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Daily rhythms in antennal protein and olfactory sensitivity in the malaria mosquito Anopheles gambiae.

Rund SS, Bonar NA, Champion MM, Ghazi JP, Houk CM, Leming MT, Syed Z, Duffield GE - Sci Rep (2013)

Bottom Line: Further, electrophysiological investigations demonstrate time-of-day specific differences in olfactory sensitivity of antennae to major host-derived odorants.The pre-dusk/dusk peaks in OBPs and takeout gene expression correspond with peak protein abundance at night, and in turn coincide with the time of increased olfactory sensitivity to odorants requiring OBPs and times of increased blood-feeding behavior.This suggests an important role for OBPs in modulating temporal changes in odorant sensitivity, enabling the olfactory system to coordinate with the circadian niche of An. gambiae.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biological Sciences and Eck Institute for Global Health, Galvin Life Science Center, University of Notre Dame, Notre Dame, IN 46556 [2].

ABSTRACT
We recently characterized 24-hr daily rhythmic patterns of gene expression in Anopheles gambiae mosquitoes. These include numerous odorant binding proteins (OBPs), soluble odorant carrying proteins enriched in olfactory organs. Here we demonstrate that multiple rhythmically expressed genes including OBPs and takeout proteins, involved in regulating blood feeding behavior, have corresponding rhythmic protein levels as measured by quantitative proteomics. This includes AgamOBP1, previously shown as important to An. gambiae odorant sensing. Further, electrophysiological investigations demonstrate time-of-day specific differences in olfactory sensitivity of antennae to major host-derived odorants. The pre-dusk/dusk peaks in OBPs and takeout gene expression correspond with peak protein abundance at night, and in turn coincide with the time of increased olfactory sensitivity to odorants requiring OBPs and times of increased blood-feeding behavior. This suggests an important role for OBPs in modulating temporal changes in odorant sensitivity, enabling the olfactory system to coordinate with the circadian niche of An. gambiae.

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Related in: MedlinePlus

Targeted quantitative proteomics method.Our experimental method is described. Briefly, adult female heads, antennae or THAs are pulverized, proteins extracted under denaturing conditions, clarified by centrifugation, digested with trypsin, quenched and desalted. Targeted quantitative proteomics was performed using multiple reaction monitoring (MRM) on a triple quadrupole (QTrap 5500). MRM data were acquired in-triplicate or quadruplicate from samples collected every 4 hr for 24 hr. Peak areas for each peptide corresponding to the protein were integrated and the data normalized. AUC response (area under curve) was graphed for each protein vs. time, shown here after median transformation of the data. Note every protein had 2–3 underlying peptides used for quantification/qualification. An example of the three peptides used to quantify protein levels of OBP1 over 24 hr is shown. Targeted (quantitative MS/MS) proteomics is advantageous as it is highly specific, sensitive and linear with respect to quantity. In this work we demonstrate relative protein abundance rather than absolute quantities. Horizontal bars indicate day/night (white/black). See Fig. S1 for a detailed methodology and example results.
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f1: Targeted quantitative proteomics method.Our experimental method is described. Briefly, adult female heads, antennae or THAs are pulverized, proteins extracted under denaturing conditions, clarified by centrifugation, digested with trypsin, quenched and desalted. Targeted quantitative proteomics was performed using multiple reaction monitoring (MRM) on a triple quadrupole (QTrap 5500). MRM data were acquired in-triplicate or quadruplicate from samples collected every 4 hr for 24 hr. Peak areas for each peptide corresponding to the protein were integrated and the data normalized. AUC response (area under curve) was graphed for each protein vs. time, shown here after median transformation of the data. Note every protein had 2–3 underlying peptides used for quantification/qualification. An example of the three peptides used to quantify protein levels of OBP1 over 24 hr is shown. Targeted (quantitative MS/MS) proteomics is advantageous as it is highly specific, sensitive and linear with respect to quantity. In this work we demonstrate relative protein abundance rather than absolute quantities. Horizontal bars indicate day/night (white/black). See Fig. S1 for a detailed methodology and example results.

Mentions: We focused our proteomics study on rhythmic olfaction genes identified in our An. gambiae transcriptome study78 in olfactory tissues. We hypothesized that rhythmic levels of these transcripts will be translated into quantifiable protein rhythms, and lead to time-of-day specific changes in olfactory sensitivity and behavior. In order to assess the quality of our protein extraction, determine which olfactory proteins were likely ‘visible' to a proteome, and generate empirical MS/MS spectra to be used for targeting, we performed data-dependent bottom-up proteomics on tryptic digests of mosquito heads, THAs and antennae (Fig. 1 and Fig. S1a). Single-dimensional nano-UHPLC MS/MS was chosen as it is identical to the level of separation analyzed by the targeted proteomics approach, thus allowing direct comparisons for verification and validation of targets. Our combined injections for bottom-up proteomics yielded a total of 1022 protein and 6157 peptide identifications at a 1% false discovery rate (Supplementary Dataset 1).


