High Enrichment Recovery Research Articles

A Two-Dimensional Metallacycle Cross-Linked Switchable Polymer for Fast and Highly Efficient Phosphorylated Peptide Enrichment.

The selective and efficient capture of phosphopeptides is critical for comprehensive and in-depth phosphoproteome analysis. Here we report a new switchable two-dimensional (2D) supramolecular polymer that serves as an ideal platform for the enrichment of phosphopeptides. A well-defined, positively charged metallacycle incorporated into the polymer endows the resultant polymer with a high affinity for phosphopeptides. Importantly, the stimuli-responsive nature of the polymer facilitates switchable binding affinity of phosphopeptides, thus resulting in an excellent performance in phosphopeptide enrichment and separation from model proteins. The polymer has a high enrichment capacity (165 mg/g) and detection sensitivity (2 fmol), high enrichment recovery (88%), excellent specificity, and rapid enrichment and separation properties. Additionally, we have demonstrated the capture of phosphopeptides from the tryptic digest of real biosamples, thus illustrating the potential of this polymeric material in phosphoproteomic studies.

A hydrophilic two-dimensional titanium-based metal-organic framework nanosheets for specific enrichment of glycopeptides

A hydrophilic two-dimensional titanium-based metal-organic framework (MOF) nanosheets were prepared and applied to improve the glycopeptides enrichment efficiency. Seventeen N-linked glycopeptides from horseradish peroxidase (HRP) tryptic digests were readily identified and the results showed highly improved signal intensities. As the molar ratio of the mixture of tryptic digests of HRP, glycoprotein (HRP), and nonglycoprotein (BSA) reached 1:800:800, good selectivity of the Ti-MOF nanosheets were still clearly observed. As to human serum samples, 41 glycoproteins and 66 glycopeptides corresponding to 68 N-glycosylation sites were finally detected, which showed the good ability of the nanosheets material to enrich N-linked glycopeptides from complex human serum samples. Moreover, the Ti-MOF nanosheets exhibited unique stability and reusability even after being regenerated and reused for five times. It also performed a high sensitivity (1 × 10−10 M HRP), a good enrichment capacity (250 mg/g) for glycopeptides enrichment and a high enrichment recovery of 78%, suggesting a great application prospect for the enrichment of N-linked glycopeptides and the analysis glycoproteomic research.

Two-Dimensional MoS2-Based Zwitterionic Hydrophilic Interaction Liquid Chromatography Material for the Specific Enrichment of Glycopeptides.

Mass spectrometry (MS)-based glycoproteomics research requires highly efficient sample preparation to eliminate interference from non-glycopeptides and to improve the efficiency of glycopeptide detection. In this work, a novel MoS2/Au-NP (gold nanoparticle)-L-cysteine nanocomposite was prepared for glycopeptide enrichment. The two-dimensional (2D) structured MoS2 nanosheets served as a matrix that could provide a large surface area for immobilizing hydrophilic groups (such as L-cysteine) with low steric hindrance between the materials and the glycopeptides. As a result, the novel nanomaterial possessed an excellent ability to capture glycopeptides. Compared to commercial zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) materials, the novel nanomaterials exhibited excellent enrichment performance with ultrahigh selectivity and sensitivity (approximately 10 fmol), high binding capacity (120 mg g-1), high enrichment recovery (more than 93%), satisfying batch-to-batch reproducibility, and good universality for glycopeptide enrichment. In addition, its outstanding specificity and efficiency for glycopeptide enrichment was confirmed by the detection of glycopeptides from an human serum immunoglobulin G (IgG) tryptic digest in quantities as low as a 1:1250 molar ratio of IgG tryptic digest to bovine serum albumin tryptic digest. The novel nanocomposites were further used for the analysis of complex samples, and 1920 glycopeptide backbones from 775 glycoproteins were identified in three replicate analyses of 50 μg of proteins extracted from HeLa cell exosomes. The resulting highly informative mass spectra indicated that this multifunctional nanomaterial-based enrichment method could be used as a promising tool for the in-depth and comprehensive characterization of glycoproteomes in MS-based glycoproteomics.

Maltose-Functionalized Hydrophilic Magnetic Nanoparticles with Polymer Brushes for Highly Selective Enrichment of N-Linked Glycopeptides.

