Identification and quantification of bipyridyl dicarboxylic acid isomers by ion mobility spectrometry.

The identification of positional isomers is of interest because different isomers have different chemical or biological functions and applications. The analysis of positional isomers is sometimes challenging since they have similar chemical structures and properties. For example, the analysis of mass cannot identify different positional isomers because they have identical mass-to-charge ratios and show a single mass peak in mass spectrometry. In this study, an efficient and simple qualitative and quantitative analytical method for differentiating 2,2'-bipyridine-3,3'-dicarboxylic acid (3,3'-BDA), 2,2'-bipyridine-4,4'-dicarboxylic acid (4,4'-BDA), and 2,2'-bipyridine-5,5'-dicarboxylic acid (5,5'-BDA) was developed by using ion mobility spectrometry (IMS). The results revealed that the three BDA isomers formed non-covalent complexes with cyclodextrins (CDs) and Mg2+ ions in the gas phase: [ß-CD+3,3'/4,4'/5,5'-BDA+Mg]2+ and [γ-CD+3,3'/4,4'/5,5'-BDA+Mg]2+, which were distinguished by measuring the mobility of the complexes because of their spatial conformational differences. The peak-to-peak resolution (Rp-p) values of the three isomers of [γ-CD+3,3'/4,4'/5,5'-BDA+Mg]2+ reached 2.983 and 2.892, respectively. The conformations of the ternary complexes simulated by the theoretical calculations revealed the different interactions and shapes of the stereoisomers, and the predicted results agreed with the experimental results. Simultaneously, further studies on the collisional dissociation of the ternary complexes revealed that the dissociation energies of the different complex ions varied were different owing to the diverse different conformations. Finally, the relative quantitative analysis of the different isomers in mixed samples was performed and satisfactory linearity results (R2 > 0.99) were obtained. Thus, an effective analytical method was proposed for the identification and quantification of BDA isomers without chemical derivatization, offering a promising approach for the identification of similar derivatives or positional isomers that could be applied in various fields including chemicals and pharmaceuticals.

Highly sensitive quantitative detection of glycans on exosomes in renal disease serums using fluorescence signal amplification strategies.

Exosomal glycoproteins play a significant role in many physiological and pathological processes. However, the detection of exosome surface glycans is currently challenged by the complexity of biological samples or the sensitivity of the methods. Herein, we prepared a novel fluorescent probe of biotin-functionalized nanocrystals (denoted as CdTe@cys-biotin) and applied it for the first time for the detection of the expression of exosomal surface glycans using a fluorescence amplification strategy. First, the dual affinity of TiO2 and CD63 aptamers of Fe3O4@TiO2-CD63 was utilized to rapidly and efficiently capture exosomes within 25 min. In this design, interference from other vesicles and soluble impurities can be avoided due to the dual recognition strategy. The chemical oxidation of NaIO4 oxidized the hydroxyl sites of exosomal surface glycans to aldehydes, which were then labeled with aniline-catalyzed biotin hydrazide. Using the high affinity between streptavidin and biotin, streptavidin-FITC and probes were successively anchored to the glycans on the exosomes. The fluorescent probe achieved the dual function of specific recognition and fluorescent labeling by modifying biotin on the surface of nanocrystals. This method showed excellent specificity and sensitivity for exosomes at concentrations ranging from 3.30 × 102 to 3.30 × 106 particles/mL, with a detection limit of 121.48 particles/mL. The fluorescent probe not only quantified exosomal surface glycans but also distinguished with high accuracy between serum exosomes from normal individuals and patients with kidney disease. In general, this method provides a powerful platform for sensitive detection of exosomes in cancer diagnosis.

In situ grown magnetic COF@MOF with a phosphoserine anchor for in-depth N-glycopeptide analysis in serum.

A hydrophilic phosphoserine-functionalized magnetic organic framework composite (termed Fe3O4@COF@MOF-PS) was synthesized by an in situ growth strategy for effective capture of N-glycopeptides. Fe3O4@COF@MOF-PS exhibited high sensitivity (0.2 fmol µL-1), outstanding exclusion of size capability (1 : 10 000), good selectivity (1 : 2000), and reusability (at least 10 times). It also exhibited remarkable performance in the N-glycopeptide analysis in complex biological samples. Via nano-LC-MS/MS analysis, a total of 223 N-glycopeptides with 161 glycosylation sites assigned to 91 glycoproteins and 331 N-glycopeptides with 243 glycosylation sites assigned to 134 glycoproteins were identified in sera from cervical cancer patients and normal controls, respectively. Biological processes and molecular functional analyses indicate that the captured glycoproteins are of significant relevance to cervical cancer, for example, gene coverage or expression of cell adhesion and extracellular matrix structural constituents. Thus, Fe3O4@COF@MOF-PS not only efficiently captures N-glycopeptides, but also has the possibility of screening potential disease markers and elucidating the process of cervical cancer development.

