RESUMEN
In this work, a composite hydrogel material consisting of chitosan-based composite hydrogel was prepared by a simple and rapid synthetic method and will be named three-dimensional (3D)-IL-COF-1@CS hydrogel. Possessing a stable 3D network structure and outstanding hydrophilicity, the novel hydrogel is capable of capturing glycopeptides. The 3D-IL-COF-1@CS hydrogel showed good sensitivity (0.1 fmol/µL) and selectivity (1:2000). In addition, 19 glycopeptides were captured in standard samples. In the analysis of human serum, 148 glycopeptides assigned to 72 glycoproteins were assayed in the serum of normal individuals, and 245 glycopeptides corresponding to 100 glycoproteins were found in the serum of colorectal cancer (CRC) patients. More importantly, several functional programs based on Gene Ontology analysis supported molecular biological processes that may be relevant to the pathogenesis of CRC, including aging, fibrinogen complex, and arylesterase activity. The low cost, simplicity, rapid synthesis, and good enrichment performance have a great future in glycoproteomics analysis and related diseases.
Asunto(s)
Neoplasias Colorrectales , Glicopéptidos , Interacciones Hidrofóbicas e Hidrofílicas , Humanos , Neoplasias Colorrectales/sangre , Glicopéptidos/sangre , Glicopéptidos/química , Hidrogeles/química , Polímeros/química , Quitosano/químicaRESUMEN
The development of facilely synthetic materials acts an essential role in glycoproteome analysis, especially for the highly efficient enrichment of N-linked glycopeptides. In this work, a facile and timesaving route was introduced in which COFTP-TAPT served as a carrier and poly (ethylenimine) (PEI) and carrageenan (Carr) were successively coated on the surface via electrostatic interaction. The resultant COFTP-TAPT@PEI@Carr showed remarkable performance in glycopeptide enrichment with high sensitivity (2 fmol µL-1), high selectivity (1:800, molar ratio of human serum IgG to BSA digests), large loading capacity (300 mg g-1), satisfactory recovery (102.4 ± 6.0%) and reusability (at least eight times). Due to the brilliant hydrophilicity and electrostatic interactions between COFTP-TAPT@PEI@Carr and positively charged glycopeptides, the prepared materials could be applied in the identification and analysis in the human plasma of healthy subjects and patients with nasopharyngeal carcinoma. As a result, 113 N-glycopeptides with 141 glycosylation sites corresponding to 59 proteins and 144 N-glycopeptides with 177 glycosylation sites corresponding to 67 proteins were enriched from 2 µL plasma trypsin digests of the control groups and patients with nasopharyngeal carcinoma, respectively. 22 glycopeptides were identified only from the normal controls and 53 glycopeptides were detected only from the other set. The results demonstrated that this hydrophilic material was promising on a large scale and further N-glycoproteome research.
Asunto(s)
Estructuras Metalorgánicas , Neoplasias Nasofaríngeas , Humanos , Glicopéptidos/análisis , Carcinoma Nasofaríngeo , Interacciones Hidrofóbicas e Hidrofílicas , Inmunoglobulina GRESUMEN
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.
Asunto(s)
Cáscara de Huevo , Glicopéptidos , Animales , Humanos , Glicopéptidos/química , Cáscara de Huevo/química , Cáscara de Huevo/metabolismo , Glicosilación , Interacciones Hidrofóbicas e Hidrofílicas , Peroxidasa de Rábano Silvestre/metabolismo , Albúmina Sérica Bovina/químicaRESUMEN
Exosomes can reflect the physiological state of parent cells and are potential disease biomarkers. In this study, we developed an innovative hydrophilic material by post-synthesis of covalent organic frameworks with dual hydrophilic groups of glutathione and cysteine (denoted as COF-S@Au@GC) to detect glycosylated exosomes in human serum. COF-S@Au@GC enriched glycosylated exosomes in human serum due to glutathione and cysteine (GC) hydrophilicity. Our results show that COF-S@Au@GC has a detection limit of 5 amol µL-1, selectivity of 1:2000, size-exclusion effect of 1:10,000, repeatability of 10 cycles, recovery of 98.3 ± 0.5%, and loading capacity of 50 mg g-1 for glycopeptides. In addition, 182 glycopeptides were detected after enrichment with COF-S@Au@GC from renal carcinoma serum, demonstrating the feasibility of enriching glycopeptides from complex biological samples. Furthermore, COF-S@Au@GC successfully captured glycosylated exosomes in the serum of renal cancer patients, with their 161 glycopeptides detected by nano liquid chromatography with tandem mass spectrometry (LC-MS/MS). This study provides a new heuristic strategy for isolating exosomes and contributes to further functional analysis of exosomes.
