Application of lectin immobilized on polyHIPE monoliths for bioprocess monitoring of glycosylated proteins.

In-process monitoring of glycosylated protein concentration becomes very important with the introduction of perfusion bioprocesses. Affinity chromatography based on lectins allows selective monitoring when carbohydrates are accessible on the protein surface. In this work, we immobilized lectin on polyHIPE type of monoliths and implemented it for bioprocess monitoring. A spacer was introduced to lectin, which increased binding kinetics toward Fc-fusion protein, demonstrated by bio-layer interferometry. Furthermore, complete desorption using 0.25 M galactose was shown. Affinity column exhibited linearity in the range between 0.5 and 8 mg/ml and flow-unaffected binding for the flow-rates between 0.5 and 8 ml/min. Long-term stability over at least four months period was demonstrated. No unspecific binding of culture media components, including host cell proteins and DNA, was detected. Results obtained by affinity column matched concentration values obtained by a reference method.

Modified secreted alkaline phosphatase as an improved reporter protein for N-glycosylation analysis.

N-glycosylation is a common posttranslational modification of proteins in eukaryotic cells. The modification is often analyzed in cells which are able to produce extracellular, glycosylated proteins. Here we report an improved method of the use of genetically modified, secreted alkaline phosphatase (SEAP) as a reporter glycoprotein which may be used for glycoanalysis. Additional N-glycosylation sites introduced by site-directed mutagenesis significantly increased secretion of the protein. An improved purification protocol of recombinant SEAP from serum or serum-free media is also proposed. The method enables fast and efficient separation of reporter glycoprotein from a relatively small amount of medium (0.5-10 ml) with a high recovery level. As a result, purified SEAP was ready for enzymatic de-glycosylation without buffer exchange, sample volume reductions or other procedures, which are usually time-consuming and may cause partial loss of the reporter glycoprotein.

Transient expression of the full-length glycoprotein from infectious hematopoietic necrosis virus in bean (Phaseolus vulgaris) leaves via agroinfiltration.

The glycoprotein of infectious hematopoietic necrosis virus (IHNV), the causative agent of acute disease in salmonids, is the only structural protein of the virus that can induce protective immunity in the fish host. Here, the reliability of bean (Phaseolus vulgaris) plant for the production of this viral protein was examined by the transient expression method. Using the syringe agroinfiltration method, leaves of bean plants were transformed with the expression construct encoding the full-length of IHNV glycoprotein (IHNV-G) gene. Furthermore, the transformation efficacy of two infiltration buffers including PBS-A (PBS+acetosyringone) and MMS-A (MES buffer + MgSO4  + sucrose + acetosyringone) was compared. The analysis of mRNA and dot-blot assay confirmed the transcription and translation of IHNV-G protein in bean leaves. Moreover, Western blotting verified the production of intact, full-length (∼57 kDa) IHNV-G protein in the agroinfiltrated plants. Of note, the production level of IHNV-G using MMS-A agroinfiltration buffer was approximately five times higher compared to PBS-A buffer (0.48 vs. 0.1% of total soluble protein), indicating the effect of infiltration buffer on the transient transformation efficiency. The recombinant protein was purified at the final yield of 0.35 µg/g of fresh leaf tissue, using nickel affinity chromatography. The present work is the first report describing the feasibility of the plant expression platform for the production of IHNV-G protein, which can be served as an oral vaccine against IHNV infection.

Characterization of low-cost glycolipoprotein biosurfactant produced by Lactobacillus plantarum 60 FHE isolated from cheese samples using food wastes through response surface methodology and its potential as antimicrobial, antiviral, and anticancer activities.

Considering the need of new lactic acid bacteria (LAB) for the production of novel biosurfactant (BS) molecules, the current study brings out a new insight on the exploration of cheese samples for BS producers and process optimization for industrial applications. In view of this, Lactobacillus plantarum 60FHE, Lactobacillus paracasei 75FHE, and Lactobacillus paracasei 77FHE were selected as the most operative strains. The biosurfactants (BSs) described as glycolipoproteins via Fourier-transform infrared spectroscopy (FTIR) exhibited antimicrobial activity against the food-borne pathogens. L. plantarum 60FHE BS showed an anticancer activity against colon carcinoma cells and had a week antiviral activity against Hepatitis A virus. Furthermore, glycolipoprotein production was enhanced by 1.42-fold through the development of an optimized process using central composite design (CCD). Emulsifying activities were stable after 60-min incubation from 4 to 120 °C, at pH 2-12, and after the addition of NaCl (2-14%). Characterization by nuclear magnetic resonance spectroscopy (1H NMR) revealed that BS produced from strain 60FHE was glycolipoprotein. L. plantarum produced mixed BSs determined by Liquid Chromatography/Mass Spectrometry (LC-MS). Thus, indicating that BS was applied as a microbial food prevention and biomedical. Also, L. plantarum 60FHE BS was achieved with the use of statistical optimization on inexpensive food wastes.

