4 research outputs found
Chemical Modulation of Protein O‑GlcNAcylation <i>via</i> OGT Inhibition Promotes Human Neural Cell Differentiation
The enzymes that
determine protein O-GlcNAcylation, O-GlcNAc transferase
(OGT) and O-GlcNAcase (OGA), act on key transcriptional and epigenetic
regulators, and both are abundantly expressed in the brain. However,
little is known about how alterations in O-GlcNAc cycling affect human
embryonic stem cell (hESC) neural differentiation. Here, we studied
the effects of perturbing O-GlcNAcylation during neural induction
of hESCs using the metabolic inhibitor of OGT, peracetylated 5-thio-<i>N</i>-acetylglucosamine (Ac<sub>4</sub>-5SGlcNAc). Treatment
of hESCs with Ac<sub>4</sub>-5SGlcNAc during induction limited protein
O-GlcNAcylation and also caused a dramatic decrease in global levels
of UDP-GlcNAc. Concomitantly, a subpopulation of neural progenitor
cells (NPCs) acquired an immature neuronal morphology and expressed
early neuronal markers such as β-III tubulin (TUJ1) and microtubule
associated protein 2 (MAP2), phenotypes that took longer to manifest
in the absence of OGT inhibition. These data suggest that chemical
inhibition of OGT and perturbation of protein O-GlcNAcylation accelerate
the differentiation of hESCs along the neuronal lineage, thus providing
further insight into the dynamic molecular mechanisms involved in
neuronal development
Analysis of the Acidic Proteome with Negative Electron-Transfer Dissociation Mass Spectrometry
We describe the first implementation of negative electron-transfer
dissociation (NETD) on a hybrid ion trap-orbitrap mass spectrometer
and its application to high-throughput sequencing of peptide anions.
NETD, coupled with high pH separations, negative electrospray ionization
(ESI), and an NETD compatible version of OMSSA, is part of a complete
workflow that includes the formation, interrogation, and sequencing
of peptide anions. Together these interlocking pieces facilitated
the identification of more than 2000 unique peptides from <i>Saccharomyces cerevisiae</i> representing the most comprehensive
analysis of peptide anions by tandem mass spectrometry to date. The
same <i>S. cerevisiae</i> samples were interrogated using
traditional, positive modes of peptide LC-MS/MS analysis (e.g., acidic
LC separations, positive ESI, and collision activated dissociation),
and the resulting peptide identifications of the different workflows
were compared. Due to a decreased flux of peptide anions and a tendency
to produce lowly charged precursors, the NETD-based LC-MS/MS workflow
was not as sensitive as the positive mode methods. However, the use
of NETD readily permits access to underrepresented acidic portions
of the proteome by identifying peptides that tend to have lower pI
values. As such, NETD improves sequence coverage, filling out the
acidic portions of proteins that are often overlooked by the other
methods
Analysis of the Acidic Proteome with Negative Electron-Transfer Dissociation Mass Spectrometry
We describe the first implementation of negative electron-transfer
dissociation (NETD) on a hybrid ion trap-orbitrap mass spectrometer
and its application to high-throughput sequencing of peptide anions.
NETD, coupled with high pH separations, negative electrospray ionization
(ESI), and an NETD compatible version of OMSSA, is part of a complete
workflow that includes the formation, interrogation, and sequencing
of peptide anions. Together these interlocking pieces facilitated
the identification of more than 2000 unique peptides from <i>Saccharomyces cerevisiae</i> representing the most comprehensive
analysis of peptide anions by tandem mass spectrometry to date. The
same <i>S. cerevisiae</i> samples were interrogated using
traditional, positive modes of peptide LC-MS/MS analysis (e.g., acidic
LC separations, positive ESI, and collision activated dissociation),
and the resulting peptide identifications of the different workflows
were compared. Due to a decreased flux of peptide anions and a tendency
to produce lowly charged precursors, the NETD-based LC-MS/MS workflow
was not as sensitive as the positive mode methods. However, the use
of NETD readily permits access to underrepresented acidic portions
of the proteome by identifying peptides that tend to have lower pI
values. As such, NETD improves sequence coverage, filling out the
acidic portions of proteins that are often overlooked by the other
methods
Analysis of the Acidic Proteome with Negative Electron-Transfer Dissociation Mass Spectrometry
We describe the first implementation of negative electron-transfer
dissociation (NETD) on a hybrid ion trap-orbitrap mass spectrometer
and its application to high-throughput sequencing of peptide anions.
NETD, coupled with high pH separations, negative electrospray ionization
(ESI), and an NETD compatible version of OMSSA, is part of a complete
workflow that includes the formation, interrogation, and sequencing
of peptide anions. Together these interlocking pieces facilitated
the identification of more than 2000 unique peptides from <i>Saccharomyces cerevisiae</i> representing the most comprehensive
analysis of peptide anions by tandem mass spectrometry to date. The
same <i>S. cerevisiae</i> samples were interrogated using
traditional, positive modes of peptide LC-MS/MS analysis (e.g., acidic
LC separations, positive ESI, and collision activated dissociation),
and the resulting peptide identifications of the different workflows
were compared. Due to a decreased flux of peptide anions and a tendency
to produce lowly charged precursors, the NETD-based LC-MS/MS workflow
was not as sensitive as the positive mode methods. However, the use
of NETD readily permits access to underrepresented acidic portions
of the proteome by identifying peptides that tend to have lower pI
values. As such, NETD improves sequence coverage, filling out the
acidic portions of proteins that are often overlooked by the other
methods