Efficient enrichment glycoproteins/glycopeptides from complex biological solutions are very important in the biomedical sciences, in particular biomarker research. In this work, the high hydrophilic polyethylenimine conjugated polymaltose polymer brushes functionalized magnetic Fe3O4 nanoparticles (NPs) denoted as Fe3O4–PEI–pMaltose were designed and synthesized via a simple two-step modification. The obtained superhydrophilic Fe3O4–PEI–pMaltose NPs displayed outstanding advantages in the enrichment of N-linked glycopeptides, including high selectivity (1:100, mass ratios of HRP and bovine serum albumin (BSA) digest), low detection limit (10 fmol), large binding capacity (200 mg/g), and high enrichment recovery (above 85%). The above-mentioned excellent performance of novel Fe3O4–PEI–pMaltose NPs was attributed to graft of maltose polymer brushes and efficient assembly strategy. Moreover, Fe3O4–PEI–pMaltose NPs were further utilized to selectively enrich glycopeptides from human renal mesangial cell (HRMC, 200 μg) tryptic digest, and 449 N-linked glycopeptides, representing 323 different glycoproteins and 476 glycosylation sites, were identified. It was expected that the as-synthesized Fe3O4–PEI–pMaltose NPs, possessing excellent performance (high binding capacity, good selectivity, low detection limit, high enrichment recovery, and easy magnetic separation) coupled to a facile preparation procedure, have a huge potential in N-glycosylation proteome analysis of complex biological samples.

PAMA-Arg brush-functionalized magnetic composite nanospheres for highly effective enrichment of phosphorylated biomolecules.

Highly effective enrichment of phosphorylated biomolecules is vital for an in depth and comprehensive phosphoproteome analysis. Among affinity materials, materials based on the immobilized metal affinity chromatography (IMAC) method are most commonly used in phosphopeptide enrichment. However, the problem of metal cation loss in IMAC materials is still a challenge. In this study, a novel strategy has been developed for both highly effective enrichment of phosphorylated biomolecules and prevention of metal cation loss using Fe3O4/polydopamine/poly(2-aminoethyl methacrylate hydrochloride)/arginine (Fe3O4/PDA/PAMA-Arg) composite nanospheres. The magnetic composite nanospheres were prepared by a facile method, possessing uniform morphology, a high saturation magnetization (45 emu g-1), and a quick magnetic response (within 10 s). Noticeably, abundant guanidyl and amino groups of the PAMA-Arg brushes endowed the nanospheres with extremely high detection sensitivity (0.2 fmol), excellent selectivity (β-casein/BSA = 1 : 500), and high recyclability (5 cycles) in phosphopeptide analysis. Furthermore, the Fe3O4/PDA/PAMA-Arg nanospheres exhibited specific separation capacity towards β-casein or ovalbumin (OVA) in the model protein mixture and remarkably selective enrichment performance for phosphorylated biomolecules in real biological samples (egg white, nonfat milk, and rat brain lysate), showing great potential for trace biological detection and proteomics analysis.

Single-Step Enrichment of N-Glycopeptides and Phosphopeptides with Novel Multifunctional Ti4+-Immobilized Dendritic Polyglycerol Coated Chitosan Nanomaterials.