Neem leaf glycoprotein reverses tumor-induced and age-associated thymic involution to maintain peripheral CD8+ T cell pool.

Aim: As tumor causes atrophy in the thymus to target effector-T cells, this study is aimed to decipher the efficacy of neem leaf glycoprotein (NLGP) in tumor- and age-associated thymic atrophy. Materials & methods: Different thymus parameters were studied using flow cytometry, reverse transcriptase PCR and immunocyto-/histochemistry in murine melanoma and sarcoma models. Results: Longitudinal NLGP therapy in tumor hosts show tumor-reduction along with significant normalization of thymic alterations. NLGP downregulates intrathymic IL-10, which eventually promotes Notch1 to rescue blockade in CD25+CD44+c-Kit+DN2 to CD25+CD44-c-Kit-DN3 transition in T cell maturation and suppress Ikaros/IRF8/Pu.1 to prevent DN2-T to DC differentiation in tumor hosts. The CD5intTCRαßhigh DP3 population was also increased to endorse CD8+ T cell generation. Conclusion: NLGP rescues tumor-induced altered thymic events to generate more effector T cells to restrain tumor.

Establishment of multiplex RT-PCR for differentiation between rabies virus with and that without mutation at position 333 of glycoprotein.

Rabid raccoon dogs (Nyctereutes procyonoides koreensis) have been responsible for animal rabies in South Korea since the 1990s. A recombinant rabies vaccine strain, designated as ERAGS, was constructed for use as a bait vaccine. Therefore, new means of differentiating ERAGS from other rabies virus (RABV) strains will be required in biological manufacturing and diagnostic service centers. In this study, we designed two specific primer sets for differentiation between ERAGS and other RABVs based on mutation in the RABV glycoprotein gene. Polymerase chain reaction analysis of the glycoprotein gene revealed two DNA bands of 383 bp and 583 bp in the ERAGS strain but a single DNA band of 383 bp in the field strains. The detection limits of multiplex reverse transcription polymerase chain reaction (RT-PCR) were 80 and 8 FAID50/reaction for the ERAGS and Evelyn-Rokitnicki-Abelseth strains, respectively. No cross-reactions were detected in the non-RABV reference viruses, including canine distemper virus, parvovirus, canine adenovirus type 1 and 2, and parainfluenza virus. The results of multiplex RT-PCR were 100% consistent with those of the fluorescent antibody test. Therefore, one-step multiplex RT-PCR is likely useful for differentiation between RABVs with and those without mutation at position 333 of the RABV glycoprotein gene.

Boronic acid modified polyoxometalate-alginate hybrid for the isolation of glycoproteins at neutral environment.

Boronate affinity is widely used for the isolation of glycoproteins at alkaline conditions. For further proteomic studies, however, it is of highly importance to perform protein adsorption in neutral medium. For this purpose, we report a novel composite material, i.e., 4-carboxyphenylboronic acid (CPBA) functionalized nickel-substituted polyoxometalate [Ni6(en)3(Tris)(H2O)2(PW9O34)]•nH2O (Ni6PW9)-sodium alginate (SA) hybrid. The abundant oxygen atoms in the hybrid (shortly termed as CPBA-Ni6PW9/SA) significantly reduce the pKa value of CPBA moiety, which well facilitates the selective adsorption of glycoproteins at neutral environment (at pH 7.0). The moiety of sodium alginate (SA) in the hybrid further improves the isolation/enrichment capacity for glycoproteins through hydrophilic interaction. The adsorption efficiency of Immunoglobulin G (IgG, 1.0 mL, 100 µg mL-1) by 1.0 mg CPBA-Ni6PW9/SA hybrid reaches up to 91%, and 1.0 mL of Tris-HCl buffer (100 mmol L-1) provides an elution efficiency of 82%. The adsorption behavior of IgG fits Langmuir adsorption model, offering a maximum adsorption capacity of 495 mg g-1. The CPBA-Ni6PW9/SA hybrid is practically applied for the enrichment of glycoproteins from human serum. SDS-PAGE assay result indicates that approximately 92% serum albumin is eliminated and high-purity IgG is obtained. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis clearly demonstrated that after enriching with CPBA-Ni6PW9/SA, 81 glycoproteins are identified and 79.4% recognition selectivity is achieved.

