphopeptide analysis.

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.

Specific enrichment of phosphopeptides by using magnetic nanocomposites of type Fe3O4@graphene oxide and Fe3O4@C coated with self-assembled oligopeptides.

Iron(III-immobilized magnetic nano-composites (MNCs) were first fabricated using one-step aqueous self-assembly of oligopeptides (Glu-Pro-Ala-Lys-Ala-Lys-Ala-Lys; EPAK-VI) for the highly selective capture of phosphopeptides from complex biological samples. Under physiological conditions, EPAK-VI can readily self-organize into a robust and complete coating layer mainly composed of ß-sheets and ß-turns on the surface of Fe3O4@GO and Fe3O4@C MNCs. Tailored by the cyclic structure of proline, the Glu-Pro motifs of EPAK-VI are vertically erected on the surface and thus serve as an effective linker to chelate Fe3+ through carboxyl (COO-) group in the glutamic acid (E) residues. The ionic hydrogen bonds between the ε-amino groups and the surface negative charges coupled with intermolecular hydrogen bonds render the EPAK-VI coating on the MNCs insusceptible to repeated extreme washing conditions. The Fe3+-EPAK-VI coated MNCs exhibit high enrichment efficiency for ß-casein tryptic digest (0.05 fmol µL-1), excellent selectivity from mixed digests (ß-casein/bovine serum albumin, mass ratio 1:500), and high recovery rate (over 80%). Graphical abstractSchematic representation of the fabrication of Fe3+-immobilized MNCs for phosphopeptide enrichment.

Core-shell magnetic bimetallic MOF material for synergistic enrichment of phosphopeptides.

In proteomics, phosphorylation is an important process for protein post-translational modification (PTM), which greatly improves the diversity of proteomes. The PTM regulates almost all physiological and pathological processes such as signal transduction, cell division, proliferation, differentiation and metabolism. The abnormal expression of protein phosphorylation is also associated with cellular metabolic disorders and a range of diseases. However, in mass spectrometry-based phosphorylated peptideomics studies, phosphorylated peptide signals were inhibited by a high abundance of non-phosphorylated peptides; thus, highly selective enrichment was required. In this study, a newly designed material named Fe3O4@MIL(Fe/Ti) was synthesized using a layer-by-layer self-assembly technique that coats the surface of magnetic oxide nanospheres with bimetallic MOF of iron and titanium. The synergistic synthetic coating of the bimetallic MOF gives the material a large surface area and excellent hydrophilicity, which endow the nanoparticles with excellent phosphopeptide enrichment ability, high selectivity (ß-casein/BSA molar ratio 1:500), a low detection limit (3 fmol), high recovery rate (85%), strong binding capacity, size exclusion ability, and ideal batch-to-batch repeatability. For comparison, we used Fe3O4@MIL(Fe/Ti) and two single-metal MOF materials Fe3O4@MIL-100(Fe) and Fe3O4@MIL-125(Ti), to enrich α-casein in the middle. Thus, the iron-titanium bimetallic MOF can not only enrich all the phosphorylated peptides enriched by Fe3O4@MIL-100(Fe) and Fe3O4@MIL-125(Ti), but can also specifically enrich four phosphorylated peptides. Encouraged by the excellent results of characterization and standard protein enrichment, we used this material to analyze human serum and found that bimetallic materials can effectively enrich all four phosphorylated peptides and exclude high molecular proteins. These experimental results indicate that the novel bimetallic MOF is a good candidate to analyze protein phosphorylation in complex samples.

Solvothermal Synthesis of Novel Magnetic Nickel Based Iron Oxide Nanocomposites for Selective Capture of Global- and Mono-Phosphopeptides.

A facile solvothermal method was developed for synthesis of magnetic nickel-based iron oxide nanocomposites (MNFOs) with different ratios of Ni2+ to Fe3+ for different reaction time. Two factors including dosage of Ni source and length of reaction were investigated for influence on the morphology and composition of MNFOs, as well as their distinct selectivity for different phosphopeptides. After thorough characterization, the possible formation mechanism of MNFOs was proposed. Very interestingly, MNFOs with Ni2+/Fe3+ ratios of 4:5 prepared for 8 h (MNFO-S) and for 24 h (MNFO-L) can selectively capture global- and monophosphopeptides at the fmol level with excellent enrichment performance. These two affinity probes have been exploited to isolate and enrich the phosphopeptides from human normal hepatic cells HL 7702 after exposure to atmospheric fine particulates (PM2.1), which revealed that the protein phosphorylation level was increased significantly in cells after stimulation by fine particulate matters. The findings could provide a new insight for the nickel-based affinity protocol to analyze the mutation of phosphopeptides during cellular signaling pathways in response to exogenous environment stimulation. Consequently, this present work proposed a promising strategy to isolate monophosphopeptides from global phosphopeptides for phosphoproteome research.

