7 research outputs found
Simultaneous Quantitation of Glycoprotein Degradation and Synthesis Rates by Integrating Isotope Labeling, Chemical Enrichment, and Multiplexed Proteomics
Protein glycosylation
is essential for cell survival and regulates
many cellular events. Reversible glycosylation is also dynamic in
biological systems. The functions of glycoproteins are regulated by
their dynamics to adapt the ever-changing inter- and intracellular
environments. Glycans on proteins not only mediate a variety of protein
activities, but also creates a steric hindrance for protecting the
glycoproteins from degradation by proteases. In this work, a novel
strategy integrating isotopic labeling, chemical enrichment and multiplexed
proteomics was developed to simultaneously quantify the degradation
and synthesis rates of many glycoproteins in human cells. We quantified
the synthesis rates of 847 N-glycoproteins and the degradation rates
of 704 glycoproteins in biological triplicate experiments, including
many important glycoproteins such as CD molecules. Through comparing
the synthesis and degradation rates, we found that most proteins have
higher synthesis rates since cells are still growing throughout the
time course, while a small group of proteins with lower synthesis
rates mainly participate in adhesion, locomotion, localization, and
signaling. This method can be widely applied in biochemical and biomedical
research and provide insights into elucidating glycoprotein functions
and the molecular mechanism of many biological events
Global and Site-Specific Analysis Revealing Unexpected and Extensive Protein SâGlcNAcylation in Human Cells
Protein glycosylation
is highly diverse and essential for mammalian
cell survival. Heterogeneous glycans may be bound to different amino
acid residues, forming multiple types of protein glycosylation. In
this work, unexpected protein S-GlcNAcylation on cysteine residues
was observed to extensively exist in human cells through global and
site-specific analysis of protein GlcNAcylation by mass spectrometry.
Three independent experiments produced similar results of many cysteine
residues bound to <i>N</i>-acetylglucosamine (GlcNAc). Among
well-localized S-GlcNAcylation sites, several motifs with an acidic
amino acid around the sites were identified, which strongly suggests
that a particular type of enzyme is responsible for this modification.
Clustering results show that glycoproteins modified with S-GlcNAc
are mainly involved in cellâcell adhesion and gene expression.
For the first time, we found that proteins were extensively bound
to GlcNAc through the side chains of cysteine residues in human cells,
and the current discovery further advances our understanding of protein
glycosylation
Global Analysis of Secreted Proteins and Glycoproteins in <i>Saccharomyces cerevisiae</i>
Protein secretion
is essential for numerous cellular activities,
and secreted proteins in bodily fluids are a promising and noninvasive
source of biomarkers for disease detection. Systematic analysis of
secreted proteins and glycoproteins will provide insight into protein
function and cellular activities. Yeast (<i>Saccharomyces cerevisiae</i>) is an excellent model system for eukaryotic cells, but global analysis
of secreted proteins and glycoproteins in yeast is challenging due
to the low abundances of secreted proteins and contamination from
high-abundance intracellular proteins. Here, by using mild separation
of secreted proteins from cells, we comprehensively identified and
quantified secreted proteins and glycoproteins through inhibition
of glycosylation and mass spectrometry-based proteomics. In biological
triplicate experiments, 245 secreted proteins were identified, and
comparison with previous experimental and computational results demonstrated
that many identified proteins were located in the extracellular space.
Most quantified secreted proteins were down-regulated from cells treated
with an N-glycosylation inhibitor (tunicamycin). The quantitative
results strongly suggest that the secretion of these down-regulated
proteins was regulated by glycosylation, while the secretion of proteins
with minimal abundance changes was contrarily irrelevant to protein
glycosylation, likely being secreted through nonclassical pathways.
Glycoproteins in the yeast secretome were globally analyzed for the
first time. A total of 27 proteins were quantified in at least two
protein and glycosylation triplicate experiments, and all except one
were down-regulated under N-glycosylation inhibition, which is solid
experimental evidence to further demonstrate that the secretion of
these proteins is regulated by their glycosylation. These results
provide valuable insight into protein secretion, which will further
advance protein secretion and disease studies
Global Analysis of Secreted Proteins and Glycoproteins in <i>Saccharomyces cerevisiae</i>
Protein secretion
is essential for numerous cellular activities,
and secreted proteins in bodily fluids are a promising and noninvasive
source of biomarkers for disease detection. Systematic analysis of
secreted proteins and glycoproteins will provide insight into protein
function and cellular activities. Yeast (<i>Saccharomyces cerevisiae</i>) is an excellent model system for eukaryotic cells, but global analysis
of secreted proteins and glycoproteins in yeast is challenging due
to the low abundances of secreted proteins and contamination from
high-abundance intracellular proteins. Here, by using mild separation
of secreted proteins from cells, we comprehensively identified and
quantified secreted proteins and glycoproteins through inhibition
of glycosylation and mass spectrometry-based proteomics. In biological
triplicate experiments, 245 secreted proteins were identified, and
comparison with previous experimental and computational results demonstrated
that many identified proteins were located in the extracellular space.
Most quantified secreted proteins were down-regulated from cells treated
with an N-glycosylation inhibitor (tunicamycin). The quantitative
results strongly suggest that the secretion of these down-regulated
proteins was regulated by glycosylation, while the secretion of proteins
with minimal abundance changes was contrarily irrelevant to protein
glycosylation, likely being secreted through nonclassical pathways.
