Fmol μL-1 Research Articles

One-Pot Preparation of Hydrophilic Glucose Functionalized Quantum Dots for Diabetic Serum Glycopeptidome Analysis

This paper reports a hydrophilic carbohydrate functionalized quantum dots material (denoted as MnZnS@G6P) synthesized through one-pot strategy and used as hydrophilic materials for the specific enrichment of glycopeptides. MnZnS@G6P had the advantages of worthy stability and low cost, and Mn-doped ZnS quantum dots offer abundant modification sites. To improve the hydrophilicity of the material, glucose-6-phosphate was grafted onto the material through the interaction between phosphate groups and metal ions. Encouraged by the excellent sensitivity (0.05 fmol μL−1) and selectivity (the molar ratio of HRP and BSA is 1:1000), MnZnS@G6P was applied to the capture of glycopeptides in actual biological samples. The outcomes of nano LC-MS/MS indicated that MnZnS@G6P could efficiently capture glycopeptides in the serum. In short, from serum tryptic digests of a healthy human and a diabetic patient, 139 N-glycopeptides corresponding to 76 glycoproteins and 100 N-glycopeptides corresponding to 54 glycoproteins were enriched and identified, respectively.

Nanoporous Substrates with Molecular-Level Perfluoroalkyl/Alkylamide Surface for Laser Desorption/Ionization Mass Spectrometry of Small Proteins.

The rapid detection of biomolecules greatly contributes to health management, clinical diagnosis, and prevention of diseases. Mass spectrometry (MS) is effective for detecting and analyzing various molecules at high throughput. However, there are problems with the MS analysis of biological samples, including complicated separation operations and essential pretreatments. In this study, a nanostructured organosilica substrate for laser desorption/ionization mass spectrometry (LDI-MS) is designed and synthesized to detect peptides and small proteins efficiently and rapidly. The surface functionality of the substrate is tuned by perfluoroalkyl/alkylamide groups mixed at a molecular level. This contributes to both lowering the surface free energy and introducing weak anchoring sites for peptides and proteins. Analyte molecules applied onto the substrate are homogeneously distributed and readily desorbed by the laser irradiation. The organosilica substrate enables the efficient LDI of various compounds, including peptides, small proteins, phospholipids, and drugs. An amyloid β protein fragment, which is known as a biomarker for Alzheimer's disease, is detectable at 0.05 fmol μL-1. The detection of the amyloid β at 0.2 fmol μL-1 is also confirmed in the presence of blood components. Nanostructured organosilica substrates incorporating a molecular-level surface design have the potential to enable easy detection of a wide range of biomolecules.

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

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

Hydrophilic arginine-functionalized mesoporous polydopamine-graphene oxide composites for glycopeptides analysis

Considering the importance of glycopeptides in the clinical diagnosis of cancer and some serious diseases, the identification of glycopeptides from complex biological samples has attracted considerable attention. Effective pre-enrichment before mass spectrometry analysis plays an important role. In this work, a kind of hydrophilic two-dimensional composites (denoted as GO@MPDA@Arg) based on mesoporous polydopamine-graphene oxide were used to selectively enrich glycopeptides in biological samples. The mesoporous polydopamine (MPDA) layer self-assembled with template Pluronic F127 provided more binding sites to load arginine, and bound arginine enhanced the hydrophilicity of the material. As a result, GO@MPDA@Arg composites exhibited excellent enrichment performance for glycopeptides, containing good selectivity (IgG digests : BSA digests = 1:50, molar ratio), low detection limit for IgG digests (10 fmol μL−1), high loading capacity for IgG digests (200 μg mg−1), and good size exclusion (IgG digests : IgG : BSA = 1:100:100, mass ratio). In addition, mouse brain tissue was selected as the actual biological sample to further study the enrichment effect of GO@MPDA@Arg composites. In three parallel experiments, a total of 401 glycopeptides belonging to 233 glycoproteins were enriched from 200 μg digestion of mouse brain extract. The enrichment results demonstrate that GO@MPDA@Arg composites have application potential for glycopeptides enrichment in protein post-translational modification research.

In Situ Controllable Fabrication of Two-Dimensional Magnetic Fe3O4/TiO2@Ti3C2Tx Composites for Highly Efficient Phosphopeptides Enrichment.