Asunto(s)
Exosomas , Estructuras Metalorgánicas , Cromatografía Liquida , Cisteína , Glutatión , Glicopéptidos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Espectrometría de Masas en TándemRESUMEN
In this work, nitrogen-rich linear porous organic polymers (POPs) were designed to improve glycopeptides enrichment efficiency in HILIC. Two kinds of novel POPs (CH-pPAL and CH-mPAL) were one-step prepared via the Schiff-base condensation reaction and intermolecular hydrogen-bonded using a flexible amine monomer (carbohydrazide, CH) to react with two aldehyde monomers, p-phthalaldehyde (pPAL) and m-phthalaldehyde (mPAL), respectively. The specific surface area of CH-pPAL and CH-mPAL was 238 and 232 m2 g-1, respectively. Based on the hydrogen-bond interaction, several narrow peaks existed in the PXRD patterns. Particularly, micron-sized spherical CH-pPAL possessed a honeycomb-like orderly tunnel structure. The contact angle of CH-pPAL and CH-mPAL severally was 35.4° and 28.8°, respectively, indicating excellent hydrophilicity of as-synthesized POPs. They both demonstrated high sensitivity and high adsorption capacity in glycopeptides enrichment. The 851 and 794 unique glycopeptides derived from 170 and 200 N-glycosylated proteins were identified after the enrichment by CH-pPAL and CH-mPAL within three independent replicates of proteins extracted from human serum, respectively. Therefore, these CH-based hydrophilic POP materials would be applied in the enrichment and identification of low-abundance N-linked glycopeptides from complicated biological samples.
Asunto(s)
Glicopéptidos , Polímeros , Glicopéptidos/química , Humanos , Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Nitrógeno , Polímeros/química , PorosidadRESUMEN
Protein glycosylation plays a vital role in many physiological activities in organisms. Due to the low abundance of glycopeptides and the interference of numerous non-glycopeptides in biological samples, selective enrichment of glycopeptides is of great significance for their successful identification. Metal organic frameworks (MOFs) materials are appropriate for glycopeptides enrichment by virtue of their large specific surface area and outstanding hydrophilic properties. However, the instability of hydrophilic MOFs in acidic solutions have severely limited their applications. In this work, a rational facile strategy was established to synthesize a stable hydrophilic hierarchical porous MOF (denoted as HP-MOF-Arg@mSiO2). This new material improved the selectivity and sensitivity of enrichment for glycopeptides via modification of arginine groups. More importantly, the mesoporous silica layer was introduced to enhance the stability of MOFs in aqueous solution and achieve the size exclusion effect of large-size proteins in complex samples. Overall, owing to the unique hierarchical porous and the hydrophilic modification, the synthesized HP-MOF-Arg@mSiO2 materials showed excellent hydrophilicity and hydrolytic stability, resulting in outstanding specific separation capacity in glycopeptides enrichment. A total of 521 and 342 glycopeptides were respectively captured from 2 µL human serum digests and mouse testis tissue digests, revealing the potential of the materials in the study of glycoproteomics in complex biological samples.
Asunto(s)
Estructuras Metalorgánicas , Nanoestructuras , Animales , Glicopéptidos , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , PorosidadRESUMEN
A novel hydrophilic porous biocomposite was fabricated by incorporating graphene oxide (GO) @chitosan (CS) foam substrate (GO@CS@ZIF-8 foam) with ZIF-8 crystals in situ via a facile stirring method for simultaneous enrichment of glycopeptides and phosphopeptides from complex biological samples. The experimental results demonstrated that GO@CS@ZIF-8 foam exhibited favorable specificity for simultaneous enrichment of N-glycopeptides and phosphopeptides under the same condition for HRP and ß-casein tryptic digest mixtures. The novel material was further applied to enriching both glycopeptides and phosphopeptides simultaneously from 4 µL complex human serum, and 423 N-glycopeptides and 40 phosphopeptides corresponding to 133 glycoproteins and 29 phosphoproteins were identified, respectively.
