8 research outputs found
Combining Amine Metabolomics and Quantitative Proteomics of Cancer Cells Using Derivatization with Isobaric Tags
Quantitative metabolomics and proteomics
technologies are powerful
approaches to explore cellular metabolic regulation. Unfortunately,
combining the two technologies typically requires different LC-MS
setups for sensitive measurement of metabolites and peptides. One
approach to enhance the analysis of certain classes of metabolites
is by derivatization with various types of tags to increase ionization
and chromatographic efficiency. We demonstrate here that derivatization
of amine metabolites with tandem mass tags (TMT), typically used in
multiplexed peptide quantitation, facilitates amino acid analysis
by standard nanoflow reversed-phase LC-MS setups used for proteomics.
We demonstrate that this approach offers the potential to perform
experiments at the MS1-level using duplex tags or at the MS2-level
using novel 10-plex reporter ion-containing isobaric tags for multiplexed
amine metabolite analysis. We also demonstrate absolute quantitative
measurements of amino acids conducted in parallel with multiplexed
quantitative proteomics, using similar LC-MS setups to explore cellular
amino acid regulation. We further show that the approach can also
be used to determine intracellular metabolic labeling of amino acids
from glucose carbons
Combining Amine Metabolomics and Quantitative Proteomics of Cancer Cells Using Derivatization with Isobaric Tags
Quantitative metabolomics and proteomics
technologies are powerful
approaches to explore cellular metabolic regulation. Unfortunately,
combining the two technologies typically requires different LC-MS
setups for sensitive measurement of metabolites and peptides. One
approach to enhance the analysis of certain classes of metabolites
is by derivatization with various types of tags to increase ionization
and chromatographic efficiency. We demonstrate here that derivatization
of amine metabolites with tandem mass tags (TMT), typically used in
multiplexed peptide quantitation, facilitates amino acid analysis
by standard nanoflow reversed-phase LC-MS setups used for proteomics.
We demonstrate that this approach offers the potential to perform
experiments at the MS1-level using duplex tags or at the MS2-level
using novel 10-plex reporter ion-containing isobaric tags for multiplexed
amine metabolite analysis. We also demonstrate absolute quantitative
measurements of amino acids conducted in parallel with multiplexed
quantitative proteomics, using similar LC-MS setups to explore cellular
amino acid regulation. We further show that the approach can also
be used to determine intracellular metabolic labeling of amino acids
from glucose carbons
Putative Hypoxia Response Element binding sites of metabolic regulatory enzymes.
<p>Putative Hypoxia Response Element binding sites of metabolic regulatory enzymes.</p
HIF1dPA and HIF2dPA are functional transcription factors.
<p>Gene expression of known HIF transcriptional targets was assessed by quantitative real time PCR. Fold change was calculated compared to the paired unrecombined cell line. (<b>A</b>) Gene expression of known joint targets <i>Egln3</i> and <i>Vegfa</i>. <i>Vhl</i> WT murine ES cells (endogenous HIFs lowly expressed) and <i>Vhl</i> null murine ES cells (endogenous HIFs highly expressed) show the increased expression of these targets when both HIF1 and HIF2 are expressed. Significant increase of <i>Egln3</i> expression in HIF1dPA+ and HIF2dPA+ cells, slight increase of <i>Vegfa</i> mRNA expression by HIF1dPA+, but significant increase of <i>Vegfa</i> by HIF2dPA+. (<b>B</b>) Expression of canonical HIF1 metabolic gene targets Lactate dehydrogenase (<i>Ldha1</i>), Pyruvate dehydrogenase kinase (<i>Pdk1</i>), and Phosphofructokinase (<i>Pfk1</i>). HIF1dPA+ cells have statistically significant increase in expression of <i>Ldha1</i>, <i>Pdk1</i> and <i>Pfk1</i>; HIF2dPA+ cells have decreased expression of the metabolic targets. (<b>C</b>) To assess the effect of HIF expression on metabolic gene expression, we analyzed gene expression by qRT-PCR of metabolic enzymes regulating entry into and progression of the TCA cycle; fold change of 4-OHT treated cells to paired unrecombined NEK cell line is shown. Both HIF1dPA+ and HIF2dPA+ cells had increased levels of Pyruvate carboxylase (<i>Pcx</i>) transcripts. HIF2dPA+ expressing cells showed increased levels of Glutamine synthetase (<i>Glul</i>) and decreased Glutaminase (<i>Gls</i>). Bars indicate average with the SEM. *pā¤0.05, **pā¤0.01, (ns) not significant.</p
Metabolic function of differentially expressed HIF1 and HIF2.