Ti4+ functionalized ß-cyclodextrin covalent organic framework as a new immobilized metal ion affinity chromatography platform for selective capture of phosphorylated peptides and exosomes.

A Ti4+ functionalized ß-cyclodextrin covalent organic framework nanoparticle (named as ß-CD-COF@Ti4+) was synthesized using a one-pot method successfully realizing the enrichment of phosphorylated peptides and exosomes based on the immobilized metal ion affinity chromatography strategy. The functionalized ß-CD-COF@Ti4+ exhibited superior performance on the enrichment of phosphopeptides, including high selectivity (1:1000), low detection limit (0.5 fmol), and loading capacity for phosphopeptides (100 mg·g-1). After treatment with ß-CD-COF@Ti4+, 9 phosphopeptides from defatted milk, 29 phosphopeptides related to 23 phosphoproteins from normal group serum, and 24 phosphopeptides related to 22 phosphoproteins from the serum of uremia patients were captured. Through the analysis of Gene Ontology, the captured phosphoprotein is closely related to kidney disease, including lipoprotein metabolism, very-low-density lipoprotein particle, high-density lipoprotein particle, and lipid binding activity process. Furthermore, western blot verification showed that this nanoparticle could successfully capture exosomes from human serum. This study demonstrates great prospects for the enrichment of phosphopeptides and exosomes from actual bio-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.

One-step preparation of boronic acid-rich hydrothermal spheres for N-glycopeptide analysis from preeclampsia serum.

In this study, we developed a green, one-step hydrothermal carbonization (HTC) method that used water as the sole solvent to create boronic acid group-rich carbonaceous spheres (BCS). When the abundant boronic acid groups on the carbonaceous spheres react with the hydroxyl groups on the glycans in an alkaline environment, the glycopeptides are specifically captured. The results showed that BCS had excellent detection limits (0.1 fmol µL-1), selectivity (1 : 1000), and stability (10 cycles). In addition, the BCS also demonstrated excellent glycopeptide enrichment capabilities in complex biological samples; 219 glycopeptides attributed to 167 glycoproteins and 235 glycopeptides related to 166 glycoproteins in PE patient and normal pregnancy control serum were identified by nano LC-MS/MS, respectively. In addition, the molecular function of heparin binding and biological process of complement activation, positive regulation of immune response, and positive regulation of tumor necrosis factor production were significantly different between PE patients and healthy pregnant women according to gene ontology analysis, indicating that these may be associated with the development of PE.

Application of Microscopic Highly Hydrophilic Silica-Based Nanocomposites with High Surface Exposure in the Efficient Identification of Intact N-Glycopeptides.

Glycosylation of proteins regulates the life activities of organisms, while abnormalities of glycosylation sites and glycan structures occur in various serious diseases such as cancer. A separation and enrichment procedure is necessary to realize the analysis of the glycoproteins/peptides by mass spectrometry, for which the surface hydrophilicity of the material is an important factor for the separation and enrichment performance. In the present work, under the premise of an obvious increase of the surface silicon exposure (79.6%), the amount of surface polar silanol is remarkably generated accompanying the introduction of the active amino groups on the surface of silica. The microscopic hydrophilicity, which is determined with water physical-adsorption measurements and can directly reflect the interaction of water molecules and the intrinsic surface of the material, maximally increases by 44%. This microscopically highly hydrophilic material shows excellent enrichment ability for glycopeptides, such as extremely low detection limits (0.01 fmol µL-1), remarkable selectivity (1:8000), and size exclusion effects (1:8000). A total of 677 quantifiable intact N-glycopeptides were identified from the serum of patients with cervical cancer, and the glycosylation site and glycan structure were analyzed in depth, indicating that this novel material can show a broad practical application in cervical cancer diagnosis.

Selective enrichment of glycopeptides using ground eggshell materials.