Expression, Purification, and Crystallization of HSV-1 Glycoproteins for Structure Determination.

Herpes simplex viruses utilize glycoproteins displayed on the viral envelope to perform a variety of functions in the viral infectious cycle. Structural and functional studies of these viral glycoproteins can benefit from biochemical, biophysical, and structural analysis of purified proteins. Here, we describe a general protocol for expression and purification of viral glycoproteins from insect cells based on those developed for the HSV-1 gB and HSV-2 gH/gL ectodomains as well as the protocol for crystallization of these glycoproteins. This protocol can be used for generating milligram amounts of wild-type (WT) or mutant gB and gH/gL ectodomains or can be adapted to produce purified ectodomains of glycoproteins from HSV or other herpesviruses for biochemical and structural studies.

L-cysteine functionalized straticulate C3N4 for the selective enrichment of glycopeptides.

The facile enrichment of glycopeptides or glycoproteins poses great challenges for glycoproteomic research. In this study, a novel hydrophilic material, named zwitterionic hydrophilic L-cysteine derivatized straticulate-C3N4 composites (LCAC), were synthesized and evaluated for the enrichment of N-glycopeptides. LCAC exhibited good biocompatibility, excellent hydrophilicity and selectivity, by virtue of the large surface of C3N4 and the zwitterionic property offered by cysteine. LCAC demonstrated excellent performance for N-glycopeptide enrichment with the sensitivity of 0.033 fmol/µL, selectivity of 1:100, and high recovery rate (∼85%). The performance of LCAC was demonstrated by the identification of 35 N-glycopeptides from IgG, as well as capturing 1809 human urine N-glycopeptides corresponding to 876 N-glycoproteins. Comparing the LCAC with our developed phenylboronic acid functionalized material showed a certain complementary due to the different binding mechanism. The simple production and enhanced hydrophilic properties make the material a promising choice for glycoproteomics researches.

Anti-inflammatory effect of Yam Glycoprotein on lipopolysaccharide-induced acute lung injury via the NLRP3 and NF-κB/TLR4 signaling pathway.

Acute lung injury (ALI) is a common lung disease accompanied by acute and persistent pulmonary inflammatory response syndrome, which leads to alveolar epithelial cells and capillary endothelial cell damage. Yam glycoprotein, separated from traditional Chinese yam, has been shown to have anti-inflammatory and immunomodulatory effects. In this experiment, we mainly studied the therapeutic effect and mechanism of a glycoprotein on the lipopolysaccharide (LPS)-induced ALI mice. An oral glycoprotein method was used to treat the mouse ALI model induced by LPS injection in the peritoneal cavity. Afterward, we measured the wet/dry (W/D) ratio, the activity of myeloperoxidase (MPO), the oxidative index superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-PX) and the production of inflammatory cytokines interleukin-1ß (IL-1ß), tumour necrosis factor-α (TNF-α), and interleukin-6 (IL-6) to evaluate the effect of yam glycoprotein on lung tissue changes. We examined the protein expression of TLR4, ASC, NF-κBp65, p-NF-κBp65, Caspase-1, IκB, NLRP3, p-IκB, and ß-actin by western blot analysis. Immunohistochemical analyses of NLRP3 and p-p65 in lung tissue were carried out to assess the mechanism of glycoprotein action. This result suggests that glycoprotein markedly depressed LPS-induced lung W/D ratio, MPO activity, MDA content SOD and GSH-Px depletion, and the contents of inflammatory cytokines IL-1ß, IL-6, and TNF-α. Moreover, glycoprotein blocked TLR4/NF-κBp65 signaling activation and NLRP3inflammasome expression in LPS-induced ALI mice. As this particular study shows, glycoprotein has a safeguarding effects on LPS-induced ALI mice, possibly via activating NLRP3inflammasome and TLR4/NF-κB signaling pathways.

