11 research outputs found
Implications of Hydrophobicity and Free Energy of Solvation for Characterization of Nucleic Acids by Electrospray Ionization Mass Spectrometry
Global Proteomic Analysis of Functional Compartments in Immature Avian Follicles Using Laser Microdissection Coupled to LC-MS/MS
Laser
microdissection (LMD) was utilized for the separation of
the yolk, follicular wall (granulosa and theca), and surrounding stromal
cells of small white follicles (SWF) obtained from reproductively
active domestic fowl. Herein, we provide an in situ proteomics-based
approach to studying follicular development through the use of LMD
and mass spectrometry. This study resulted in a total of 2889 proteins
identified from the three specific isolated compartments. White yolk
from the smallest avian follicles resulted in the identification of
1984 proteins, while isolated follicular wall and ovarian stroma yielded
2470 and 2456 proteins, respectively. GO annotations highlighted the
functional differences between the compartments. Among the three compartments
examined, the relative abundance of vitellogenins, steroidogenic enzymes,
anti-Mullerian hormone, transcription factors, and proteins involved
in retinoic acid receptors/retinoic acid synthesis, transcription
factors, and cell surface receptors such as EGFR and their associated
signaling pathways reflected known cellular function of the ovary.
This study has provided a global proteome for SWF, white yolk, and
ovarian stroma of the avian ovary that can be used as a baseline for
future studies and verifies that the coupling of LMD with proteomic
analysis can be used to evaluate proteins from small, physiologically
functional compartments of complex tissue
Dual Electrospray Ionization Source for Confident Generation of Accurate Mass Tags Using Liquid Chromatography Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
Compartment Proteomics Analysis of White Perch (<i>Morone americana</i>) Ovary Using Support Vector Machines
Compartment
proteomics enable broad characterization of target
tissues. We employed a simple fractionation method and filter-aided
sample preparation (FASP) to characterize the cytosolic and membrane
fractions of white perch ovary tissues by semiquantitative tandem
mass spectrometry using label-free quantitation based on normalized
spectral counts. FASP depletes both low-molecular-weight and high-molecular-weight
substances that could interfere with protein digestion and subsequent
peptide separation and detection. Membrane proteins are notoriously
difficult to characterize due to their amphipathic nature and association
with lipids. The simple fractionation we employed effectively revealed
an abundance of proteins from mitochondria and other membrane-bounded
organelles. We further demonstrate that support vector machines (SVMs)
offer categorical classification of proteomics data superior to that
of parametric statistical methods such as analysis of variance (ANOVA).
Specifically, SVMs were able to perfectly (100% correct) classify
samples as either membrane or cytosolic fraction during cross-validation
based on the expression of 242 proteins with the highest ANOVA <i>p</i>-values (i.e., those that were not significant for enrichment
in either fraction). The white perch ovary cytosolic and membrane
proteomes and transcriptome presented in this study can support future
investigations into oogenesis and early embryogenesis of white perch
and other members of the genus <i>Morone</i>
Compartment Proteomics Analysis of White Perch (<i>Morone americana</i>) Ovary Using Support Vector Machines
Compartment
proteomics enable broad characterization of target
tissues. We employed a simple fractionation method and filter-aided
sample preparation (FASP) to characterize the cytosolic and membrane
fractions of white perch ovary tissues by semiquantitative tandem
mass spectrometry using label-free quantitation based on normalized
spectral counts. FASP depletes both low-molecular-weight and high-molecular-weight
substances that could interfere with protein digestion and subsequent
peptide separation and detection. Membrane proteins are notoriously
difficult to characterize due to their amphipathic nature and association
with lipids. The simple fractionation we employed effectively revealed
an abundance of proteins from mitochondria and other membrane-bounded
organelles. We further demonstrate that support vector machines (SVMs)
offer categorical classification of proteomics data superior to that
of parametric statistical methods such as analysis of variance (ANOVA).
Specifically, SVMs were able to perfectly (100% correct) classify
samples as either membrane or cytosolic fraction during cross-validation
based on the expression of 242 proteins with the highest ANOVA <i>p</i>-values (i.e., those that were not significant for enrichment
in either fraction). The white perch ovary cytosolic and membrane
proteomes and transcriptome presented in this study can support future
investigations into oogenesis and early embryogenesis of white perch
and other members of the genus <i>Morone</i>
C/EBPα regulates CRL4<sup>Cdt2</sup>-mediated degradation of p21 in response to UVB-induced DNA damage to control the G<sub>1</sub>/S checkpoint
<div><p>The bZIP transcription factor, C/EBPα is highly inducible by UVB and other DNA damaging agents in keratinocytes. C/EBPα-deficient keratinocytes fail to undergo cell cycle arrest in G<sub>1</sub> in response to UVB-induced DNA damage and mice lacking epidermal C/EBPα are highly susceptible to UVB-induced skin cancer. The mechanism through which C/EBPα regulates the cell cycle checkpoint in response to DNA damage is unknown. Here we report untreated C/EBPα-deficient keratinocytes have normal levels of the cyclin-dependent kinase inhibitor, p21, however, UVB-treated C/EBPα-deficient keratinocytes fail to up-regulate nuclear p21 protein levels despite normal up-regulation of <i>Cdkn1a</i> mRNA levels. UVB-treated C/EBPα-deficient keratinocytes displayed a 4-fold decrease in nuclear p21 protein half-life due to the increased proteasomal degradation of p21 via the E3 ubiquitin ligase CRL4<sup>Cdt2</sup>. Cdt2 is the substrate recognition subunit of CRL4<sup>Cdt2</sup> and <i>Cdt2</i> mRNA and protein levels were up-regulated in UVB-treated C/EBPα-deficient keratinocytes. Knockdown of Cdt2 restored p21 protein levels in UVB-treated C/EBPα-deficient keratinocytes. Lastly, the failure to accumulate p21 in response to UVB in C/EBPα-deficient keratinocytes resulted in decreased p21 interactions with critical cell cycle regulatory proteins, increased CDK2 activity, and inappropriate entry into S-phase. These findings reveal C/EBPα regulates G<sub>1</sub>/S cell cycle arrest in response to DNA damage via the control of CRL4<sup>Cdt2</sup> mediated degradation of p21.</p></div