The current research of protein glycosylation is focused on develop various functionalized hydrophilic materials that can effectively enrich glycopeptides. However, most of these materials require complex synthesis steps, plenty of chemical reagents, and high cost. In this study, we employed the natural eggshell for glycopeptides enrichment for the first time. Using horseradish peroxidase (HRP) tryptic digest as a standard sample, eggshell exhibited excellent sensitivity (0.05 fmol µL-1), good selectivity [HRP tryptic digest:bovine serum albumin (BSA) tryptic digest = 1:1000], excellent size-exclusion effect (HRP tryptic digest:BSA protein = 1:10,000), good loading capacity (75 mg g-1), and recovery (97.6 ± 0.3%). In addition, 153 and 114 glycopeptides were captured by eggshell from the serum tryptic digests of normal humans and diabetic patients, respectively. Benefiting from the singular porous structure and abundant biomass, eggshell performed excellently in the capture and separation of glycopeptides. These results demonstrated the potential of environmentally friendly eggshell in glycosylation proteomics analysis.

Surface functionalization modification of ultra-hydrophilic magnetic spheres with mesoporous silica for specific identification of glycopeptides in serum exosomes.

Protein glycosylation of human serum exosomes can reveal significant physiological information, and the development of large-scale identification strategies is crucial for the in-depth investigation of the serum exosome glycoproteome. In this study, using surface functionalization techniques, an ultra-hydrophilic mesoporous silica magnetic nanosphere (denoted as Fe3O4-CG@mSiO2) was synthesized for the quick and accurate detection of glycopeptides from HRP digests. The Fe3O4-CG@mSiO2 nanospheres demonstrated outstanding enrichment capability, high sensitivity (5 amol/µL), good size exclusion effect (HRP digests/BSA proteins, 1:10,000), stable reusability (at least 10 times), and an excellent recovery rate (108.6 ± 5.5%). Additionally, after enrichment by Fe3O4-CG@mSiO2, 156 glycopeptides assigned to 64 proteins derived from human serum exosomes were successfully identified, which demonstrates that the nanospheres have great potential for the research of the large-scale serum exosome glycoproteome.

Magnetic guanidyl-functionalized covalent organic framework composite: a platform for specific capture and isolation of phosphopeptides and exosomes.

A guanidine-functionalized (GF) covalent organic framework (COF) nanocomposite has been developed by a post-synthetic approach for specific capture and separation of phosphopeptides and exosomes. The abundant binding sites on COF can immobilize a large number of gold nanoparticles (AuNPs), which can be used to react with amino groups to graft polyethyleneimine (PEI). Finally, Fe3O4@COF@Au@PEI-GF is obtained through the reaction of PEI and guanidyl group for phosphopeptides and exosomes detection. This composite shows a low detection limit (0.02 fmol), size exclusion effect (ß-casein digests:Albumin from bovine serum protein = 1:10,000), good reusability (10 cycles), and high selectivity (ß-casein digests:Albumin from bovine serum digests = 1:10,000). For complex biological sample, 4 phosphopeptides can be successfully identified from human serum. Furthermore, for the first time, we used guanidyl-functionalized probe to capture exosomes in human serum, providing a new method for enriching exosomes. The above experiments showed that Fe3O4@COF@Au@PEI-GF not only effectively enrich phosphopeptides and remove macromolecular proteins, but also successfully separate and capture exosomes. This demonstrates the great potential of this composite for the specific enrichment of phosphopeptides and isolation of exosomes.

Construction of boric acid-functionalized metal-organic frameworks for glycopeptide recognition in the serum of cervical cancer patients.

RATIONALE: Cervical cancer is one of the most common malignant tumors in women, and it is essential to explore potential biomarkers such as glycopeptides closely related to cancer in physiological samples of cervical cancer patients. Sample pretreatment is required before direct detection using mass spectrometry because there are certain limitations. Meanwhile, it is still highly desired to promote the functionalization and application of metal-organic framework (MOF)-derived materials. METHODS: Using a post-synthesis modification method, a novel type of boric acid-functionalized MOF probe (designated as UiO-66@PEI@Au@B(OH)2 ) is prepared for recognition of glycopeptides. The results are obtained using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nano-liquid chromarography-tandem mass spectrometry. RESULTS: The UiO-66@PEI@Au@B(OH)2 probe exhibits a low detection limit (0.6 fmol µL-1 ), an excellent recovery rate, comparatively good reusability and selectivity (HRP digests:BSA digests = 1:500). When UiO-66@PEI@Au@B(OH)2 is used to selectively capture glycopeptides from the serum of a healthy person and a cervical cancer patient, 101 glycopeptides corresponding to 54 glycoproteins and 108 glycopeptides corresponding to 57 glycoproteins are detected, respectively. CONCLUSIONS: The successful preparation of UiO-66@PEI@Au@B(OH)2 provides a path for the investigation of the functionalization of MOF-derived materials. The excellent performance of UiO-66@PEI@Au@B(OH)2 not only demonstrates the huge potential of functionalized MOFs in the glycoproteome, but also opens up new phases of the application of MOF-based materials.