Highly efficient enrichment of phosphopeptides is of great significance for phosphoproteomics-related biological and pathological processes research, but it remains challenging due to the lack of affinity materials which hold high enrichment efficiency and capacity. Ti3C2Tx MXene, a novel two-dimensional material with outstanding physicochemical properties, has attracted wide research interests for application in various fields. However, there are few reports on the use of MXene-derived materials for phosphopeptides separation in the biomedical field. In this work, we proposed a facile one-pot method that in situ oxidation and modification of Ti3C2Tx MXene, to prepare two-dimensional (2D) magnetic Fe3O4/TiO2@Ti3C2Tx composites for potential application in phosphopeptides enrichment. Benefiting from the outstanding magnetic responsiveness and multiaffinity sites (Ti-O, Fe-O, and NH2 groups), the Fe3O4/TiO2@Ti3C2Tx composites possessed excellent enrichment performance with high sensitivity (0.1 fmol μL-1), excellent selectivity (β-casein: bovine serum albumin = 1:5000, molar ratio), good repeatability (5 times), and high enrichment capacity (200 mg g-1). Moreover, the results of selective enrichment of phosphopeptides from nonfat milk, human saliva, human serum, and rat brain lysates indicated the great potential of Fe3O4/TiO2@Ti3C2Tx composites in low-abundance phosphopeptides enrichment from complex biological samples. This work has put forward a versatile method to prepare magnetic MXene composites and promoted the use of MXene composites in phosphoproteome in biomedicine.

Facile synthesis of Ti4+-immobilized magnetic covalent organic frameworks for enhanced phosphopeptide enrichment

In this work, core-shell structured Ti4+-immobilized magnetic covalent organic frameworks (denoted as Fe3O4@TAPTDHTA-Ti4+ composites) were prepared for enhanced phosphopeptide enrichment by one-pot synthesis of COFs shell with inherent bifunctional groups on Fe3O4 NPs and further Ti4+ immobilization. The widely distributed bifunctional groups could provide abundant chelating sites for Ti4+ immobilizing. Combining with the high specific surface area and mesoporous structure, the Fe3O4@TAPTDHTA-Ti4+ composites exhibited excellent enrichment efficiency for phosphopeptides, such as low detection limit (0.05 fmol μL−1), high selectivity (1:5000 of molar ratio of β-casein/bovine serum albumin (BSA) tryptic digests), high adsorption capacity (62.9 μg mg−1) and strong size-exclusive effect (1:250:250 of molar ratio of β-casein tryptic digest/β-casein/BSA). In addition, this method was general for immobilizing other metal ions (Zr4+ and Fe3+). Notably, the Fe3O4@TAPTDHTA-Fe3+ composites exhibited controllable affinity towards mono-phosphopeptides and multi-phosphopeptides. Furthermore, the Fe3O4@TAPTDHTA-Ti4+ composites were successfully applied to selectively capture phosphopeptides from complex biological samples including the tryptic digest of nonfat milk, human serum and human saliva. More significantly, 3333 phosphopeptides derived from 1409 phosphoproteins with 3492 phosphorylation sites were clearly identified from the tryptic digest of HeLa cell lysate. In addition to providing a potential excellent enrichment probe for comprehensive phosphoproteomic analysis, this study also offers a new perspective for the functionalization of COFs.

Flexible and hierarchical metal-organic framework composite as solid-phase media for facile affinity-tip fabrication to selectively enrich glycopeptides and phosphopeptides

Micro-tip-based solid-phase microextraction is considered as one of the green and powerful analytical sample preparation techniques, but its efficiency is severely hampered by some basic issues such as tedious fabrication, instability of sorbent bed, and blocking of the tip, especially for biological samples due to low permeability. These issues are tackled by introducing a flexible and hierarchical substrate in the microtip, having good mechanical strength and specific functionality to capture the desired biomolecules. Considering the well-ordered and flexible structure of melamine foam, it was used as a substrate and for hydrophilic interaction chromatography (HILIC). Metal-organic framework, due to its excellent characteristics, was grafted on its surface anchored by self-assembling polydopamine. The resulting material was characterized and packed in the tip by just pressing the material in the conical structure of the tip. This affinity tip established good and tunable permeability and was used to selectively enrich glycopeptides as well as phosphopeptides. The affinity tip demonstrated excellent performance to enrich glycopeptides and phosphopeptides with a low limit of detection up to 0.5 fmol μL−1 from tryptic digests of horseradish peroxidase and β-Casein, respectively, and was stable up to 5 rounds of enrichment. Moreover, this affinity-tip also exhibited high selectivity up to up to 1:1000 (HRP digest to BSA digest) for glycopeptides and 1:200 (β-Casein digest to BSA digest) for phosphopeptides and demonstrated several other fascinating characteristics such as; excellent size exclusion effect for the omission of large-sized proteins, modest backpressure, reproducibility, reusability, smooth enrichment, and successfully applied to a human saliva sample.