Orthogonal dual molecularly imprinted polymer-based plasmonic immunosandwich assay: A double characteristic recognition strategy for specific detection of glycoproteins.

Sensitive and specific detection methods are critical to the detection of glycoproteins. Immunoassay has been a powerful tool for this purpose, in which antibodies or their mimics particularly molecularly imprinted polymers (MIPs) are used for specific recognition. Epitope and glycan are two structure features of a glycoprotein. However, immunoassays based on simultaneous recognition towards the two characteristics have been scarcely explored so far. Herein we present a new strategy called orthogonal dual molecularly imprinted polymer-based plasmonic immunosandwich assay (odMIP-PISA). It relies on double recognition towards a target glycoprotein by two different types of MIPs, using epitope-imprinted gold nanoparticles (AuNPs)-coated slide as capturing substrate to recognize the peptide epitope and glycans-imprinted Raman-active silver nanoparticles as labeling nanotags to recognize the glycans. Carcinoembryonic antigen (CEA), a routinely used marker for colon cancer, was used as a test glycoprotein. The orthogonal double recognition apparently improved the specificity, reducing the maximum cross-reactivity from 14.4% for epitope recognition and 15.2% for glycan recognition to 8.2% for double recognition. Meanwhile, the plasmonic nanostructure-based Raman detection provided ultrahigh sensitivity, yielding a limit of detection of 5.56 × 10-14 M (S/N = 10). Through measuring the CEA level in human serum, this method permitted differentiation of colon cancer patient from healthy individual. Compared with the traditional immunoassay, odMIP-PISA exhibited multiple advantages, including simplified procedure (6 steps), speed (30 min), reduced cost, and so on. Therefore, this new approach holds great promise in many applications particularly clinical diagnosis.

Glycopatterns and Glycoproteins Changes in MCN and SCN: A Prospective Cohort Study.

Background. Advances in imaging improve the detection of malignant pancreatic cystic including mucinous cystic neoplasm (MCN), intraductal papillary mucinous neoplasm (IPMN), and mucinous cystic adenocarcinoma (MCA), but the distinction between benign and malignant lesions remains a problem. In an effort to establish glycopatterns as potential biomarkers for differential diagnosis between MCN and SCN, we systematically investigated the alterations of glycopatterns in cystic fluids for both SCN and MCN. Methods. Among the 75 patients enrolled, 37 were diagnosed as MCN and 38 as SCN based on histology. Lectin microarray analysis was performed on each sample, and the fluorescence intensity was used to obtain the fold-change. Then, mixed cyst fluids of MCN group and SCN group were cross bonded with magnetic particles coupled by Lectin STL and WGA, respectively. Hydrophilic interaction liquid chromatography (HILIC) enrichment was performed, liquid chromatography (LC)/mass spectrometry (MS) analysis and bioinformatical analysis was conducted to find the differential glycoproteins between MCNs and SCNs. Results. Through analysis of lectin microarray between MCNs and SCNs, stronger lectin signal patterns were assigned to Lectin WFA, DBA, STL, WGA, and BPL; and weaker signal patterns were assigned to Lectin PTL-I, Con A, ACA, and MAL-I. The glycoproteins were enriched by STL or WGA-coupled magnetic particles. Furthermore, the 10 identified correspondding genes were found to be significantly elevated in the mucinous cystadenoma: CLU, A2M, FGA, FGB, FGG, PLG, SERPINA1, SERPING1, C5, C8A, and C9. Bioinformatics analysis revealed that the above genes may activate the KEGG pathway: immune complement system. Conclusion. This study shows changes in glycopatterns and glycoproteins are associated with MCNs and SCNs.

Anticancer activity of a novel glycoprotein from Camellia oleifera Abel seeds against hepatic carcinoma in vitro and in vivo.