Magnetite nanoparticles coated with chitosan and polyethylenimine as anion exchanger for sorptive enrichment of phosphopeptides.

An anion exchange solid-phase sorbent is described. Chitosan coated magnetite nanoparticles were modified with polyethylenimine which is positively charged at pH 3 and therefore can be used for the magnet-supported enrichment of phosphopeptides which are negatively charged at this pH value. A 2-step strategy was used to synthesize the sorbent. The materials were characterized by transmission electron microscopy, scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, thermogravimetry and magnetic moment analysis. The anion exchanger was applied to extract phosphopeptides from a ß-casein digest. Characteristic analytical figures include (a) a loading buffer of pH 3, (b) and elution buffer of pH 11, (c) a loading time of 5 min, (d) good selectivity (the ß-casein to BSA ratio is 1:1000), and (e) excellent sensitivity (1 fmol). The optimized method was applied to egg yolk digest, non-fat milk digest, and diluted human serum. Graphical abstractSchematic representation of synthesis of PEI@chitosan@Fe3O4 nanoparticles, and of the enrichment of phosphopeptides by magnetic solid phase extraction prior to the determination of the peptides by MALDI-MS analysis.

Highly Selective Capture of Monophosphopeptides by Two-Dimensional Metal-Organic Framework Nanosheets.

Separation of monophosphopeptides from multi-phosphopeptides in complex biological samples is significant in the study of protein kinase signal transduction pathways. To the best of our knowledge, very few materials have been reported that could selectively enrich monophosphopeptides because of the chemical difficulty in retaining the intermediate monophosphopeptides and excluding both non-phosphopeptides and multi-phosphopeptides in acidic conditions, which requires unique interactions to balance the metallic affinity and the hydrophobicity. With the large surface area, abundant accessible active sites, and ultrathin structures, two-dimensional (2-D) metal-organic framework (MOF) Hf-1,3,5-tris(4-carboxyphenyl)benzene (BTB) nanosheets were rationally selected. Due to the elongated organic ligands and the balance between metallic affinity of clusters and hydrophobicity from ligands, the 2-D Hf-BTB nanosheets exhibited unique enrichment selectivity toward monophosphopeptides. The 2-D MOF nanosheets demonstrated excellent sensitivity (detection limit of 0.4 fmol µL-1) and selectivity [1:1000 molar ratios of ß-casein/BSA (bovine serum albumin)] in model phosphopeptides enrichment. The nanosheets were implemented for the analysis of nonfat milk and human saliva samples as well as in situ isotope labeling for dysregulated phosphopeptides from patients' serum with anal canal inflammation, exhibiting 6.6-fold upregulation of serum phosphopeptide HS4 (ADpSGEGDFLAEGGGVR) compared to the control healthy serum. The proteomics analysis of mouse brain cortical samples associated with Alzheimer's disease, which were from Akt (protein kinase B) conditional knockout mouse and littermate control mouse, was further established with 2-D Hf-BTB nanosheets. With high capture efficiency for monophosphopeptides, this method was capable of distinguishing the difference of monophosphopeptides from microtubule-associated protein τ (MAPT/τ) between the Akt knockout sample and control sample.

Instrument-free enrichment and detection of phosphopeptides using paper-based Phos-PAD.

The facile detection of phosphopeptides is important for clinical screening and phosphoproteomic research. This work develops an instrument-free, cost-effective, convenient paper-based method for quantitative analysis of phosphorylated peptides. With a novel portable device, Phos-PAD, this method can achieve selective enrichment and colorimetric detection of phosphopeptides within 15 min TiO2 nanoparticle-based chemisorption and tetrabromophenol blue-based colorimetric assay were integrated into the single paper-based analytical device. The color change can indicate the presence of phosphopeptides and the mean pixel intensity of the red channel can be used for phosphopeptide quantification. With capability of quantifying phosphopeptides in serum samples, this Phos-PAD assisted phosphopeptide assay may attract significant attention to clinical analysis of endogenous serum phosphopeptides.