Two-dimensional guanidinium-based covalent organic nanosheets for controllable recognition and specific enrichment of global/multi-phosphopeptides

Highly specific capture of phosphopeptides, especially multi-phosphopeptides, from complex biological samples is critical for comprehensive phosphoproteomic analysis, but it still poses great challenges due to the lack of affinity material with ideal enrichment efficiency. Here, two-dimensional (2D) covalent organic framework (COFs) nanosheets was applied for selective separation of phosphopeptides for the first time. Particularly, by incorporating guanidinium units, the 2D guanidinium-based COF nanosheets (denoted as TpTGCl CONs) exhibited controllable and specific enrichment performance towards global/multi-phosphopeptides. TpTGCl CONs was easy to prepare and showed large surface area, low steric hindrance, abundant accessible interaction sites and high chemical stability. Taking these merits together, TpTGCl CONs exhibited excellent efficiency for phosphopeptide enrichment, such as low detection limits (0.05 fmol μL−1 for global phosphopeptides and 0.1 fmol μL−1 for multi-phosphopeptides), high selectivity (1:5000 of molar ratios of β-casein/BSA for both global and multi-phosphopeptides), high adsorption capacity (100 mg g−1 for global phosphopeptides and 50 mg g−1 for multi-phosphopeptides). Furthermore, TpTGCl CONs could be reused due to the high chemical stability. In addition, TpTGCl CONs were successfully applied to controllable and specific capture of endogenous global/multi-phosphopeptides from human serum and human saliva, indicating its good potential in rapid and sensitive detection of biomarkers from biological fluid. Finally, rat liver protein digest was used to confirm the high specificity of TpTGCl CONs towards multi-phosphopeptides and demonstrated its potential as an ideal enrichment probe for comprehensive phosphoproteomic analysis.

Bowl-like mesoporous polydopamine with size exclusion for highly selective recognition of endogenous glycopeptides

It has been confirmed that endogenous glycopeptide plays an important role in a variety of pathological and physiological processes. However, direct analysis of endogenous glycopeptide is still a great challenge owing to the low abundance of endogenous glycopeptides and the presence of a large number of interfering substances such as large-sized proteins and heteropeptides in complex biological sample. Herein, we reported a novel bowl-like mesoporous polydopamine nanoparticle modified by carrageenan (denoted as MPDA@PEI@CA) with strong hydrophilicity and size-exclusion effect for high specificity enrichment of endogenous glycopeptides. Thanks to the suitable pore channel structure as well as strong hydrophilic surface, the as-prepared MPDA@PEI@CA nanoparticles exhibited prominent performance in enrichment of N-linked glycopeptide with ultrahigh selectivity (1:5000 M ratio of horseradish peroxidase (HRP) digests/bovine serum albumin (BSA) digests), low detection limit (5 fmol μL−1), outstanding size-exclusion ability (1:1000 mass of HRP/BSA), and unique reusability (five times). 125 N-glycosylation sites of 134 glycopeptides from 65 glycoproteins were identified from 2 μL sample of human serum treated with the MPDA@PEI@CA nanoparticles, which manifested the ability to enrich endogenous N-linked glycopeptides from complex biological samples. These results indicated that the bowl-like MPDA@PEI@CA nanoparticles with novel structure prepared in this work had great potential for glycopeptidome analysis.

