Post-synthesis of a titanium-rich magnetic COF nanocomposite with flexible branched polymers for efficient enrichment of phosphopeptides from human saliva and serum.

A Ti4+-functionalized magnetic covalent organic framework material with flexible branched polymers (mCOF@ε-PL@THBA-Ti4+) built via an immobilized metal ion affinity chromatography (IMAC) enrichment strategy was proposed through post-synthesis modification. Hydrophilic ε-poly-L-lysine (ε-PL) rich in amino active groups was first introduced in the fabrication of the phosphopeptide enrichment material to increase the hydrophilicity while providing more functional modification pathways of the material. 2,3,4-Trihydroxy-benzaldehyde (THBA) provides abundant binding sites for the immobilization of numerous Ti4+, which is advantageous for the subsequent efficient phosphopeptide enrichment. The magnetic nanocomposite exhibited outstanding performance of phosphopeptide enrichment with good selectivity (1 : 5000), a low detection limit (2 fmol), and relatively high loading capacity (66.7 mg g-1). What's more, after treatment with mCOF@ε-PL@THBA-Ti4+, 16 endogenous phosphopeptides from fresh saliva of healthy people were recognized by MALDI-TOF MS, and 50 phosphopeptides belonging to 35 phosphoproteins from the serum of uremia patients were detected by nano-LC-MS/MS. Proteomics data analysis for the differential protein selection between uremia and normal controls was conducted using R software, and four down-regulated and three up-regulated proteins were obtained. The results suggested that the prepared material has potential applications in biomarker discovery.

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

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

Efficient separation of phosphopeptides employing a Ti/Nb-functionalized core-shell structure solid-phase extraction nanosphere.

A strategy for effectively enriching global phosphopeptides was successfully developed by using ammonia methyl phosphate (APA) as a novel chelating ligand and Ti4+ and Nb5+ as double functional ions (referred to as Fe3O4@mSiO2@APA@Ti4+/Nb5+). With the advantage of large specific surface area (151.1 m2/g), preeminent immobilized ability for metal ions (about 8% of total atoms), and unbiased enrichment towards phosphopeptides, Fe3O4@mSiO2@APA@Ti4+/Nb5+ displays high selectivity (maximum mass ratio ß-casein to BSA is 1:1500), low limit of detection (LOD, as low as 0.05 fmol), good relative standard deviation (RSD, lower than 7%), recovery rate of 87% (18O isotope labeling method), outstanding phosphopeptide loading capacity (330 µg/mg), and at least five times re-use abilities. In the examination of the actual sample, 24 phosphopeptides were successfully detected in saliva and 4 phosphopeptides were also selectively extracted from human serum. All experiments have shown that Fe3O4@mSiO2@APA@Ti4+/Nb5+ exhibits exciting potential in view of the challenge of low abundance of phosphopeptides. Graphical abstract.

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

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

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

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

Postsynthesis of zwitterionic hydrophilic composites for enhanced enrichment of N-linked glycopeptides from human serum.

RATIONALE: Glycosylation of proteins plays an important role in life activities, but the concentration of naturally occurring glycopeptides is usually relatively low, and glycosylation has microfacies heterogeneity, so direct mass spectrometry is not feasible. Therefore, selective enrichment of glycopeptides before mass spectrometry has turned into an urgent problem to be resolved. METHODS: Herein, the zwitterionic L-cysteine functionalized hydrophilic graphene oxide composite (GO@PDA@MIL-125-NH2 @Au@L-Cys) was prepared via a postsynthetic method. The obtained material was used for glycopeptide enrichment. The enriched peptides were then detected using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) to demonstrate the enrichment performance of the material. RESULTS: In the actual enrichment process, GO@PDA@MIL-125-NH2 @Au@L-Cys nanomaterials exhibited high selectivity (1:1000), outstanding sensitivity (0.5 fmol), and excellent repeatability for the enrichment of glycopeptides. In addition, the proposed material showed good performance in the enrichment of glycopeptides from complex biosamples; 56 glycopeptides were detected from 2 µL of human serum using MALDI-TOFMS. CONCLUSIONS: The experimental results showed that GO@PDA@MIL-125-NH2 @Au@L-Cys exhibited excellent performance on glycopeptide analysis. It has great potential in the enrichment of glycopeptides and provides new ideas for synthetic materials with better enrichment properties in the future.

Binary magnetic metal-organic frameworks composites: a promising affinity probe for highly selective and rapid enrichment of mono- and multi-phosphopeptides.

A binary magnetic metal-organic framework (MOF)-functionalized material (magG@PDA@Ni-MOF@Fe-MOF) was prepared through grafting Ni-MOF and Fe-MOF on magnetic (Fe3O4) graphene with polydopamine (PDA) as a middle layer. Compared with single MOFs functionalized materials (magG@PDA@Ni-MOF and magG@PDA@Fe-MOF) under the same conditions, magG@PDA@Ni-MOF@Fe-MOF not only displays lower detection limits (4 fmol) and selectivity (1:1000), but also has better enrichment efficiency for both multi- and monophosphopeptides. Other than this, magG@PDA@Ni-MOF@Fe-MOF exhibits fine reusability (five cycles) and rapid enrichment property (1 min), and 24 phosphopeptides were detected when it was applied to the analysis of human saliva. Graphical abstractThe strategy of preparing a binary magnetic metal-organic framework (MOF)-functionalized material (magG@PDA@Ni-MOF@Fe-MOF) through grafting Ni-MOF and Fe-MOF on magnetic graphene with polydopamine (PDA) as a middle layer.