Asunto(s)
Quitosano , Estructuras Metalorgánicas , Quitosano/química , Glicopéptidos/química , Grafito , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Estructuras Metalorgánicas/química , Fosfopéptidos/químicaRESUMEN
Herein, a hydrophilic graphene composite functionalized with glutathione (GSH) and L(+)-Cysteine (Cys) was prepared via a simple and fast synthesis route, which was named G@S@Au@GC. The combination attack with two different zwitterionic polymers resulted in enhanced adsorption sites for glycopeptides. The obtained G@S@Au@GC exhibited excellent performance on a low limit of detection (0.2 fmol), a high selectivity (HRP: bovine serum albumin = 1:1500), a good load capacity (250 µgâ¢mg-1) and recovery rate (93%), which was also evaluated with IgG. Subsequently, 60 glycopeptides from complex biological sample (human saliva) were identified by Nano-LC-MS/MS. The advantages of combination attack, low-cost, simple and fast synthesis, and superior enrichment performance make G@S@Au@GC composite a bright future on glycoproteomics analysis and related diseases.
Asunto(s)
Glicopéptidos , Grafito , Aminoácidos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Espectrometría de Masas en TándemRESUMEN
The pretreatment of complicated biological samples to eliminate the interference of nonglycopeptides and improve the efficiency of glycopeptides detection is crucial in glycoproteomics research. Hydrophilic interaction chromatography (HILIC) has been adopted for enrichment of glycosylated peptides following identification with mass spectrometry, but it is still urgent to develop novel hydrophilic materials to save cost and improve enrichment efficiency. Scientists are pursuing to fabricate freestanding intelligent artificial materials. One promising approach is to use biomimic material. In our case, "one-pot" strategy was developed to prepare bioinspired nano-core-shell silica microspheres (CSSMs), employing tetrapropylorthosilicate as the silicon source and phenolic resin as the soft template. The pore structure of the obtained microspheres diverged from the center to the outside with diameter ranged from 150 to 340 nm, and shell layer ranged from 25 to 83 nm by adjusting the preparation parameters. Some of them showed dandelion-like morphology. After hydrophilic modification, these CSSMs exhibited great hydrophilicity and could be used as sorbents for enriching N-glycopeptides from complicated biological samples in HILIC. Up to 594 unique N-glycopeptides and 367 N-glycosylation sites from 182 N-glycoproteins were unambiguously identified from 2 µL of human serum, which was superior to the enrichment performance of many HILIC materials in reported papers, demonstrating great potential advantages in proteomic application.
Asunto(s)
Glicopéptidos , Nanopartículas , Glutatión , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Proteómica , Dióxido de SilicioRESUMEN
A polyacrylamide-based hydrophilic microsphere with a lot of hydroxyl groups on surface (PAM-OH HMS) was prepared in one step. The synthetic process was simple reverse suspension polymerization without any chemical derivation or grafting steps. The properties of obtained HMS were characterized by scanning electron microscopy (SEM), static water contact angle measurement, and FT-IR. The abundant hydroxyl groups on the surface make the material highly good hydrophilic and thus it was utilized for N-glycopeptides enrichment. The enrichment efficiency of PAM-OH HMSs was demonstrated by capturing N-linked glycopeptides from tryptic digest of human immunoglobulin G (IgG). The detection sensitivity for N-glycopeptides identification by MALDI-TOF MS was as low as 10 fmol for tryptic digest of standard human IgG. The selectivity of the HMS towards N-glycopeptides had almost no decrease when the molar ratio of BSA tryptic digest to IgG tryptic digest was increased from 10:1 to 100:1. Moreover, in the LC-MS/MS analysis of real biological sample, a total of 344 unique N-glycosites in 598 unique N-glycopeptides from 172 N-glycoproteins were identified from 2 µL human serum after deglycosylated by PNGase F, and 825 intact N-glycopeptides with different types of glycoform were detected when directly analyzed the N-glycopeptides enriched by PAM-OH HMS. To show the potential of our material in solving real biological issues, N-glycopeptides in the serum from hepatocelluar carcinoma (HCC) patient and health control were enriched and quantified. All the experiments demonstrated that this polyacrylamide-based hydrophilic microsphere shows a great potential to enrich the low-abundance N-glycopeptides for glycoproteome analysis of real complicated biological samples.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Cromatografía Liquida , Glicopéptidos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Microesferas , Espectroscopía Infrarroja por Transformada de Fourier , Espectrometría de Masas en TándemRESUMEN