Dual-functionalized magnetic bimetallic metal-organic framework composite for highly specific enrichments of phosphopeptides and glycopeptides

In this work, a dual-functionalized magnetic bimetallic metal-organic framework composite denoted as Fe3O4@SiO2@(Zr–Ti-MOF)10-NH2 was ingeniously designed and fabricated by a facial layer-by-layer assembly technique. The composite not only exhibited strong affinity for phosphopeptide due to the coexistence of Zr–O clusters and Ti–O clusters, but also owned great hydrophilicity for glycopeptides relying on abundant hydrophilic NH2 groups, meeting the demand for simultaneously enrichment and sequential elution of phosphopeptides and glycopeptides. As expected, the synthesized composite showed great selectivity (1:2000 M ratio of β-casein:BSA; 1:50 M ratio of IgG:BSA), good sensitivity (1 fmol μL−1 for both α-casein and IgG), and good capacity (80 mg g−1 for α-casein and 200 mg g−1 for IgG). By using sequential elution strategy, 29 phosphopeptides and 24 glycopeptides from α-casein and IgG digests mixture could be simultaneously enriched and respectively detected through a single-step enrichment and sequential elution method. Furthermore, the composite was successfully applied to the analysis of intricate biological samples. 4 endogenous phosphopeptides and 20 phosphopeptides were trapped from human serum and non-fat milk tryptic digest respectively. From 0.5 mg of tryptic digest of rat brain, 141 N-linked glycopeptides corresponding to 127 glycoproteins and 918 phosphopeptides corresponding to 397 phosphoproteins were enriched simultaneously and identified respectively, proving the Fe3O4@SiO2@(Zr–Ti-MOF)10-NH2 to be a dependable candidate for the simultaneously enrichment of trace phosphopeptides and glycopeptides in intricate biological samples.

Hollow MnFe2O4@C@APBA Nanospheres with Size Exclusion and pH Response for Efficient Enrichment of Endogenous Glycopeptides

Enrichment and detection of glycopeptides are an important clinical measure for the diagnosis of complex diseases. Enrichment materials play a key role in this process; they must have an effective sample-screening ability to eliminate the interference of nonglycopeptides. In this work, novel hollow MnFe2O4@C@APBA nanospheres (HMCAs) with magnetic and pH responsiveness were prepared for glycopeptide enrichment. The as-prepared composites have a suitable hollow structure and large specific surface area, and the boron hydroxyl group in their cavities can fix or disconnect the hydrophilic groups of the glycopeptides at different pH, so the glycopeptides can be adsorbed or desorbed in a controllable way. Enrichment results showed that the HMCAs exhibited an excellent enrichment performance: ultralow limit of detection (approximately 0.5 fmol μL-1), perfect size-exclusion effect (HRP/BSA, 1:800, w/w), favorable universality (HRP, IgG, and RNase B), and high binding capacity (150 mg/g). In order to verify the application of materials in practice, the HMCAs were used for the analysis of complex samples and it was found that 474 glycopeptides were identified from 210 glycoproteins in three replicate analyses of 2 μL of human serum. The results showed that the HMCAs could be used as a promising enrichment material for glycopeptide characterization in MS-based glycoproteomics and related fields.

Hydrophilic polydopamine-derived mesoporous channels for loading Ti(IV) ions for salivary phosphoproteome research

Salivary phosphoproteome holds great promise in clinic diagnosis. For profiling of salivary phosphoproteome, it is essential to develop efficient enrichment methods prior to mass spectrum (MS). Among developed enrichment strategies, immobilized metal ions affinity chromatography (IMAC) has exhibited outstanding performance. In this work, we report a coherent approach where polydopamine (PDA) is first utilized to form mesoporous structure through soft templating method, then chelated with Ti4+ to construct hydrophilic polydopamine-derived magnetic mesoporous nanocomposite (denoted Fe3O4@mPDA@Ti4+). In virtue of the merits including ordered mesoporous channels, appropriate superparamagnetism, and abundant Ti4+, the enrichment strategy based on Fe3O4@mPDA@Ti4+ combined with MS is employed for accurate identification of phosphopeptides in β-casein digest and human saliva. As expected, Fe3O4@mPDA@Ti4+ revealed a great selectivity (1:200) and a low detection limit (0.1 fmol μL−1) toward phosphopeptides. More importantly, the further successful capture of phosphopeptides from human saliva indicated the prominent potential of this method for seeking phosphopeptide biomarkers in further analysis.