Metal organic frameworks as advanced adsorbent materials for separation and analysis of complex samples.

Sample pre-treatment is of great significance for study protein phosphorylation and glycosylation. Due to the low concentration and suppression of matrices, the direct detection of phosphoprotein and glycoprotein by mass spectrometry (MS) are still facing many challenges. The demand of efficient and specific enrichment of phosphoprotein and glycoprotein promotes the sample preparation methods based on metal-organic frameworks (MOFs). In this review, recent advances in MOFs-based samples pre-treatment, including the enrichment of protein, endogenous peptides, glycopeptides, phosphopeptides and glycans are summarized and discussed. In addition, the design and synthesis of different types of MOFs according to different enrichment mechanisms and principles are discussed. Furthermore, the potential problems of sample pre-treatment in proteomics is prospected.

Bi-amino acid functionalized biomimetic honeycomb chitosan membrane as a multifunctional hydrophilic probe for specific capture of N-linked glycopeptides in nasopharyngeal carcinoma's disease patient's serum.

In this work, a novel porous bifunctionalized composite material was synthesized via a simple method. Gold nanoparticles are uniformly dispersed on the surface of the biomimetic honeycomb chitosan membrane through the interaction between amino and Au, and then cysteine and glutathione are successfully grafted onto the surface of the Au by the Au-S bond. The modification of cysteine and glutathione makes this bifunctionalized composite material have significant advantages of superhydrophilicity and small steric hindrance simultaneously. This material manifests excellent property in glycopeptides enrichment, with high selectivity (1:5000), low detection limit (0.1 fmol·µL-1 ), high recovery rate (99.4 ± 0.5%), and good repeatability. In addition, with the help of nano-flow liquid chromatography tandem mass spectrometry, this composite achieved excellent performance in efficiently enriching glycopeptides in the serum of healthy people and nasopharyngeal carcinoma's disease patient. More excitingly, further gene ontology analysis of molecular function and biological process indicated that 41 original glycoproteins of the identified glycopeptides from serum of nasopharyngeal carcinoma's disease patient significantly partake in numerous cancer-associated events, including protease binding, calcium ion binding, enzyme binding, extracellular matrix organization, cellular response to tumor necrosis factor, and inflammatory response.

Post-synthesis of boric acid-functionalized magnetic covalent organic framework as an affinity probe for the enrichment of N-glycopeptides.

A novel type of boric acid-functionalized magnetic covalent organic framework (mCOF) with polyethyleneimine (PEI) as a linker (denoted as mCOF@PEI@B(OH)2) has been prepared through a post-synthesis strategy, which points out an achievable path for the construction of boronic acid-functionalized COFs. Based on the boric acid chemistry, the obtained core-shell structured mCOF@PEI@B(OH)2 can selectively isolate glycopeptides through the modified boronic acid groups. The mCOF@PEI@B(OH)2 exhibits excellent performance with good reusability (ten cycles), low detection limit (0.5 fmol·µL-1), size-exclusion effect, and relatively high loading capacity (80 µg·mg-1), recovery yield (94.9 ± 2.8%), and selectivity (HRP digests:BSA digests = 1:500). Detection is done by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In addition, 37 endogenous glycopeptides are captured from human saliva with mCOF@PEI@B(OH)2, providing effective proofs for its capability to capture low-abundance glycopeptides from actual biological samples.

Clinical application of individualized total arterial coronary artery bypass grafting in coronary artery surgery.

