9 research outputs found
In-Depth Proteome Coverage by Improving Efficiency for Membrane Proteome Analysis
Although great achievement
has been made in the mapping of human
proteome, the efficiency of sample preparation still needs to be improved,
especially for membrane proteins. Herein, we presented a novel method
to deepen proteome coverage by the sequential extraction of proteins
using urea and 1-dodecyl-3- methylimidazolium chloride (C12Im-Cl).
With such a strategy, the commonly lost hydrophobic proteins by 8
M urea extraction could be further recovered by C12Im-Cl, as well
as the suppression effect of high abundance soluble proteins could
be decreased. Followed by the in situ sample preparation and separation
with different stationary phases, more than 9810 gene products could
be identified, covering 8 orders of magnitude in abundance, which
was, to the best of our knowledge, the largest data set of HeLa cell
proteome. Compared with previous work, not only the number of proteins
identified was obviously increased, but also the analysis time was
shortened to a few days. Therefore, we expect that such a strategy
has great potential applications to achieve unprecedented coverage
for proteome analysis
In-Depth Proteome Coverage by Improving Efficiency for Membrane Proteome Analysis
Although great achievement
has been made in the mapping of human
proteome, the efficiency of sample preparation still needs to be improved,
especially for membrane proteins. Herein, we presented a novel method
to deepen proteome coverage by the sequential extraction of proteins
using urea and 1-dodecyl-3- methylimidazolium chloride (C12Im-Cl).
With such a strategy, the commonly lost hydrophobic proteins by 8
M urea extraction could be further recovered by C12Im-Cl, as well
as the suppression effect of high abundance soluble proteins could
be decreased. Followed by the in situ sample preparation and separation
with different stationary phases, more than 9810 gene products could
be identified, covering 8 orders of magnitude in abundance, which
was, to the best of our knowledge, the largest data set of HeLa cell
proteome. Compared with previous work, not only the number of proteins
identified was obviously increased, but also the analysis time was
shortened to a few days. Therefore, we expect that such a strategy
has great potential applications to achieve unprecedented coverage
for proteome analysis
In-Depth Proteome Coverage by Improving Efficiency for Membrane Proteome Analysis
Although great achievement
has been made in the mapping of human
proteome, the efficiency of sample preparation still needs to be improved,
especially for membrane proteins. Herein, we presented a novel method
to deepen proteome coverage by the sequential extraction of proteins
using urea and 1-dodecyl-3- methylimidazolium chloride (C12Im-Cl).
With such a strategy, the commonly lost hydrophobic proteins by 8
M urea extraction could be further recovered by C12Im-Cl, as well
as the suppression effect of high abundance soluble proteins could
be decreased. Followed by the in situ sample preparation and separation
with different stationary phases, more than 9810 gene products could
be identified, covering 8 orders of magnitude in abundance, which
was, to the best of our knowledge, the largest data set of HeLa cell
proteome. Compared with previous work, not only the number of proteins
identified was obviously increased, but also the analysis time was
shortened to a few days. Therefore, we expect that such a strategy
has great potential applications to achieve unprecedented coverage
for proteome analysis
Hydrophobic Tagging-Assisted N‑Termini Enrichment for In-Depth N‑Terminome Analysis
The analysis of protein
N-termini is of great importance for understanding
the protein function and elucidating the proteolytic processing. Herein,
we develop a negative enrichment strategy, termed as hydrophobic tagging-assisted
N-termini enrichment (HYTANE) to achieve a global N-terminome analysis.
The HYTANE strategy showed a high efficiency in hydrophobic tagging
and C18 material-assisted depletion using bovine serum albumin (BSA)
as the sample. This strategy was applied to N-termini profiling from <i>S. cerevisiae</i> cell lysates and enabled the identification
of 1096 protein N-termini, representing the largest N-terminome data
set of <i>S. cerevisiae</i>. The identified N-terminal
peptides accounted for 99% of all identified peptides, and no deficiency
in acidic, histidine (His)-containing, and His-free N-terminal peptides
was observed. The presented HYTANE strategy is therefore a highly
selective, efficient, and unbiased strategy for the large scale N-terminome
analysis. Furthermore, using the HYTANE strategy, we identified 329
cleavage sites and 291 substrates of caspases in Jurkat cells, demonstrating
the great promise of HYTANE strategy for protease research. Data are
available via ProteomeXchange with identifier PXD004690
Biphasic Microreactor for Efficient Membrane Protein Pretreatment with a Combination of Formic Acid Assisted Solubilization, On-Column pH Adjustment, Reduction, Alkylation, and Tryptic Digestion
Combining good dissolving ability
of formic acid (FA) for membrane
proteins and excellent complementary retention behavior of proteins
on strong cation exchange (SCX) and strong anion exchange (SAX) materials,
a biphasic microreactor was established to pretreat membrane proteins
at microgram and even nanogram levels. With membrane proteins solubilized
by FA, all of the proteomics sample processing procedures, including
protein preconcentration, pH adjustment, reduction, and alkylation,
as well as tryptic digestion, were integrated into an “SCX-SAX”
biphasic capillary column. To evaluate the performance of the developed
microreactor, a mixture of bovine serum albumin, myoglobin, and cytochrome
c was pretreated. Compared with the results obtained by the traditional
in-solution process, the peptide recovery (93% vs 83%) and analysis