C18-functionalized magnetic nanocomposites fabricated by one-step aqueous coating of tailored oligopeptides for enrichment of low-abundance peptides

Screening and monitoring endogenous peptides from complicated biosamples is still a major challenge in mass spectrometry-based proteomics research, mainly due to their low concentration and the interference of high-abundance proteins and other contaminants in biological samples. Herein, a facile and novel approach was described for rapid fabrication of C18-functionalized magnetic nanocomposites (C18-MNCs) based on one-step aqueous coating of C18-Val-Lys-Val-Lys-Val-Lys (C18-VK-VI) for the highly selective enrichment of low-abundance endogenous peptides from biological samples. C18-VK-VI can readily self-assemble into complete monolayers mainly composed of β-sheets with C18 hydrophobic chains erecting on the surface of GO@Fe3O4 MNCs under the physiological conditions. The resulting C18VK-VI-GO@Fe3O4 MNCs exhibited good performance for peptides enrichment from digests of standard protein (myoglobin, MYO) and human serum, such as high sensitivity (0.05 fmol μL−1) and selectivity (mass ratio of MYO digests and MYO = 1:500), rapid separation, and good reproducibility. Such a simple mild and rapid one-step aqueous coating method on the basis of oligopeptides self-assembly showed great potential in surface functionalization of various nanoadsorbents for proteome/peptidome researches.

Glycosylation heterogeneity and low abundant serum glycoproteins MS analysis by boronic acid immobilized Fe3O4@1,2-Epoxy-5-Hexene/DVB magnetic core shell nanoparticles

Glycosylation in biological systems has been studied by enriching glyco- moieties followed by their mass spectrometric (MS) analyses. Complexity of glycoproteins heterogeneity is addressed in this study. Mono- and multi-glycosylated peptides after proteolysis from complex biological samples are enriched by newly synthesized boronic acid functionalized magnetic 1,2-epoxy-5-hexene/DVB core shell nanoparticles under optimized pH conditions. The composition, surface area, structure and morphology are found out by X-ray diffraction, scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy and nitrogen adsorption porosimetry. Glycopeptides enrichment efficiency is assessed by using tryptic digests of Horseradish Protease with 8 and Immunoglobulin G with single glycosylation site, identifying 17 and 26 glycopeptides respectively by MALDI-mass spectrometry representing complete glycosylation sites coverage. The material shows low sensitivity (0.1 fmol µL−1), high selectivity (1:250) using bovine serum albumin digest, high adsorption capacity (332 mg g−1 of horseradish peroxidase) and reusability (up to three times), providing cost-effective in-house solution to study complex proteolytic samples. The application of magnetic polymeric core shell NPs to digested human serum helps identifying 347 glycopeptides by nano-LC-MS/MS, which also include less abundant glycoproteins like ceruloplasmin, complement C4 and acid alpha-1-glycoprotein. The ease of sample preparation because of magnetic nature, high selectivity and sensitivity promise a tool to study glycoproteins of clinical value.

Construction of Magnetic Covalent Organic Frameworks with Inherent Hydrophilicity for Efficiently Enriching Endogenous Glycopeptides in Human Saliva.

In this work, a magnetic covalent organic framework (COF) with inherent hydrophilicity (denoted mCTpBD) was synthesized through interface deposition of a hydrophilic COF shell on amino group-functionalized magnetite particles via the reaction between a carboxyl group-containing monomer and benzidine. Thanks to the superior hydrophilicity, appropriate porous structure, and easy magnetic separation, the resulting mCTpBD exhibited excellent performance in conveniently enriching glycopeptides from standard samples with a high sensitivity of 0.5 fmol μL-1 and strong size-exclusion effect of up to 1:1000 (w/w). Furthermore, by using the mCTpBD adsorbent, endogenous glycopeptides in saliva of healthy people and patients with inflammatory bowel disease were successfully enriched and identified by the combined liquid chromatography-mass spectrometry/mass spectrometry technology, which indicates a promising prospective of core-shell magnetic composite microspheres with a hydrophilic COF shell in glycoproteomics research.

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 β-sheets and β-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 verticallyerected 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 β-casein tryptic digest (0.05 fmol μL-1), excellent selectivity from mixed digests (β-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.

Glutathione-Functionalized Magnetic Covalent Organic Framework Microspheres with Size Exclusion for Endogenous Glycopeptide Recognition in Human Saliva.

