9 research outputs found
Capillary Electrophoresis-Electrospray Ionization-Mass Spectrometry for Quantitative Analysis of Glycans Labeled with Multiplex Carbonyl-Reactive Tandem Mass Tags
Recently developed carbonyl-reactive
aminoxy tandem mass tag (aminoxyTMT)
reagents enable multiplexed characterization and quantitative comparison
of structurally complex glycans between different biological samples.
Compared to some previously reported isotopic labeling strategies
for glycans, the use of the aminoxyTMT method features a simple labeling
procedure, excellent labeling efficiency, and reduced spectral complexity
at the MS<sup>1</sup> level. Presence of the tertiary amine functionality
in the reporter region of the aminoxyTMT labels leads to increased
ionization efficiency of the labeled glycans thus improving electrospray
ionization (ESI)-mass spectrometry (MS) detection sensitivity. The
use of the labeling reagent also makes electrophoretic separation
of the labeled neutral and acidic glycans feasible. In this work,
we characterized the ESI and collision induced dissociation (CID)
behavior of the aminoxyTMT-labeled neutral and sialylated glycans.
For the high-mannose N-glycans and small sialylated oligosaccharides,
CID fragmentation of [M + Na + H]<sup>2+</sup> provides the most informative
MS<sup>2</sup> spectra for both quantitative and qualitative analysis.
For complex N-glycans, MS<sup>3</sup> of the protonated Y<sub>1(H)</sub> ion can be used for relative quantification without interference
from the HexNAc fragments. Online capillary electrophoresis (CE)-ESI-MS/MS
analyses of multiplexed aminoxyTMT-labeled human milk oligosaccharides
(HMOs) and different types of N-glycans released from glycoprotein
standards were demonstrated. Improved resolution and quantification
accuracy of the labeled HMO isomers was achieved by coupling CE with
traveling wave ion mobility (TWIM)-CID-MS/MS. N-Glycans released from
human serum protein digests were labeled with six-plex aminoxyTMT
and subjected to CE-ESI-MS/pseudo-MS<sup>3</sup> analysis, which demonstrated
the potential utility of this glycan relative quantification platform
for more complex biological samples
Carbonyl-Reactive Tandem Mass Tags for the Proteome-Wide Quantification of N-Linked Glycans
N-Linked protein glycosylation is one of the most prevalent
post-translational
modifications and is involved in essential cellular functions such
as cell–cell interactions and cellular recognition as well
as in chronic diseases. In this study, we explored stable isotope
labeled carbonyl-reactive tandem mass tags (glyco-TMTs) as a novel
approach for the quantification of N-linked glycans. Glyco-TMTs bearing
hydrazide- and aminooxy-functionalized groups were compared for glycan
reducing end derivatization efficiency and quantification merits.
Aminooxy TMTs outperform the hydrazide reagents in terms of labeling
efficiency (>95% vs 65% at 0.1 μM) and mass spectrometry
based
quantification using heavy/light-TMT labeled glycans enabled accurate
quantification in MS1 spectra (CV < 15%) over a broad dynamic range
(up to 1:40). In contrast, isobaric TMT labeling with quantification
of reporter ions in tandem mass spectra suffered from severe ratio
compression already at low sample ratios. To demonstrate the practical
utility of the developed approach, we characterized the global N-linked
glycosylation profiles of the isogenic human colon carcinoma cell
lines SW480 (primary tumor) and SW620 (metastatic tumor). The data
revealed significant down-regulation of high-mannose glycans in the
metastatic cell line
Carbonyl-Reactive Tandem Mass Tags for the Proteome-Wide Quantification of N-Linked Glycans
N-Linked protein glycosylation is one of the most prevalent
post-translational
modifications and is involved in essential cellular functions such
as cell–cell interactions and cellular recognition as well
as in chronic diseases. In this study, we explored stable isotope
labeled carbonyl-reactive tandem mass tags (glyco-TMTs) as a novel
approach for the quantification of N-linked glycans. Glyco-TMTs bearing
hydrazide- and aminooxy-functionalized groups were compared for glycan
reducing end derivatization efficiency and quantification merits.