Daily rhythms in antennal protein and olfactory sensitivity in the malaria mosquito Anopheles gambiae.

Rund SS, Bonar NA, Champion MM, Ghazi JP, Houk CM, Leming MT, Syed Z, Duffield GE - Sci Rep (2013)

Targeted quantitative proteomics method.Our experimental method is described. Briefly, adult female heads, antennae or THAs are pulverized, proteins extracted under denaturing conditions, clarified by centrifugation, digested with trypsin, quenched and desalted. Targeted quantitative proteomics was performed using multiple reaction monitoring (MRM) on a triple quadrupole (QTrap 5500). MRM data were acquired in-triplicate or quadruplicate from samples collected every 4 hr for 24 hr. Peak areas for each peptide corresponding to the protein were integrated and the data normalized. AUC response (area under curve) was graphed for each protein vs. time, shown here after median transformation of the data. Note every protein had 2–3 underlying peptides used for quantification/qualification. An example of the three peptides used to quantify protein levels of OBP1 over 24 hr is shown. Targeted (quantitative MS/MS) proteomics is advantageous as it is highly specific, sensitive and linear with respect to quantity. In this work we demonstrate relative protein abundance rather than absolute quantities. Horizontal bars indicate day/night (white/black). See Fig. S1 for a detailed methodology and example results.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3756343&req=5

f1: Targeted quantitative proteomics method.Our experimental method is described. Briefly, adult female heads, antennae or THAs are pulverized, proteins extracted under denaturing conditions, clarified by centrifugation, digested with trypsin, quenched and desalted. Targeted quantitative proteomics was performed using multiple reaction monitoring (MRM) on a triple quadrupole (QTrap 5500). MRM data were acquired in-triplicate or quadruplicate from samples collected every 4 hr for 24 hr. Peak areas for each peptide corresponding to the protein were integrated and the data normalized. AUC response (area under curve) was graphed for each protein vs. time, shown here after median transformation of the data. Note every protein had 2–3 underlying peptides used for quantification/qualification. An example of the three peptides used to quantify protein levels of OBP1 over 24 hr is shown. Targeted (quantitative MS/MS) proteomics is advantageous as it is highly specific, sensitive and linear with respect to quantity. In this work we demonstrate relative protein abundance rather than absolute quantities. Horizontal bars indicate day/night (white/black). See Fig. S1 for a detailed methodology and example results.
Mentions: We focused our proteomics study on rhythmic olfaction genes identified in our An. gambiae transcriptome study78 in olfactory tissues. We hypothesized that rhythmic levels of these transcripts will be translated into quantifiable protein rhythms, and lead to time-of-day specific changes in olfactory sensitivity and behavior. In order to assess the quality of our protein extraction, determine which olfactory proteins were likely ‘visible' to a proteome, and generate empirical MS/MS spectra to be used for targeting, we performed data-dependent bottom-up proteomics on tryptic digests of mosquito heads, THAs and antennae (Fig. 1 and Fig. S1a). Single-dimensional nano-UHPLC MS/MS was chosen as it is identical to the level of separation analyzed by the targeted proteomics approach, thus allowing direct comparisons for verification and validation of targets. Our combined injections for bottom-up proteomics yielded a total of 1022 protein and 6157 peptide identifications at a 1% false discovery rate (Supplementary Dataset 1).

Bottom Line: Further, electrophysiological investigations demonstrate time-of-day specific differences in olfactory sensitivity of antennae to major host-derived odorants.The pre-dusk/dusk peaks in OBPs and takeout gene expression correspond with peak protein abundance at night, and in turn coincide with the time of increased olfactory sensitivity to odorants requiring OBPs and times of increased blood-feeding behavior.This suggests an important role for OBPs in modulating temporal changes in odorant sensitivity, enabling the olfactory system to coordinate with the circadian niche of An. gambiae.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biological Sciences and Eck Institute for Global Health, Galvin Life Science Center, University of Notre Dame, Notre Dame, IN 46556 [2].

ABSTRACT
We recently characterized 24-hr daily rhythmic patterns of gene expression in Anopheles gambiae mosquitoes. These include numerous odorant binding proteins (OBPs), soluble odorant carrying proteins enriched in olfactory organs. Here we demonstrate that multiple rhythmically expressed genes including OBPs and takeout proteins, involved in regulating blood feeding behavior, have corresponding rhythmic protein levels as measured by quantitative proteomics. This includes AgamOBP1, previously shown as important to An. gambiae odorant sensing. Further, electrophysiological investigations demonstrate time-of-day specific differences in olfactory sensitivity of antennae to major host-derived odorants. The pre-dusk/dusk peaks in OBPs and takeout gene expression correspond with peak protein abundance at night, and in turn coincide with the time of increased olfactory sensitivity to odorants requiring OBPs and times of increased blood-feeding behavior. This suggests an important role for OBPs in modulating temporal changes in odorant sensitivity, enabling the olfactory system to coordinate with the circadian niche of An. gambiae.

Show MeSH
Related in: MedlinePlus