23 research outputs found
Trypsin Immobilization on Hairy Polymer Chains Hybrid Magnetic Nanoparticles for Ultra Fast, Highly Efficient Proteome Digestion, Facile <sup>18</sup>O Labeling and Absolute Protein Quantification
In recent years, quantitative proteomic research attracts
great
attention because of the urgent needs in biological and clinical research,
such as biomarker discovery and verification. Currently, mass spectrometry
(MS) based bottom up strategy has become the method of choice for
proteomic quantification. In this strategy, the amount of proteins
is determined by quantifying the corresponding proteolytic peptides
of the proteins, therefore highly efficient and complete protein digestion
is crucial for achieving accurate quantification results. However,
the digestion efficiency and completeness obtained using conventional
free protease digestion is not satisfactory for highly complex proteomic
samples. In this work, we developed a new type of immobilized trypsin
using hairy noncross-linked polymer chains hybrid magnetic nanoparticle
as the matrix aiming at ultra fast, highly efficient proteomic digestion
and facile <sup>18</sup>O labeling for absolution protein quantification.
The hybrid nanoparticle is synthesized by in situ growth of hairy
polymer chains from the magnetic nanoparticle surface using surface
initiated atom transfer radical polymerization technique. The flexible
noncross-linked polymer chains not only provide large amount of binding
sites but also work as scaffolds to support three-dimensional trypsin
immobilization which leads to increased loading amount and improved
accessibility of the immobilized trypsin. For complex proteomic samples,
obviously increased digestion efficiency and completeness was demonstrated
by 27.2% and 40.8% increase in the number of identified proteins and
peptides as well as remarkably reduced undigested proteins residues
compared with that obtained using conventional free trypsin digestion.
The successful application in absolute protein quantification of enolase
from <i>Thermoanaerobacter tengcongensis</i> protein extracts
using <sup>18</sup>O labeling and MRM strategy further demonstrated
the potential of this hybrid nanoparticle immobilized trypsin for
high throughput proteome quantification
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A Highly Efficient and Visualized Method for Glycan Enrichment by Self-Assembling Pyrene Derivative Functionalized Free Graphene Oxide
Protein glycosylation plays key roles in many biological
processes,
such as cell growth, differentiation, and cell–cell recognition.
Therefore, global structure profiling of glycans is very important
for investigating the biological significance and roles of glycans
in disease occurrence and development. Mass spectrometry (MS) is currently
the most powerful technique for structure analysis of oligosaccharides,
but the limited availability of glycan/glycoproteins from natural
sources restricts the wide adoption of this technique in large-scale
glycan profiling. Though various enrichment methods have been developed,
most methods relay on the weak physical affinity between glycans and
adsorbents that yields insufficient enrichment efficiency. Furthermore,
the lack of monitoring the extent/completeness of enrichment may lead
to incomplete enrichment unless repeated sample loading and prolonged
incubation are adopted, which limits sample handling throughput. Here,
we report a rapid, highly efficient, and visualized approach for glycan
enrichment using 1-pyrenebutyryl chloride functionalized free graphene
oxide (PCGO). In this approach, glycan capturing is achieved by reversible
covalent bond formation between the hydroxyl groups of glycans and
the acyl chloride groups on graphene oxide (GO) introduced by π–π
stacking of 1-pyrenebutyryl chloride on the GO surface. The multiple
hydroxyl groups of glycans lead to cross-linking and self-assembly
of free PCGO sheets into visible aggregation within 30 s, therefore
achieving simple visual monitoring of the enrichment process. Improved
enrichment efficiency is achieved by the large specific surface area
of free PCGO and heavy functionalization of highly active 1-pyrenebutyryl
chloride. Application of this method in enrichment of standard oligosaccharides
or <i>N</i>-glycans released from glycoproteins results
in remarkably increased MS signal intensity (approximately 50 times),
S/N, and number of glycoform identified
Two-Dimensional MoS<sub>2</sub>‑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 MoS<sub>2</sub>/Au-NP (gold nanoparticle)–L-cysteine
nanocomposite was prepared for glycopeptide enrichment. The two-dimensional
(2D) structured MoS<sub>2</sub> 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<sup>–1</sup>), 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
Two-Dimensional MoS<sub>2</sub>‑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 MoS<sub>2</sub>/Au-NP (gold nanoparticle)–L-cysteine
nanocomposite was prepared for glycopeptide enrichment. The two-dimensional
(2D) structured MoS<sub>2</sub> 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<sup>–1</sup>), 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
Two-Dimensional MoS<sub>2</sub>‑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 MoS<sub>2</sub>/Au-NP (gold nanoparticle)–L-cysteine
nanocomposite was prepared for glycopeptide enrichment. The two-dimensional
(2D) structured MoS<sub>2</sub> 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<sup>–1</sup>), 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
Two-Dimensional MoS<sub>2</sub>‑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 MoS<sub>2</sub>/Au-NP (gold nanoparticle)–L-cysteine
nanocomposite was prepared for glycopeptide enrichment. The two-dimensional
(2D) structured MoS<sub>2</sub> 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<sup>–1</sup>), 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
Two-Dimensional MoS<sub>2</sub>‑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 MoS<sub>2</sub>/Au-NP (gold nanoparticle)–L-cysteine
nanocomposite was prepared for glycopeptide enrichment. The two-dimensional
(2D) structured MoS<sub>2</sub> 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<sup>–1</sup>), 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
Two-Dimensional MoS<sub>2</sub>‑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 MoS<sub>2</sub>/Au-NP (gold nanoparticle)–L-cysteine
nanocomposite was prepared for glycopeptide enrichment. The two-dimensional
(2D) structured MoS<sub>2</sub> 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<sup>–1</sup>), 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
Two-Dimensional MoS<sub>2</sub>‑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 MoS<sub>2</sub>/Au-NP (gold nanoparticle)–L-cysteine
nanocomposite was prepared for glycopeptide enrichment. The two-dimensional
(2D) structured MoS<sub>2</sub> 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<sup>–1</sup>), 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