Enrichment of glycoproteins has been important because of their dynamicity and role in biological systems. Study of glycoproteins is complex because of the simultaneous glycosylation and deglycosylation inside the body. Often employed affinities for glycopeptides are hydrazide, boronic acid, or physiosorbed lectin on support materials. Cellulose, a natural polysaccharide, has rich surface chemistry, stable structure, low cost and availability in different variants. In present study, fibrous cellulose is oxidized using periodate to modify with boronic acid. Attachment of boronic acid is confirmed by Fourier transform infrared spectroscopy. Particle size and morphology of boronic acid@fibrous cellulose is studied by scanning electron microscopy. The enrichment efficiency is evaluated by using horseradish peroxidase as model protein. Boronic acid@fibrous cellulose is selective up to 1:250 for spiked horseradish peroxidase in bovine serum albumin digest, sensitive down to 0.1 femtomol and recovering 88.15% glycopeptides. Moreover, protein binding capacity is determined as 213 mg/g and 41% sequence coverage of horseradish peroxidase protein with all eight glycosylation sites detected. Total of 18 glycopeptides are enriched from immunoglobulin digest showing ability of boronic acid@fibrous cellulose to enrich glycoproteins from multiglycoforms. Enrichment from human serum recovers 18% extracellular and 72% secreted glycoproteins via bottom-up approach and online tools.
Asunto(s)
Ácidos Borónicos/metabolismo , Celulosa/metabolismo , Glicopéptidos/metabolismo , Adsorción , Animales , Ácidos Borónicos/sangre , Ácidos Borónicos/química , Bovinos , Celulosa/sangre , Celulosa/química , Glicopéptidos/sangre , Glicopéptidos/química , Peroxidasa de Rábano Silvestre/metabolismo , Humanos , Inmunoglobulinas/metabolismo , Albúmina Sérica Bovina/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Magnetic nanoparticles (MNPs) have been widely explored in enrichment of low-abundance glycoproteins/glycopeptides prior to mass spectrometry analysis in glycoproteomics. Currently, most functional groups for recognizing glycoproteins/glycopeptides are usually immobilized on the nanomaterial surface based on covalent modification, which suffers from multistep treatment, surface-dependence, and harsh conditions. In this work, we first report a facile and rapid method for surface functionalization and subsequent glycopeptides enrichment via one-step assembly of maltose-modified oligopeptides with a sequence of Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys (AEK8-maltose). In physiological conditions, AEK8-maltose readily self-organized into a complete coating layer dominated by ß-sheets on the surface of SiO2@Fe3O4 and C@Fe3O4 MNPs, which remain intact to repeat washing with acidic organic and aqueous solutions extensively used in the sample enrichment treatments. Thus, the resulting AEK8-maltose functionalized MNPs show excellent performance in enrichment of glycopeptides in standard glycoprotein digests (24 glycopeptides from horseradish peroxidase (HRP), 31 glycopeptides from immunoglobulin (IgG)) and human serum digests (282 glycopeptides), including rapid enrichment speed (5â¯min), high detection sensitivity (0.001â¯ng/µL HRP), high selectivity (mass ratios of HRP and bovine serum albumin (BSA) digests up to 1:150), good enrichment recovery (over 86.3%), remarkable stability (repeatable for more than 8 times), and excellent renewability, which are better than or comparable with the literature results reported to date. The current work based on self-assembling oligopeptides provides a mild, economic and nontoxic procedure for one-step surface functionalization of various nanomaterials.