Protein glycosylation and phosphorylation, two of the most important post-translational modifications (PTMs) in the proteome, play a vital role in regulating a number of complex biological processes and involvement in a variety of diseases. Comprehensive characterization of the phosphoproteome and glycoproteome requires highly specific and sensitive enrichment methods of purification of phosphopeptides and glycopeptides because many glycoproteins and phosphoproteins naturally occur at low abundances and substoichiometry. Here, we reported a facile route to fabricate a novel multifunctional Ti4+-mmobilized dentritic polyglycerol CS@PGMA@IDA (CS, chitosan; PGMA, poly(glycidyl methacrylate); IDA, iminodiacetic acid) nanomaterials. The polymer surface endows the nanomaterials with biocompatibility, excellent hydrophilic property, and a large amount of Ti 4+ which have the property of immobilized metal ion affinity chromatography (IMAC)- and hydrophilic interaction liquid chromatography (HILIC)-based functional materials. The CS@PGMA@IDA-Ti4+ nanomaterials demonstrate an outstanding ability for N-glycopeptides and phosphopeptides enrichment simultaneously, evaluated by the extremely high binding capacity (150 mg g-1), sensitivity (above 0.1 fmol), and high enrichment recovery (above 75.4%). Its outstanding specificity and efficiency for purification of phosphopeptides is reflected in quantities as low as 1:5000 molar ratios of phosphopeptides which can be detected. Furthermore, we used CS@PGMA@IDA-Ti4+ to enrich for N-glycopeptides and phosphopeptides followed by PNGase F treatment, fractionated and separated N-glycopeptides and phosphopeptides with different eluents, and then analyzed by MS, a total of 423 (84.4 ID/μg, 3.525 ID/min) N-glycopeptides in 235 different glycoproteins and 422 (84.4 ID/μg, 3.517 ID/min) phosphopeptides in 256 different phosphoproteins which were finally identified in two independent LC-MS/MS runs (with a total time of 120 min) from 50 μg of mouse liver. The results demonstrated that the method based on CS@PGMA@IDA-Ti4+ to single-step enrichment of N-glycopeptides and phosphopeptides is simple, efficient, specific, and compatible to MS. It can be expected that CS@PGMA@IDA-Ti4+ would hold great applicability of modification-based proteomics to the precious and low amounts of clinical samples.

Facile synthesis of guanidyl-functionalized magnetic polymer microspheres for tunable and specific capture of global phosphopeptides or only multiphosphopeptides.

The highly selective and efficient capture of heterogeneous types of phosphopeptides is critical for comprehensive and in-depth phosphoproteome analysis, but it still remains a challenge since the lack of affinity material with large binding capacity and controllable specificity. Here, a new affinity material was prepared to improve the enrichment capacity and endue the tunable specificity by introducing guanidyl onto poly(glycidyl methacrylate) (PGMA) modified Fe3O4 microsphere (denoted as Fe3O4@PGMA-Guanidyl). The thick polymer shell endows the composite microsphere with large amount of guanidyl and is beneficial to enhancing the affinity interaction between phosphopeptides and the material. Interestingly, the Fe3O4@PGMA-Guanidyl possesses tunable enriching ability for global phosphopeptides or only multiphosphopeptides through simple regulation of buffer composition. The composite has large enrichment capacity (200 mg g(-1)), extremely high detection sensitivity (0.5 fmol), high enrichment recovery (91.30%), great specificity, and rapid magnetic separation. Moreover, the result of the application to capture of phosphopeptides from tryptic digest of nonfat milk has demonstrated the great potential of Fe3O4@PGMA-Guanidyl in detection and identification of low-abundance phosphopeptides of interest in biological sample.

Two-in-One Strategy for Effective Enrichment of Phosphopeptides Using Magnetic Mesoporous γ-Fe2O3 Nanocrystal Clusters

Designed with a two-in-one strategy, the magnetic mesoporous γ-Fe(2)O(3) nanocrystal clusters (m-γ-Fe(2)O(3)) have been successfully prepared for integrating the functions of effective enrichment and quick separation of phosphopeptides into a single architecture. First, the mesoporous Fe(3)O(4) nanocrystal clusters (mFe(3)O(4)) were synthesized by solvothermal reaction and then were subjected to calcination in air to form m-γ-Fe(2)O(3). The obtained m-γ-Fe(2)O(3) have spherical morphology with uniform particle size of about 200 nm and mesoporous structure with the pore diameter of about 9.7 nm; the surface area is as large as 117.8 m(2)/g, and the pore volume is 0.34 cm(3)/g. The m-γ-Fe(2)O(3) possessed very high magnetic responsiveness (Ms = 78.8 emu/g, magnetic separation time from solution is less than 5 s) and were used for the selective enrichment of phosphopeptides for the first time. The experimental results demonstrated that the m-γ-Fe(2)O(3) possessed high selectivity for phosphopeptides at a low molar ratio of phosphopeptides/nonphosphopeptides (1:100), high sensitivity (the detection limit was at the fmol level), high enrichment recovery (as high as 89.4%), and excellent speed (the enrichment can be completed in 10 min). Moreover, this material is also quite effective for enrichment of phosphopeptides from the real sample (drinking milk), showing great potential in the practical application.