Glycoproteins are naturally occurring biological macromolecule that possess known pharmacological properties. This study investigated the anticancer potential of a new glycoprotein, COG2a with the weight-average molecular weight of 25.9 kDa, isolated from Camellia oleifera Abel seeds by multiple column chromatography and the mechanism of anticancer action. MTT assay showed that the maximum inhibition rate of COG2a on HepG2 cells was 92.1%. Electron microscopic and fluorescence microscopy observation indicated that the HepG2 cells treated with COG2a exhibited typical apoptotic morphological character. Flow cytometry manifested that COG2a induced the cell cycle arrest at the G2 phases and decreased in mitochondrial membrane potential on the HepG2 cells. Western blotting exhibited that COG2a increased the expressions of the pro-apoptotic protein caspase-3 and Bax and decreased the expression of the anti-apoptotic proteins Bcl-2. Additionally, COG2a exerted an obvious anticancer effect on tumor-bearing mice and the maximum tumor inhibition rate is 72.77%. It also can promote the mouse thymus and pancreas index, the number of T lymphocytes cell subset and interferon-gamma to increase. These results indicate that COG2a exhibits in vitro and in vivo anticancer activities that associate with its immunopotentiation properties and may serve as a potential anticancer agent.

Identification and functional characterization of lactadherin, an agglutinating glycoprotein from the chordate Styela clava.

Lactadherin is an extracellular matrix glycoprotein with stimulating agglutination ability that plays crucial roles in animal immunology. In the present study, a novel lactadherin, Sc-lactadherin, was identified from the marine invertebrate chordate, Styela clava. Its full-length cDNA consisted of 579 bps, encoding 193 amino acids with a coagulation FA58C domain. Recombinant Sc-lactadherin via a prokaryotic expression system showed strong hemocyte fusion activity. Therefore, we further examined its effects on cell behaviors using human umbilical vein endothelial cells (HUVECs) and human cervical cancer (HeLa) cells. Recombinant Sc-lactadherin significantly increased the proliferation rate of HUVECs and HeLa cells and improved the cell migration rate of HUVECs. These results demonstrated that the lactadherin identified from the marine ascidian displayed the agglutinating activity. Functional characterization of the recombinant protein showed that it promoted cell proliferation and migration, indicating the potential roles of Sc-lactadherin in immunology and organogenesis in marine ascidians.

A new glycoprotein SPG-8700 isolated from sweet potato with potential anti-cancer activity against colon cancer.

A new small molecule glycoprotein SPG-8700 with potential anti-colorectal cancer activity was firstly separated by tracking of bioactivity from a new sweet potato variety Zhongshu-1. Matrix-assisted laser desorption ionization mass spectrometry, high-performance liquid chromatography and amino acid analyzer were applied separately to determine the molecular weight and compositions of this glycoprotein. Flow cytometry analysis and western blotting analysis were employed to explore it's mechanism of the anti-colorectal cancer. The molecular weight of glycoprotein was 8703.8D (SPG-8700). Relative sugar and protein contents in SPG-8700 were 73.4 and 26.6%, comprising more than 6 types of sugars (mannose, rhamnose, glucuronic acid, glucose, galactose and arabinose with a proportion of 1:6.9:7.3:1.5:46:21). Further results indicated that SPG-8700 promoted apoptosis in HCT-116 cells through regulating the expression of Bcl-2 and Bax and had no effect on the growth of normal cells.

Anti-α-galactoside and Anti-ß-glucoside Antibodies are Partially Occupied by Either of Two Albumin-bound O-glycoproteins and Circulate as Ligand-binding Triplets.

Two heavily O-glycosylated proteins and albumin co-purified with anti-α-galactoside (anti-Gal), the chief xenograft-rejecting antibody and anti-ß-glucan (ABG) antibody isolated from human plasma by affinity chromatography on respective ligand-bearing matrices. Both antibodies and O-glycoproteins co-purified with plasma albumin eluted from albumin-specific matrix. Using components of affinity-purified antibody samples separated by electrophoresis binding of either albumin or antibody to the affinity matrix of the other or binding of O-glycoprotein to either matrix was ruled out. Enzyme-linked immunoassay and ligand-induced fluorescence enhancement of fluorolabeled antibody showed that O-glycoproteins occupied sugar-binding sites of anti-Gal and ABG. Neither antibody recognized albumin. O-Glycoprotein-albumin complexes free in plasma, or released from antibodies by specific sugars, were captured on microwell-coated O-glycan-specific lectin jacalin and detected using labeled anti-albumin. We conclude that circulating anti-Gal and ABG form protein triplets in which either O-glycoprotein bridges between antibody and albumin by binding simultaneously to both. Bound albumin restricted O-glycoprotein occupation on antibodies enabling triplets to bind other ligands using spared binding sites. Free anti-Gal and ABG were undetectable in plasma. Jacalin treatment, but not de-O-glycosylation of O-glycoproteins abolished their recognition by anti-Gal or ABG indicating that antibodies recognized serine- and threonine-rich peptide sequences that underlie the O-glycans and are reported surrogate ligands for anti-Gal. The albumin- and antibody-binding O-glycoproteins AOP1 and AOP2 were single polypeptide proteins of size 107 kDa and 98 kDa, containing 54% and 51% carbohydrate respectively and conformed to no known plasma protein in properties. Prospects of triplet-mediated modulations in autologous tissues expressing antibody ligands are discussed.