Asunto(s)
Glicopéptidos/química , Nanopartículas de Magnetita/química , Maltosa/química , Oligopéptidos/química , Animales , Dióxido de Silicio/química , Especificidad por SustratoRESUMEN
A hydrophilic interaction liquid chromatography (HILIC) material with application in glycoproteomics was obtained by sequential deposition of polyethyleneimine (PEI) and hyaluronic acid (HA) on a negatively charged substrate by means of electrostatic self-assembly. This kind of surface modification endows the material with excellent hydrophilicity and warrants efficient glycopeptides enrichment. The feasibility of this enrichment was verified by using dendritic mesoporous silica nanoparticles (DMSNs) and magnetic graphene oxide (MagG) as negatively charged substrates for PEI and HA adhesion. The two final products (DMSNs@PEI@HA and MagG@PEI@HA) exhibit high enrichment selectivity (molar ratios of IgG and BSA digests = 1:500 and 1:1000), sensitivity (detection limit, 2 fmol/µL), recovery (>90%) and enrichment capacity (300 mg/g). When using DMSNs@PEI@HA, 419 N-glycopeptides derived from 105 glycoproteins were identified. When using MagG@PEI@HA, 376 N-glycopeptides derived from 102 glycoproteins were identified, both from a 2 µL serum sample. This is better than by methods described in previous reports. Graphical abstract Schematic representation of hydrophilic modification of negatively charged nanomaterial substrates by electrostatic self-assembly techniques to obtain hydrophilic interaction liquid chromatography (HILIC) materials for enrichment of N-glycopeptides.
Asunto(s)
Glicopéptidos/análisis , Ácido Hialurónico/química , Polietileneimina/química , Electricidad Estática , Cromatografía Liquida , Grafito/química , Interacciones Hidrofóbicas e Hidrofílicas , Estructura Molecular , Nanopartículas/química , Tamaño de la Partícula , Porosidad , Dióxido de Silicio/química , Propiedades de SuperficieRESUMEN
A mercaptosuccinic acid functionalized hydrophilic magnetic metal-organic framework nanocomposite (denoted as mMOF@Au-MSA) was proposed and synthesized to provide an excellent platform for glycopeptide analysis. The novel nanomaterial integrated favorable advantages such as robust magnetic response from Fe3O4 magnetic nanoparticles, large surface area contributed by MOF, abundant ultra-high hydrophilic carboxylic groups from mercaptosuccinic acid, as well as unbiased affinity toward different types of glycopeptides. This nanocomposite was successfully utilized to capture glycopeptides from standard protein digests with the high selectivity and great sensitivity of 0.5â¯fmol⯵L-1. Notably, 307 glycopeptides assigned to 96 glycoproteins were identified from only 2⯵L serum of breast cancer patient. The satisfying achievement indicated that the as-prepared nanopartical had promising potential in exploring the knowledge of glycoproteins in breast cancer.
Asunto(s)
Glicopéptidos/sangre , Estructuras Metalorgánicas/química , Secuencia de Aminoácidos , Neoplasias de la Mama/sangre , Secuencia de Carbohidratos , Oro/química , Humanos , Nanopartículas de Magnetita/química , Nanocompuestos/química , Proteómica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Tiomalatos/químicaRESUMEN
Owing to their multiscale pore size regimes and unique properties, the materials with hierarchically porous structures have become an important family of functional materials in recent years. They have been applied from energy conversion and storage, catalysis, separation to drug delivery, etc. The synthesis of them is difficult by the need to employ multiple templates and take complicated steps. Herein, we successfully prepared epoxy-functionalized hierarchically porous hybrid monoliths (HPHMs) with micro/meso/macro-structures in an easy way. Firstly, a bulk monolithic material was formed via free radical polymerization between polyhedral oligomeric vinylsilsesquioxanes (vinylPOSS) and allyl glycidyl ether (AGE) in the presence of polycaprolactone (PCL). Then PCL was degraded with hydrochloric acid solution, and the epoxy-functionalized HPHM was obtained. This approach was very simple and suitable for large-scale preparation. Hybrid monoliths with different specific surface area (from 5.4 to 636.7â¯m2/g) were prepared by adjusting the mole ratio of vinylPOSS to AGE and the content of PCL. The results of several characterization methods, including nitrogen adsorption/desorption measurements, scanning electron microscopy (SEM) and mercury intrusion porosimetry (MIP), showed that these materials contained not only micropores and mesopores but also macropores. The materials were further modified with penicillamine to be used as hydrophilic interaction chromatography (HILIC) adsorbents for enriching N-glycopeptides in IgG and serum protein tryptic digests. Up to 23â¯N-glycopeptides were identified from IgG digest, and 385â¯N-glycopeptides and 283â¯N-glycosylation sites were identified from human serum digest.