throughput (3.5 vs 14 h) were obviously improved. The microreactor
was further applied for the pretreatment of 14 ÎĽg of membrane
proteins extracted from rat cerebellums, and 416 integral membrane
proteins (IMPs) (43% of total protein groups) and 103 transmembrane
peptides were identified by two-dimensional nanoliquid chromatography-electrospray
ionization tandem mass spectrometry (2D nano-LC-ESI-MS/MS) in triplicate
analysis. With the starting sample preparation amount decreased to
as low as 50 ng, 64 IMPs and 17 transmembrane peptides were identified
confidently, while those obtained by the traditional in-solution method
were 10 and 1, respectively. All these results demonstrated that such
an “SCX-SAX” based biphasic microreactor could offer
a promising tool for the pretreatment of trace membrane proteins with
high efficiency and throughput
Biphasic Microreactor for Efficient Membrane Protein Pretreatment with a Combination of Formic Acid Assisted Solubilization, On-Column pH Adjustment, Reduction, Alkylation, and Tryptic Digestion
Combining good dissolving ability
of formic acid (FA) for membrane
proteins and excellent complementary retention behavior of proteins
on strong cation exchange (SCX) and strong anion exchange (SAX) materials,
a biphasic microreactor was established to pretreat membrane proteins
at microgram and even nanogram levels. With membrane proteins solubilized
by FA, all of the proteomics sample processing procedures, including
protein preconcentration, pH adjustment, reduction, and alkylation,
as well as tryptic digestion, were integrated into an “SCX-SAX”
biphasic capillary column. To evaluate the performance of the developed
microreactor, a mixture of bovine serum albumin, myoglobin, and cytochrome
c was pretreated. Compared with the results obtained by the traditional
in-solution process, the peptide recovery (93% vs 83%) and analysis
throughput (3.5 vs 14 h) were obviously improved. The microreactor
was further applied for the pretreatment of 14 ÎĽg of membrane
proteins extracted from rat cerebellums, and 416 integral membrane
proteins (IMPs) (43% of total protein groups) and 103 transmembrane
peptides were identified by two-dimensional nanoliquid chromatography-electrospray
ionization tandem mass spectrometry (2D nano-LC-ESI-MS/MS) in triplicate
analysis. With the starting sample preparation amount decreased to
as low as 50 ng, 64 IMPs and 17 transmembrane peptides were identified
confidently, while those obtained by the traditional in-solution method
were 10 and 1, respectively. All these results demonstrated that such
an “SCX-SAX” based biphasic microreactor could offer
a promising tool for the pretreatment of trace membrane proteins with
high efficiency and throughput
In-Depth Proteomic Quantification of Cell Secretome in Serum-Containing Conditioned Medium
Secreted proteins play key roles
during cellular communication,
proliferation, and migration. The comprehensive profiling of secreted
proteins in serum-containing culture media is technically challenging.
Most studies have been performed under serum-free conditions. However,
these conditions might alter the status of the cells. Herein, we describe
an efficient strategy that avoids the disturbance of serum by combining
metabolic labeling, protein “equalization,” protein
fractionation, and filter-aided sample preparation, called MLEFF,
enabling the identification of 534 secreted proteins from HeLa conditioned
media, including 31 cytokines, and growth factors. This MLEFF strategy
was also successfully applied during a comparative secretome analysis
of two human hepatocellular carcinoma cell lines with differentially
metastatic potentials, enabling the quantification of 61 significantly
changed proteins involved in tumor invasion and metastasis
In-Depth Proteomic Quantification of Cell Secretome in Serum-Containing Conditioned Medium
Secreted proteins play key roles
during cellular communication,
proliferation, and migration. The comprehensive profiling of secreted
proteins in serum-containing culture media is technically challenging.
Most studies have been performed under serum-free conditions. However,
these conditions might alter the status of the cells. Herein, we describe
an efficient strategy that avoids the disturbance of serum by combining
metabolic labeling, protein “equalization,” protein
fractionation, and filter-aided sample preparation, called MLEFF,
enabling the identification of 534 secreted proteins from HeLa conditioned
media, including 31 cytokines, and growth factors. This MLEFF strategy
was also successfully applied during a comparative secretome analysis
of two human hepatocellular carcinoma cell lines with differentially
metastatic potentials, enabling the quantification of 61 significantly
changed proteins involved in tumor invasion and metastasis
Aptamer Modified Organic–Inorganic Hybrid Silica Monolithic Capillary Columns for Highly Selective Recognition of Thrombin
A novel
kind of aptamer modified organic–inorganic hybrid
silica monolithic capillary column has been developed, via the covalent
bonding of 5′-NH<sub>2</sub>-modified aptamer for human α-thrombin
on hybrid silica monolith, prepared by sol–gel method, with
tetraethoxysilane and 3-aminopropyltriethoxysilane as precursors.
Due to the large specific surface area of the hybrid matrix, the average
coverage density of aptamer reached 568 pmol/ÎĽL, and the thrombin
binding capacity was 1.15 ÎĽg/ÎĽL, 14 times higher than
that of aptamer modified open tubular capillaries. By such an affinity
capillary column, the limit of detection of thrombin was decreased
to 3.4 nM with a UV detector. Furthermore, even when thrombin was
mixed with 1000 times more concentrated human serum, it could be selectively
enriched and detected with the signal-to-noise ratio as ca.10. These
results indicate that the developed preparation strategy for aptamer
based hybrid silica monolithic capillary column might provide an effective
method to achieve highly selective recognition of trace targets