Aminooxy TMTs outperform the hydrazide reagents in terms of labeling
efficiency (>95% vs 65% at 0.1 μM) and mass spectrometry
based
quantification using heavy/light-TMT labeled glycans enabled accurate
quantification in MS1 spectra (CV < 15%) over a broad dynamic range
(up to 1:40). In contrast, isobaric TMT labeling with quantification
of reporter ions in tandem mass spectra suffered from severe ratio
compression already at low sample ratios. To demonstrate the practical
utility of the developed approach, we characterized the global N-linked
glycosylation profiles of the isogenic human colon carcinoma cell
lines SW480 (primary tumor) and SW620 (metastatic tumor). The data
revealed significant down-regulation of high-mannose glycans in the
metastatic cell line
Increasing the Multiplexing Capacity of TMTs Using Reporter Ion Isotopologues with Isobaric Masses
Quantitative mass spectrometry methods offer near-comprehensive
proteome coverage; however, these methods still suffer with regards
to sample throughput. Multiplex quantitation via isobaric chemical
tags (e.g., TMT and iTRAQ) provides an avenue for mass spectrometry-based
proteome quantitation experiments to move away from simple binary
comparisons and toward greater parallelization. Herein, we demonstrate
a straightforward method for immediately expanding the throughput
of the TMT isobaric reagents from 6-plex to 8-plex. This method is
based upon our ability to resolve the isotopic shift that results
from substituting a <sup>15</sup>N for a <sup>13</sup>C. In an accommodation
to the preferred fragmentation pathways of ETD, the TMT-127 and -129
reagents were recently modified such that a <sup>13</sup>C was exchanged
for a <sup>15</sup>N. As a result of this substitution, the new TMT
reporter ions are 6.32 mDa lighter. Even though the mass difference
between these reporter ion isotopologues is incredibly small, modern
high-resolution and mass accuracy analyzers can resolve these ions.
On the basis of our ability to resolve and accurately measure the
relative intensity of these isobaric reporter ions, we demonstrate
that we are able to quantify across eight samples simultaneously by
combining the <sup>13</sup>C- and <sup>15</sup>N-containing reporter
ions. Considering the structure of the TMT reporter ion, we believe
this work serves as a blueprint for expanding the multiplexing capacity
of the TMT reagents to at least 10-plex and possibly up to 18-plex
Proteomic Quantification and Site-Mapping of <i>S</i>‑Nitrosylated Proteins Using Isobaric iodoTMT Reagents
<i>S</i>-Nitrosylation is a redox-based protein post-translational
modification in response to nitric oxide signaling and is involved
in a wide range of biological processes. Detection and quantification
of protein <i>S</i>-nitrosylation have been challenging
tasks due to instability and low abundance of the modification. Many
studies have used mass spectrometry (MS)-based methods with different
thiol-reactive reagents to label and identify proteins with <i>S</i>-nitrosylated cysteine (SNO-Cys). In this study, we developed
a novel iodoTMT switch assay (ISA) using an isobaric set of thiol-reactive
iodoTMTsixplex reagents to specifically detect and quantify protein <i>S</i>-nitrosylation. Irreversible labeling of SNO-Cys with the
iodoTMTsixplex reagents enables immune-affinity detection of <i>S</i>-nitrosylated proteins, enrichment of iodoTMT-labeled peptides
by anti-TMT resin, and importantly, unambiguous modification site-mapping
and multiplex quantification by liquid chromatography–tandem
MS. Additionally, we significantly improved anti-TMT peptide enrichment
efficiency by competitive elution. Using ISA, we identified a set
of SNO-Cys sites responding to lipopolysaccharide (LPS) stimulation
in murine BV-2 microglial cells and revealed effects of <i>S</i>-allyl cysteine from garlic on LPS-induced protein <i>S</i>-nitrosylation in antioxidative signaling and mitochondrial metabolic
pathways. ISA proved to be an effective proteomic approach for quantitative
analysis of <i>S</i>-nitrosylation in complex samples and
will facilitate the elucidation of molecular mechanisms of nitrosative
stress in disease
Proteomic Quantification and Site-Mapping of <i>S</i>‑Nitrosylated Proteins Using Isobaric iodoTMT Reagents
<i>S</i>-Nitrosylation is a redox-based protein post-translational
modification in response to nitric oxide signaling and is involved
in a wide range of biological processes. Detection and quantification
of protein <i>S</i>-nitrosylation have been challenging
tasks due to instability and low abundance of the modification. Many
studies have used mass spectrometry (MS)-based methods with different
thiol-reactive reagents to label and identify proteins with <i>S</i>-nitrosylated cysteine (SNO-Cys). In this study, we developed
a novel iodoTMT switch assay (ISA) using an isobaric set of thiol-reactive
iodoTMTsixplex reagents to specifically detect and quantify protein <i>S</i>-nitrosylation. Irreversible labeling of SNO-Cys with the
iodoTMTsixplex reagents enables immune-affinity detection of <i>S</i>-nitrosylated proteins, enrichment of iodoTMT-labeled peptides
by anti-TMT resin, and importantly, unambiguous modification site-mapping
and multiplex quantification by liquid chromatography–tandem
MS. Additionally, we significantly improved anti-TMT peptide enrichment
efficiency by competitive elution. Using ISA, we identified a set
of SNO-Cys sites responding to lipopolysaccharide (LPS) stimulation