Asunto(s)
Cromatografía Liquida/métodos , Glicopéptidos/sangre , Compuestos de Organosilicio/química , Polivinilos/química , Adsorción , Animales , Bovinos , Compuestos Epoxi/química , Glicopéptidos/química , Glicosilación , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Inmunoglobulina G/química , Compuestos de Organosilicio/síntesis química , Penicilamina/química , Poliésteres/síntesis química , Poliésteres/química , Polimerizacion , Polivinilos/síntesis química , Porosidad , Proteolisis , Albúmina Sérica Bovina/química , Tripsina/químicaRESUMEN
In this work, magnetic mesoporous silica microspheres (Mag-MSMs) with ordered radial mesochannels were fabricated by a self-assembly synthesis in chlorobenzene-water mixed system. Then, the obtained Mag-MSMs were modified with polyethyleneimine (PEI), phytic acid (PA) and Ti4+ (denoted as Mag-MSMs@PEI-PA-Ti4+) via layer by layer (LbL) assembly. Due to the excellent hydrophilicity of PEI and PA and the large amount of Ti4+, the Mag-MSMs@PEI-PA-Ti4+ possessed combined properties of hydrophilic interaction liquid chromatography (HILIC)- and immobilized metal ion affinity chromatography (IMAC)-based materials, which could be used as a dual-purpose material for N-glycopeptides or phosphopeptides enrichment. The proposed Mag-MSMs@PEI-PA-Ti4+ exhibited an outstanding performance for N-glycopeptides enrichment (selectivity: IgG/BSAâ¯=â¯1:1000; sensitivity: 0.5 fmol/µL IgG) and phosphopeptides enrichment (selectivity: α-casein/BSA=1:5000; sensitivity: 0.2 fmol/µL α-casein). Furthermore, after enrichment with Mag-MSMs@PEI-PA-Ti4+, a total of 276 N-glycopeptides assigned to 132 glycoproteins were identified from 2⯵L human serum and 1645 phosphopeptides corresponding to 704 phosphoproteins were identified from 200⯵g HeLa cell extracts.
Asunto(s)
Glicopéptidos/análisis , Nanosferas/química , Fosfopéptidos/análisis , Ácido Fítico/química , Dióxido de Silicio/química , Titanio/química , Interacciones Hidrofóbicas e Hidrofílicas , Fenómenos Magnéticos , Estructura Molecular , Tamaño de la Partícula , Porosidad , Propiedades de SuperficieRESUMEN
Hydrophilic interaction chromatography (HILIC) utilizing zwitterion-modified material as solid phase has attracted extensive attention for selective enrichment of glycopeptides. However, a tedious synthesis and low specificity for glycopeptides have restricted its application. Herein, a facile and effective approach was developed to synthesize a zwitterionic (ZIC) polymer-coated magnetic composites (denoted as Fe3O4@PMSA) with a zwitterion ((2-(methacryloyloxy)ethyl)-dimethyl-(3-sulfopropyl) ammonium hydroxide, MSA) via one-step distillation-precipitation polymerization (DPP). The well-designed composites presented clearly ZIC-polymer shell and superior hydrophilicity (water contact angle 30.2), and the performance for selective enrichment of glycopeptides were investigated with standard and real samples, respectively. Owning to the abundant of ZIC molecules with multi-charge and polar groups on the surface of resulting polymer coating, the Fe3O4@PMSA demonstrated high selectivity for glycopeptides enrichment with IgG digest (twenty glycopeptides identified, S/Nâ¯≥â¯20) and a mixture of IgG and BSA at the mass ratio of 1:230 (sixteen glycopeptides identified, S/Nâ¯≥â¯20). Besides, the detection limit as low as 0.67â¯fmol for IgG (S/Nâ¯≥â¯10) and satisfied recovery yield more than 74% were achieved by the proposed sorbents. Finally, the Fe3O4@PMSA was applied for enriching N-linked glycopeptides from human serum, and 348 unique N-glycosylation sites and 419 glycopeptides from 158 glycoproteins were strictly identified from 1⯵L human serum. The results demonstrated that the proposed Fe3O4@PMSA showed a promising potential in glycoproteomics analysis of real biological samples.