<p>(<b>A</b>) Glycolytic function of NEK HIFdPA cells was quantified by measuring real time proton excretion, extracellular acidification rate (ECAR), using the Seahorse system. HIF1dPA+ cells incubated in complete media showed increased basal levels of glycolysis compared to HIF1dPA cells. Following treatment with a glycolytic inhibitor, 10 mM 2-deoxy-d-glucose (2-DG), levels are decreased, but a significant difference remains. (<b>B</b>) Basal and post treatment levels of glycolytic activity in HIF2dPA and HIF2dPA+ cells show no significant difference. (<b>C</b>) Glycolytic flux was measured in a separate cell line, human embryonic kidney cells (HEK 293) stably expressing either HIF1dPA or HIF2dPA. HEK HIF1dPA+ cells showed significantly increased levels of glycolytic flux. Results show the average of two independent runs. (<b>D</b>) Real time oxygen consumption rate (OCR) measurement in HIFdPA cells using the Seahorse system. HIF1dPA+ and (<b>E</b>) HIF2dPA+ cells have significantly higher levels of basal OCR over the paired unrecombined cell line. Following treatment with 750nM rotenone, an oxidative phosphorylation (OxPhos) inhibitor, OCR levels are greatly reduced. (<b>F</b>) Fold change in basal OCR levels show a significantly greater fold change in HIF2dPA+ over HIF2dPA, compared to the paired HIF1dPA+/HIF1dPA cells. Graphs indicate average with the SEM. **pā¤0.01, ā§pā¤0.001, (ns) not significant.</p
HIF1 regulation of glucose utilization dependent on Pdk1 expression.
<p>(<b>A</b>) qRT-PCR for the targeted transcript confirmed knockdown of <i>Pdk1</i> in HIF1dPA+ cells was ā¼90% by shRNA. (<b>B</b>) HIF1dPA+ cells expressing shPdk1 showed decreased levels of ECAR following administration of 10 mM Glucose, even with the addition of 5 uM Oligomycin A. ECAR levels decreased upon 20 mM 2-DG treatment. (<b>C</b>) An increase in OCR levels in HIF1dPA+ shPdk1 cells was observed following Glucose treatment. OCR levels fell with the addition of 5 uM Oligomycin A and following 2 uM Antimycin A and 2 uM Rotenone co-treatment. Lines indicate average with the SEM. ā§pā¤0.001.</p
Conditional stable expression of Hypoxia Inducible Factor in a murine derived primary cell culture system.
<p>(<b>A</b>) The previously described transgenic mouse model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098705#pone.0098705-Kim1" target="_blank">[25]</a> contains a HA-tagged human HIF1 or HIF2 insertion with double proline to alanine (dPA) substitutions in the endogenous Rosa 26 locus with inducible loxP-stop-loxP sites. These mice were crossed with a mouse containing a tamoxifen-inducible cre estrogen receptor ligand binding domain under control of a human ubiquitin C promoter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098705#pone.0098705-Ruzankina1" target="_blank">[29]</a>. Upon recombination, stable expression of HIF1dPA and HIF2dPA is induced. Recombination is denoted as ādPA+ā throughout the paper. (<b>B</b>) Primary neonatal epithelial kidney cells were derived using both kidneys from a mouse 0ā3 days post birth. The tissue was dissociated in collagenase IV and cultured in serum free (SF) media supplemented with EGF. NEK cells were recombined using 4-hydroxy tamoxifen (4-OHT). (<b>C</b>) Recombination and loss of the Rosa26-Lox-Stop-Lox alleles was confirmed in two newly isolated and recombined NEK HIF1dPA cells and (<b>D</b>) HIF2dPA cells by PCR primers specific to the R26LSL allele followed by gel electrophoresis. (<b>E</b>) qRT-PCR for the human <i>HIF1Ī±</i> transcript was significantly increased in HIF1dPA+ cells. (<b>F</b>) Similarly, gene expression levels for the human <i>HIF2Ī±</i> transcript was increased in HIF2dPA+ cells. (<b>G</b>) Immunoblot of nuclear extracts showed an increase in HIF1Ī± expression following 4-OHT treatment and quantification of immunoblot for HIF1 protein expression displayed increased expression following recombination. Expression was normalized to an internal Lamin B control. (<b>H</b>) Immunocytochemistry for HIF2 protein expression in HIF2dPA+ cells compared to unrecombined HIF2dPA cells was used to quantify levels of staining across several areas. Corrected Total Cell Staining indicated significantly higher HIF2 protein levels in HIF2dPA+ cells. Bars indicate average with the SEM. **pā¤0.01, ā§pā¤0.001.</p
HIF2 glutamine utilization is deterred by Glul expression.
<p>(<b>A</b>) <i>Glul</i> knockdown by siRNA in HIF2dPA+ cells was ā¼15% compared to untreated cells as confirmed by qRT-PCR. (<b>B</b>) Knockdown of <i>Glul</i> in HIF2dPA+ cells in 2 mM L-glutamine-supplemented media showed an increase in OCR over HIF2dPA+ cells. OCR levels decreased following 2 uM Antimycin A and 2 uM Rotenone co-treatment. Lines indicate average with the SEM. *pā¤0.05.</p