in murine BV-2 microglial cells and revealed effects of <i>S</i>-allyl cysteine from garlic on LPS-induced protein <i>S</i>-nitrosylation in antioxidative signaling and mitochondrial metabolic
pathways. ISA proved to be an effective proteomic approach for quantitative
analysis of <i>S</i>-nitrosylation in complex samples and
will facilitate the elucidation of molecular mechanisms of nitrosative
stress in disease
Proteomic Quantification and Site-Mapping of <i>S</i>‑Nitrosylated Proteins Using Isobaric iodoTMT Reagents
<i>S</i>-Nitrosylation is a redox-based protein post-translational
modification in response to nitric oxide signaling and is involved
in a wide range of biological processes. Detection and quantification
of protein <i>S</i>-nitrosylation have been challenging
tasks due to instability and low abundance of the modification. Many
studies have used mass spectrometry (MS)-based methods with different
thiol-reactive reagents to label and identify proteins with <i>S</i>-nitrosylated cysteine (SNO-Cys). In this study, we developed
a novel iodoTMT switch assay (ISA) using an isobaric set of thiol-reactive
iodoTMTsixplex reagents to specifically detect and quantify protein <i>S</i>-nitrosylation. Irreversible labeling of SNO-Cys with the
iodoTMTsixplex reagents enables immune-affinity detection of <i>S</i>-nitrosylated proteins, enrichment of iodoTMT-labeled peptides
by anti-TMT resin, and importantly, unambiguous modification site-mapping
and multiplex quantification by liquid chromatography–tandem
MS. Additionally, we significantly improved anti-TMT peptide enrichment
efficiency by competitive elution. Using ISA, we identified a set
of SNO-Cys sites responding to lipopolysaccharide (LPS) stimulation
in murine BV-2 microglial cells and revealed effects of <i>S</i>-allyl cysteine from garlic on LPS-induced protein <i>S</i>-nitrosylation in antioxidative signaling and mitochondrial metabolic
pathways. ISA proved to be an effective proteomic approach for quantitative
analysis of <i>S</i>-nitrosylation in complex samples and
will facilitate the elucidation of molecular mechanisms of nitrosative
stress in disease
Proteomic Quantification and Site-Mapping of <i>S</i>‑Nitrosylated Proteins Using Isobaric iodoTMT Reagents
<i>S</i>-Nitrosylation is a redox-based protein post-translational
modification in response to nitric oxide signaling and is involved
in a wide range of biological processes. Detection and quantification
of protein <i>S</i>-nitrosylation have been challenging
tasks due to instability and low abundance of the modification. Many
studies have used mass spectrometry (MS)-based methods with different
thiol-reactive reagents to label and identify proteins with <i>S</i>-nitrosylated cysteine (SNO-Cys). In this study, we developed
a novel iodoTMT switch assay (ISA) using an isobaric set of thiol-reactive
iodoTMTsixplex reagents to specifically detect and quantify protein <i>S</i>-nitrosylation. Irreversible labeling of SNO-Cys with the
iodoTMTsixplex reagents enables immune-affinity detection of <i>S</i>-nitrosylated proteins, enrichment of iodoTMT-labeled peptides
by anti-TMT resin, and importantly, unambiguous modification site-mapping
and multiplex quantification by liquid chromatography–tandem
MS. Additionally, we significantly improved anti-TMT peptide enrichment
efficiency by competitive elution. Using ISA, we identified a set
of SNO-Cys sites responding to lipopolysaccharide (LPS) stimulation
in murine BV-2 microglial cells and revealed effects of <i>S</i>-allyl cysteine from garlic on LPS-induced protein <i>S</i>-nitrosylation in antioxidative signaling and mitochondrial metabolic
pathways. ISA proved to be an effective proteomic approach for quantitative
analysis of <i>S</i>-nitrosylation in complex samples and
will facilitate the elucidation of molecular mechanisms of nitrosative
stress in disease
Proteomic Quantification and Site-Mapping of <i>S</i>‑Nitrosylated Proteins Using Isobaric iodoTMT Reagents
<i>S</i>-Nitrosylation is a redox-based protein post-translational
modification in response to nitric oxide signaling and is involved
in a wide range of biological processes. Detection and quantification
of protein <i>S</i>-nitrosylation have been challenging
tasks due to instability and low abundance of the modification. Many
studies have used mass spectrometry (MS)-based methods with different
thiol-reactive reagents to label and identify proteins with <i>S</i>-nitrosylated cysteine (SNO-Cys). In this study, we developed
a novel iodoTMT switch assay (ISA) using an isobaric set of thiol-reactive
iodoTMTsixplex reagents to specifically detect and quantify protein <i>S</i>-nitrosylation. Irreversible labeling of SNO-Cys with the
iodoTMTsixplex reagents enables immune-affinity detection of <i>S</i>-nitrosylated proteins, enrichment of iodoTMT-labeled peptides
by anti-TMT resin, and importantly, unambiguous modification site-mapping
and multiplex quantification by liquid chromatography–tandem
MS. Additionally, we significantly improved anti-TMT peptide enrichment
efficiency by competitive elution. Using ISA, we identified a set
of SNO-Cys sites responding to lipopolysaccharide (LPS) stimulation
in murine BV-2 microglial cells and revealed effects of <i>S</i>-allyl cysteine from garlic on LPS-induced protein <i>S</i>-nitrosylation in antioxidative signaling and mitochondrial metabolic
pathways. ISA proved to be an effective proteomic approach for quantitative
analysis of <i>S</i>-nitrosylation in complex samples and
will facilitate the elucidation of molecular mechanisms of nitrosative
stress in disease