BACKGROUND: Total arterial revascularization is associated with increased patency and long-term efficacy and decreased perioperative morbidity and mortality and incidence of cardiac-related events and sternal wound infection compared with conventional coronary artery bypass surgery (CABG), in which the left internal mammary artery (LIMA) is typically grafted to the left anterior descending artery with additional saphenous vein grafts often used. This study determined whether these favorable clinical results could be realized at the authors' institute. AIM: To summarize the early efficacy and clinical experience of individualized total arterial coronary artery bypass grafting surgery. METHODS: CABG was performed on 35 patients with non-single-vessel coronary artery disease by adopting total arterial grafts at Fourth Affiliated Hospital of Harbin Medical University between April 2016 and December 2019. LIMA was used in 35 patients, radial artery (RA) was used in 35 patients, and right gastroepiploic artery (RGEA) was used in 9 patients. Perioperative complications were observed, short-term graft patency rate was followed-up, and quality of life was assessed. RESULTS: All patients underwent off-pump coronary artery bypass and the surgeries were successful. All of them were discharged without any complications or deaths. During the follow-up, it was found that patients' angina symptoms were relieved and New York Heart Association classification for cardiac function was class I to class II. A total of 90 vessels were grafted with no occlusion for internal mammary artery, three occlusions for RA, and one occlusion for RGEA. CONCLUSION: The individualized total arterial strategy based on the vessels targeting individual anatomic characteristics can achieve complete revascularization with satisfactory short-term grafting patency rate.

Effects of chromium stress on the rhizosphere microbial community composition of Cyperus alternifolius.

Wetland plants are often used as the main body of soil, and the rhizosphere is a hot spot migration and transformation. Response mechanism to rhizosphere microorganisms on chromium(Cr) stressing could help improve the phytoremediation system. Cyperus alternifolius(CA) is selected as the research object by Cr-stress treatments and uncontaminated treatments with different cultivated pattern, included sole cultivated pattern(CAI), two-cultivated pattern (CAII), three-cultivated pattern (CAIII), and the un-planted blank samples (CK). 16s rRNA gene sequencing and metagenomic sequencing are performed to measure rhizosphere microbial community. And Five common enzymes in rhizosphere soils were observed: ß-1,4-glucosidase (BG), ß-N-acetylglucosaminidase (NAG), ß-1,4-xylosidase (BX), cellobiohydrolase (CBH) and Leucine amino peptidase (LAP) in the rhizosphere. The results show that Gammaproteobacteria, Actinobacteria, Alphaproteobacteria, Gemmatimonadetes, Deltaproteobacteria are top five (63.97%) of the total sequence number. Wetland plants enriched a large amount of soil Cr in themselves, and the rhizosphere microorganisms don't show significant difference in community structure after affecting. 10.48% variation of microbial community is caused by Cr-stress. Acidovorax showed a great potential for chromium resistance. BX involvement in tolerance processes indirectly affects microbial communities (P < 0.01), there is a strong linear relationship between enzyme activity and the plants accumulating Cr and microbial community within 15.58% variant. The material accumulation and microbial quantity of CAIII are relatively low, but high biodiversity remains after affecting. These results provide references for in-depth understanding of rhizosphere microbial response to heavy metal pollution in wetland phytoremediation and interaction between wetland plants and rhizosphere microorganisms.

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.

Simple transabdominal minimally invasive direct coronary artery bypass surgery with right gastroepiploic artery.

For three patients with isolated right coronary artery disease who had drug resistance and were intolerant to interventional therapy, simple transabdominal small incision bypass grafting of the right gastroepiploic artery and the posterior descending branch of the right coronary artery was conducted without cardiopulmonary. All three patients were discharged smoothly without complications, and were followed up for three months, during which time the myocardial bridges were unobstructed and the cardiac functions were good. The surgery needs no thoracotomy and the injury is small, and avoids influences of sternum and pericardium adhesion on other cardiac surgery in the future. The risk of median sternotomy can be avoided for patients undergoing reoperation for coronary artery bypass surgery.

Mass filter with phase modulation of radio frequency voltage.

In this work, the parametric quadrupole resonance caused by the phase modulation of waveform potential is studied. Based on analytical and numerical description of the ion motion in the quadrupole mass filter with the phase modulation, a stability island is found with good ion optical properties such as high ion transmission efficiency (16%), high mass resolution (peak width measured at 10% of peak height, R0.1 = 6000), the required separation time (100-150 radio frequency [RF] cycles), and good peak shape of trapezoid form. Furthermore, the analysis of the frequency spectrum of the applied potential and the quadrupole mass filter (QMF) acceptance are also presented; a resonance frequency is found from this spectrum. Finally, a suitable stability X-islands with relative modulation frequency, ν = 2 ± ß , ß = 1 P , 10 ≤ P ≤ 40 , is established and studied in detail. Here, ß is the imaginary part of the characteristic exponent of stable solutions to the Mathieu equation.

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

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