Glycoproteins in the yeast secretome were globally analyzed for the
first time. A total of 27 proteins were quantified in at least two
protein and glycosylation triplicate experiments, and all except one
were down-regulated under N-glycosylation inhibition, which is solid
experimental evidence to further demonstrate that the secretion of
these proteins is regulated by their glycosylation. These results
provide valuable insight into protein secretion, which will further
advance protein secretion and disease studies
Site-Specific Quantification of Surface NâGlycoproteins in Statin-Treated Liver Cells
The
frequent modification of cell-surface proteins by N-linked
glycans is known to be correlated with many biological processes.
Aberrant glycosylation on surface proteins is associated with different
cellular statuses and disease progression. However, it is extraordinarily
challenging to comprehensively and site-specifically analyze glycoproteins
located only on the cell surface. Currently mass spectrometry (MS)-based
proteomics provides the possibility to analyze the N-glycoproteome,
but effective separation and enrichment methods are required for the
analysis of surface glycoproteins prior to MS measurement. The introduction
of bio-orthogonal groups into proteins accelerates research in the
robust visualization, identification, and quantification of proteins.
Here we have comprehensively evaluated different sugar analogs in
the analysis of cell-surface N-glycoproteins by combining copper-free
click chemistry and MS-based proteomics. Comparison of three sugar
analogs, N-azidoacetylgalactosamine (GalNAz), N-azidoacetylglucosamine
(GlcNAz), and N-azidoacetylmannosamine (ManNAz), showed that metabolic
labeling with GalNAz resulted in the greatest number of glycoproteins
and glycosylation sites in biological duplicate experiments. GalNAz
was then employed for the quantification experiment in statin-treated
HepG2 liver cells, and 280 unique N-glycosylated sites were quantified
from 168 surface proteins. The quantification results demonstrated
that many glycosylation sites on surface proteins were down-regulated
in statin-treated cells compared to untreated cells because statin
prevents the synthesis of dolichol, which is essential for the formation
of dolichol-linked precursor oligosaccharides. Several glycosylation
sites in proteins that participate in the Alzheimerâs disease
pathway were down-regulated. This method can be extensively applied
for the global analysis of the cell-surface N-glycoproteome
Systematic Investigation of Cellular Response and Pleiotropic Effects in Atorvastatin-Treated Liver Cells by MS-Based Proteomics
For
decades, statins have been widely used to lower cholesterol
levels by inhibiting the enzyme HMG Co-A reductase (HMGCR). It is
well-known that statins have pleiotropic effects including improving
endothelial function and inhibiting vascular inflammation and oxidation.
However, the cellular responses to statins and corresponding pleiotropic
effects are largely unknown at the proteome level. Emerging mass spectrometry-based
proteomics provides a unique opportunity to systemically investigate
protein and phosphoprotein abundance changes as a result of statin
treatment. Many lipid-related protein abundances were increased in
HepG2 cells treated by atorvastatin, including HMGCR, FDFT, SQLE,
and LDLR, while the abundances of proteins involved in cellular response
to stress and apoptosis were decreased. Comprehensive analysis of
protein phosphorylation demonstrated that several basic motifs were
enriched among down-regulated phosphorylation sites, which indicates
that kinases with preference for these motifs, such as protein kinase
A and protein kinase C, have attenuated activities. Phosphopeptides
on a group of G-protein modulators were up-regulated, which strongly
suggests that cell signal rewiring was a result of the effect of protein
lipidation by the statin. This work provides a global view of liver
cell responses to atorvastatin at the proteome and phosphoproteome
levels, which provides insight into the pleiotropic effects of statins
Simultaneous Time-Dependent Surface-Enhanced Raman Spectroscopy, Metabolomics, and Proteomics Reveal Cancer Cell Death Mechanisms Associated with Gold Nanorod Photothermal Therapy
In
cancer plasmonic photothermal therapy (PPTT), plasmonic nanoparticles
are used to convert light into localized heat, leading to cancer cell
death. Among plasmonic nanoparticles, gold nanorods (AuNRs) with specific
dimensions enabling them to absorb near-infrared laser light have
been widely used. The detailed mechanism of PPTT therapy, however,
still remains poorly understood. Typically, surface-enhanced Raman
spectroscopy (SERS) has been used to detect time-dependent changes
in the intensity of the vibration frequencies of molecules that appear
or disappear during different cellular processes. A complete proven
assignment of the molecular identity of these vibrations and their
biological importance has not yet been accomplished. Mass spectrometry
(MS) is a powerful technique that is able to accurately identify molecules
in chemical mixtures by observing their <i>m</i>/<i>z</i> values and fragmentation patterns. Here, we complemented
the study of changes in SERS spectra with MS-based metabolÂomics
and proteomics to identify the chemical species responsible for the
observed changes in SERS band intensities during PPTT. We observed
an increase in intensity of the bands at around 1000, 1207, and 1580
cm<sup>â1</sup>, which were assigned in the literature to phenylÂalanine,
albeit with dispute. Our metabolÂomics results showed increased
levels of phenylÂalanine, its derivatives, and phenylÂalanine-containing
peptides, providing evidence for more confidence in the SERS peak
assignments. To better understand the mechanism of phenylÂalanine
increase upon PPTT, we combined metabolÂomics and proteomics
results through network analysis, which proved that phenylÂalanine
metabolism was perturbed. Furthermore, several apoptosis pathways
were activated via key proteins (e.g., HADHA and ACAT1), consistent
with the proposed role of altered phenylÂalanine metabolism in
inducing apoptosis. Our study shows that the integration of the SERS
with MS-based metabolÂomics and proteomics can assist the assignment
of signals in SERS spectra and further characterize the related molecular
mechanisms of the cellular processes involved in PPTT