One-step fabrication of strongly hydrophilic mesoporous silica for comprehensive analysis of serum glycopeptidome.

Glycopeptidome represents reliable predictors of physiological and pathological status. Obstructions mainly including low abundance of endogenous glycopeptides and varied interference necessitate glycopeptide enrichment prior to MS analysis. Inspired by the prevalence of hydrophilic interaction chromatography for glycopeptide enrichment, a novel magnetic mesoporous silica nanomaterial (Fe3O4@mSiO2-TSG) with strongly hydrophilic property was developed through a one-pot method. In this work, the gluconamide-containing organosilane is innovatively proposed to directly serve as the strongly hydrophilic silica source for fabrication of hydrophilic mesoporous silica nanomaterial for glycopeptidomics research. Apart from excellent hydrophilicity, Fe3O4@mSiO2-TSG also was equipped with large specific surface area, ordered mesopore channels and great magnetic responsiveness. With all the advantages, Fe3O4@mSiO2-TSG displayed remarkable size-exclusion effect and considerable reusability. Moreover, combined with nano-LC-MS/MS, the glycopeptidome of serum from breast cancer patients was analyzed comprehensively, which showed noteworthy difference from healthy serum through gene ontology analysis, indicating great potential of the approach for glycopeptidomics research.

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.

Profiling of Endogenously Intact N-Linked and O-Linked Glycopeptides from Human Serum Using an Integrated Platform.

Endogenous glycopeptides in serum are an invaluable resource for biomarker discovery. Due to the low abundance and the poor fragmentation in tandem mass spectrometry, the identification of endogenously intact glycopeptides still faces many challenges. Herein, an integrated platform is fabricated for the identification of N-linked and O-linked endogenously intact glycopeptides. In this platform, the high-temperature acid denaturation, ultrafiltration, and hydrophilic interaction chromatography steps are combined together for the highly efficient extraction of the endogenously intact glycopeptides from a small amount of serum. Additionally, the twin-spectra scheme and in silico deglycosylation strategy were applied for the identification of N-linked and O-linked endogenous glycopeptides, respectively. In total, 223 intact N-glycopeptides and 51 intact O-glycopeptides are identified from only 40 µL of the human serum sample. This is the first study reporting the identification of endogenously intact N-linked and O-linked glycopeptide and is also the largest data set of endogenously intact glycopeptides reported so far. The distributions of glycans among peptides and proteins and cleavage sites on peptides are further analyzed to seek the regulation of endogenous glycosylation for disease mechanism. The developed strategy provides a novel platform for the disease biomarker discovery.

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.

Fabrication of hydrophilic multilayer magnetic probe for salivary glycopeptidome analysis.

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.

Core-shell structured magnetic metal-organic framework composites for highly selective enrichment of endogenous N-linked glycopeptides and phosphopeptides.

In this work, core-shell structured magnetic metal-organic framework composites denoted as Fe3O4 @MIL-100(Fe) were synthesized by means of a layer-by-layer assembly method selecting Fe as metal center and 1,3,5-benzenetricarboxylic acid as organic ligand. The as-prepared material exhibited outstanding sensitivity (0.1 fmol/µL), good selectivity (1:20 and 1:50 respectively), excellent ability of size-exclusion (1: 500), fine reusability (six cycles) and great stability (two months) in enriching N-linked glycopeptides and phosphopeptides from tryptic digests of standard proteins by combining HILIC and IMAC. Moreover, it was applied into the enrichment of endogenous N-linked glycopeptides and phosphopeptides in human saliva and achieved great results (43 phosphopeptides and 39 N-linked glycopeptides), revealing its promising potential in enrichment of low-abundance endogenous N-linked glycopeptides and phosphopeptides in practical samples.

Hydrophilic probe in mesoporous pore for selective enrichment of endogenous glycopeptides in biological samples.

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.