Endogenous glycopeptides have been confirmed to play a significant role in multifarious pathological and physiological processes. The low abundance of endogenous glycopeptides and abundant interferents (e.g., large-size proteins and heteropeptides) in complex biological matrices render the direct analysis of endogenous glycopeptides difficult. Reported here is a novel glutathione-functionalized magnetic covalent organic framework microsphere (denoted as MCNC@COF@GSH) endowed with size-exclusion effect and strong hydrophilicity for selective and efficient enrichment of N-linked glycopeptides. The as-prepared MCNC@COF@GSH microspheres possessed fast magnetic responsiveness, regular porosity, large surface areas, and good hydrophilicity, resulting in remarkable performances in N-linked glycopeptide enrichment with low detection limit (0.01 fmol μL-1), high selectivity (1:5000, human immunoglobulin G (IgG) digests to bovine serum albumin digests), excellent size-exclusion effect (IgG digests/IgG/bovine serum albumin (BSA), 1:500:500), and reusability (at least five times). More excitingly, 143 endogenous N-linked glycopeptides were clearly identified from 10 μL sample of human saliva treated with the MCNC@COF@GSH microspheres, which is the unprecedented high efficiency in endogenous N-linked glycopeptide enrichment from human saliva. In addition to providing a strategy for versatile functionalization of magnetic covalent organic frameworks (COFs), this study may be used to develop application of endogenous glycoproteome analysis.

Preparation of zwitterionic cysteine-modified silica microsphere capillary packed columns for the on-column enrichment and analysis of glycopeptides in human saliva

In this work, for the first time, L-cysteine (L-Cys), a zwitterionic hydrophilic compound containing abundant amino and carboxyl was modified on silica microspheres for the fabrication of SiO2@L-Cys capillary packed column. Since the SiO2@L-Cys capillary packed column possessed great hydrophilicity brought by L-cysteine, they could be utilized to enrich glycopeptides with a low detection limit of 5 fmol μL−1 HRP digests as well as a good selectivity of the mixture of HRP and BSA digests up to a molar ratio of 1:50. The capillary packed column were further applied into the glycosylation analysis of human saliva and 69 glycopeptides were identified from 2 μL human saliva digests.

Polycarboxyl metal–organic framework UiO-66-(COOH)2 as efficient desorption/ionization matrix of laser desorption/ionization mass spectrometry for selective enrichment and detection of phosphopeptides

Specific enrichment and efficient detection of phosphopeptides are important for phosphoproteomic analysis. Metal–organic frameworks (MOFs) have been proved to be effective enrichment materials combined with the analysis of mass spectrometry. In this work, we have developed a polycarboxylic group MOF-UiO-66-(COOH)2 used as both efficient enrichment material for phosphopeptides and efficient desorption/ionization matrix of laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS) without elution. The crystal is spherical and the particle size is about 200 nm. When using it as enrichment material and MS matrix, phosphopeptides of β-casein can be efficiently detected under superior low detection limits of 1 fmol μL−1, and at an extremely low molar ratio of phosphoprotein/non-phosphoprotein (1:2000) mixtures, there are still four phosphopeptides that can be observed. When detecting real samples, most phosphopeptides of non-fat milk and human saliva can be observed, which is comparable with the literature.

Magnetic mesoporous silica of loading copper metal ions for enrichment and LC-MS/MS analysis of salivary endogenous peptides

Peptidomics research is of great significance for discovering potential biomarkers and monitoring human diseases. As a kind of common clinical biofluid, saliva known for its noninvasive collection and easy accessibility has been widely used in peptidomics research. In this article, we combined immobilized metal ions affinity chromotography (IMAC) with mesoporous material and proposed the copper ion doped magnetic mesoporous silica material (denoted as Fe3O4@mSiO2-Cu2+) which had a large surface area of 221 m2 g−1 and pore volume of 0.20 cm3 g−1. By immobilizing copper ions onto the mesopore walls, the standard peptide Angiotensin II could be identified in an extremely low concentration of 0.1 fmol μl−1 and in a mass ratio of 1:500 (Angiotensin II:BSA, m/m), which indicated significant sensitivity and a great size-exclusive ability. In addition, the introduction of polydopamine (PDA) made Fe3O4@mSiO2-Cu2+ more hydrophilic and biocompatible which could improve the profiling of endogenous peptides in bio-sample. Finally, 131 endogenous peptides were identified in human saliva after enrichment with Fe3O4@mSiO2-Cu2+. Therefore, Fe3O4@mSiO2-Cu2+ nanoparticles provided a promising candidate protocol for biomarker discovery.