Biomedical Applications of Lectins from Traditional Chinese Medicine.

Lectins are proteins or glycoproteins of non-immune origin which have at least one noncatalytic domain that bind reversibly to specific mono or oligosaccharides. Traditional Chinese Medicine (TCM) involves a broad range of medicinal practices sharing common concepts which have been developed in China and are based on a tradition of more than thousands of years. Plant materials which are commonly used in TCM as a complementary or alternative for Western medical treatments contain a considerable number of important lectins. These lectins have been reported to have various applications and uses such as cancer treatment, glycoconjugate research, biomarker development, and others. Here, we summarize the available literature related to lectins from TCM and recent trends in their potential biomedical applications.

Analysis of Protein Glycation Using Phenylboronate Acrylamide Gel Electrophoresis.

Carbohydrate modification of proteins adds complexity and diversity to the proteome. However, undesired carbohydrate modifications also occur in the form of glycation, which have been implicated in diseases such as diabetes, Alzheimer's disease, autoimmune diseases, and cancer. The analysis of glycated proteins is challenging due to their complexity and variability. Numerous analytical techniques have been developed that require expensive specialized equipment and complex data analysis. In this chapter, we describe two easy-to-use electrophoresis-based methods that will enable researchers to detect, identify, and analyze these posttranslational modifications. This new cost-effective methodology will aid the detection of unwanted glycation products in processed foods and may lead to new diagnostics and therapeutics for age-related chronic diseases.

Glycoproteomic Alterations in Drug-Resistant Nonsmall Cell Lung Cancer Cells Revealed by Lectin Magnetic Nanoprobe-Based Mass Spectrometry.

Understanding the functional role of glycosylation-mediated pathogenesis requires deep characterization of glycoproteome, which remains extremely challenging due to the inherently complex nature of glycoproteins. We demonstrate the utility of lectin-magnetic nanoprobe (MNP@lectin) coupled to Orbitrap HCD-CID-MS/MS for complementary glycotope-specific enrichment and site-specific glycosylation analysis of the glycoproteome. By three nanoprobes, MNP@ConA, MNP@AAL, and MNP@SNA, our results revealed the first large-scale glycoproteome of nonsmall cell lung cancer (NSCLC) with 2290 and 2767 nonredundant glycopeptides confidently identified (Byonic score ≥100) in EGFR-TKI-sensitive PC9 and -resistant PC9-IR cells, respectively, especially with more fucosylated and sialylated glycopeptides in PC9-IR cells. The complementary enrichment was demonstrated with only five glycopeptides commonly enriched in three MNP@lectins. Glycotope specificity of 79 and 62% for enrichment was achieved using MNP@AAL and MNP@SNA, respectively. Label-free quantitation revealed predominant fucosylation in PC9-IR cells, suggesting its potential role associated with NSCLC resistance. Moreover, without immunoprecipitation, this multilectin nanoprobe allows the sensitive identification of 51 glycopeptides from 10 of 12 reported sites from onco-protein EGFR. Our results not only demonstrated a sensitive approach to study the vastly under-represented N-glycoprotome but also may pave the way for a glycoproteomic atlas to further explore the site-specific function of glycoproteins associated with drug resistance in NSCLC.

Novel functionalized nanomaterials for the effective enrichment of proteins and peptides with post-translational modifications.

Low abundance proteins/peptides usually suffer strong interference with highly abundant proteins/peptides as well as other contaminants, resulting in low ionization efficiency in MS analysis. Hence, the enrichment and separation of proteins/peptides from complex mixtures are of great value to their successful identification. In this review we present an overview of the application of different nanocomposites for the specific enrichment of peptides/proteins with post-translational modifications (PTMs), such as phosphorylation and glycosylation. Furthermore, examples of the selective enrichment of cysteine-containing peptides, histidine-tagged proteins and the isolation of N-terminal peptides will be analyzed.