Asunto(s)
Glicopéptidos/química , Glicopéptidos/aislamiento & purificación , Polimerizacion , Precipitación Química , Óxido Ferrosoférrico/química , Glicopéptidos/metabolismo , Proteolisis , Tripsina/metabolismoRESUMEN
Variations in salivary components are closely associated with the predisposition and state of disease, the abnormal changes of salivary glycopeptidome are usually discovered as perilous singals of serious disease. Therefore, the monitoring and analyzing of salivary glycopeptidome are of even more overriding importance. In this work, a low-cost layer-by-layer assembly strategy was adopted to fabricate a hydrophilic multilayer magnetic probe (dubbed Mag-m-G6P) for salivary glycopeptidome analysis. The successful construction of multilayer structure not only guaranteed the good dispersal of probe by protecting magnetic core from itself aggregation tendency, but also endowed the probe with multiple advantages including the good hydrophilicity, uniform mesopore size and strong magnetic responsiveness, etc. As expected, with the optimized experimental conditions, the multifunctional probe showed high enrichemnt sensitivity, unbiased enrichment ability, excellent size-exclusion ability and reusability and so on in the process of standard sample analysis. At last, the Mag-m-G6P was successfully applied to salivary glycopeptidome analysis on further combination with LC-MS/MS analysis, a total of 53 endogenous glycopeptides were identified from human saliva.
Asunto(s)
Biología Computacional/métodos , Glicopéptidos/análisis , Magnetismo , Saliva/química , Cromatografía Liquida , Glicopéptidos/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Espectrometría de Masas en TándemRESUMEN
As one of the most important post-translational modifications (PTMs) of peptidome, glycopeptidome is closely related to serious disease, especially to cancer. In order to specifically discover and analyze glycopeptidome biomarkers for clinical diagnosis of cancer on early-stage, it is crucial to develop efficient technique to analyze low-abundance of endogenous glycopeptides in biological samples. In this report, a hydrophilic probe in mesoporous pore (denoted as Fe3O4@mSiO2@G6P) was designed and prepared. By taking advantage of the excellent hydrophilicity and size-exclusion ability, we applied Fe3O4@mSiO2@G6P to capture glycopeptides from both horseradish peroxidase (HRP) and immunoglobulin (IgG) digests successfully. Moreover, a total of 39 and 25 endogenous glycopeptides were identified from healthy saliva and gastric saliva, respectively, indicating the great potential of this probe for the exploration of glycopeptidome biomarkers.
Asunto(s)
Glicopéptidos/análisis , Interacciones Hidrofóbicas e Hidrofílicas , Sondas Moleculares/química , Proteoma/análisis , Biomarcadores/análisis , Biomarcadores/química , Compuestos Férricos/química , Glucosa-6-Fosfato/química , Glicopéptidos/química , Peroxidasa de Rábano Silvestre/química , Humanos , Inmunoglobulina G/química , Nanopartículas de Magnetita/química , Sondas Moleculares/síntesis química , Porosidad , Proteoma/química , Saliva/química , Dióxido de Silicio/químicaRESUMEN
A macroporous hydrophilic organic-silica hybrid monolithic column was synthesized via photoinitiated thiol-ene click polymerization reaction of 1-thioglycerol-modified polyhedral oligomeric vinylsilsesquioxane (vinylPOSS) and dithiothreitol (DTT) in a binary porogenic system consisting of tetrahydrofuran (THF) and dodecanol. 1-Thioglycerol was used to modify vinylPOSS in order to form a precursor with good solubility in the binary porogenic system. The influences of both the ratio of 1-thioglycerol/vinylPOSS and the porogenic solvents on the morphology and permeability of hybrid monoliths were studied in detail. The physical properties of hybrid monolith were characterized by scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy and nitrogen adsorption/desorption measurement. The chromatographic performance was evaluated by separation of neutral polar compounds in capillary liquid chromatography (cLC). The resulting column possessed homogeneous macroporous structure and showed hydrophilic interaction liquid chromatography (HILIC) separation mechanism with high efficiency of 65,000Nm-1 for formamide. Ultimately, the hybrid matrix was grafted with hydrazine groups and then exhibited the ability of glycopeptides enrichment.