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Identification of cryptic Anopheles mosquito species by molecular protein profiling.

Müller P, Pflüger V, Wittwer M, Ziegler D, Chandre F, Simard F, Lengeler C - PLoS ONE (2013)

Bottom Line: The approach also classifies specimens from different laboratory colonies; hence proving also very promising for its use in colony authentication as part of quality assurance in laboratory studies.While being exceptionally accurate and robust, MALDI-TOF MS has several advantages over other typing methods, including simple sample preparation and short processing time.As the method does not require DNA sequence information, data can also be reviewed at any later stage for diagnostic or functional patterns without the need for re-designing and re-processing biological material.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Services and Diagnostic, Swiss Tropical and Public Health Institute, Basel, Switzerland. pie.mueller@unibas.ch

ABSTRACT
Vector control is the mainstay of malaria control programmes. Successful vector control profoundly relies on accurate information on the target mosquito populations in order to choose the most appropriate intervention for a given mosquito species and to monitor its impact. An impediment to identify mosquito species is the existence of morphologically identical sibling species that play different roles in the transmission of pathogens and parasites. Currently PCR diagnostics are used to distinguish between sibling species. PCR based methods are, however, expensive, time-consuming and their development requires a priori DNA sequence information. Here, we evaluated an inexpensive molecular proteomics approach for Anopheles species: matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). MALDI-TOF MS is a well developed protein profiling tool for the identification of microorganisms but so far has received little attention as a diagnostic tool in entomology. We measured MS spectra from specimens of 32 laboratory colonies and 2 field populations representing 12 Anopheles species including the A. gambiae species complex. An important step in the study was the advancement and implementation of a bioinformatics approach improving the resolution over previously applied cluster analysis. Borrowing tools for linear discriminant analysis from genomics, MALDI-TOF MS accurately identified taxonomically closely related mosquito species, including the separation between the M and S molecular forms of A. gambiae sensu stricto. The approach also classifies specimens from different laboratory colonies; hence proving also very promising for its use in colony authentication as part of quality assurance in laboratory studies. While being exceptionally accurate and robust, MALDI-TOF MS has several advantages over other typing methods, including simple sample preparation and short processing time. As the method does not require DNA sequence information, data can also be reviewed at any later stage for diagnostic or functional patterns without the need for re-designing and re-processing biological material.

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Related in: MedlinePlus

Examples of MALDI-TOF MS spectra for Anopheles gambiae sensu stricto and A. arabiensis.Examples of representative MALDI-TOF MS spectra measured from 3 A. arabiensis (blue) and 3 A. gambiae s.s. (red) colonies. The spectra were taken from crude suspensions of heads and thoraces in SA solution. The vertical, dashed lines indicate peaks that are characteristic (but not exclusive) for one or the other species. The left panels show the whole spectra between 2 and 14 kDa, while the right panels zoom into two peaks. The two peaks are separated by only a few Daltons. While the left peak is more common in A. arabiensis, the right peak is more common in A. gambiae s.s. In this representation the peak intensities were normalised against the highest intensity measured in each spectrum.
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pone-0057486-g001: Examples of MALDI-TOF MS spectra for Anopheles gambiae sensu stricto and A. arabiensis.Examples of representative MALDI-TOF MS spectra measured from 3 A. arabiensis (blue) and 3 A. gambiae s.s. (red) colonies. The spectra were taken from crude suspensions of heads and thoraces in SA solution. The vertical, dashed lines indicate peaks that are characteristic (but not exclusive) for one or the other species. The left panels show the whole spectra between 2 and 14 kDa, while the right panels zoom into two peaks. The two peaks are separated by only a few Daltons. While the left peak is more common in A. arabiensis, the right peak is more common in A. gambiae s.s. In this representation the peak intensities were normalised against the highest intensity measured in each spectrum.

Mentions: Raw spectra were acquired with an Axima™ Confidence MALDI-TOF mass spectrometer (Shimadzu-Biotech Corp., Kyoto, Japan) in the linear, positive mode over a m/z range of 2–30 kDa for a total of 1,000 laser shots per spotted sample. The machine’s parameters were chosen by setting the ion source at 20 kV and the extraction delay time at 200 ns. The spectra obtained by the spectrometer were then loaded into Launchpad™ 2.8 software (Shimadzu-Biotech Corp., Kyoto, Japan) to create a peak list for each sample spotted on the plate (i.e. four lists for each mosquito specimen). The peak lists with size (m/z value) and intensity values (arbitrary units) were then saved as ASCII text files for further data analysis. The peak lists and additional information are provided in Dataset S1 and Table S1. The software parameters were set to the following values: parent peak cleanup = “advanced scenario”; peak width = “80 chans”; smoothing filter width = “50 chans”; baseline filter width = “500 chans”; peak detection method = “threshold apex”. For the “threshold apex” peak detection, the threshold type was set as dynamic and the threshold offset to 0.02 mV with a response factor of 1.2. For external calibration purposes the software also used the spectra of the Escherichia coli DH5 alpha strain which was spotted alongside the mosquito samples on each 48-well plate. In addition, internal reference peaks that appear to be highly conserved in mosquitoes (i.e. m/z 2670.5, 4554.4, 5115.2, 5217.4, 5328.7, 5345.6, 5371.2, 5551.8, 5591.3, 6560.1, 7683.7, 8560.0, 9109.6, 9234.2, 9453.3, 10255.6 and 16724.8, see also results Figure 1) were used for internal calibration within a range of ±700 ppm.


Identification of cryptic Anopheles mosquito species by molecular protein profiling.

Müller P, Pflüger V, Wittwer M, Ziegler D, Chandre F, Simard F, Lengeler C - PLoS ONE (2013)

Examples of MALDI-TOF MS spectra for Anopheles gambiae sensu stricto and A. arabiensis.Examples of representative MALDI-TOF MS spectra measured from 3 A. arabiensis (blue) and 3 A. gambiae s.s. (red) colonies. The spectra were taken from crude suspensions of heads and thoraces in SA solution. The vertical, dashed lines indicate peaks that are characteristic (but not exclusive) for one or the other species. The left panels show the whole spectra between 2 and 14 kDa, while the right panels zoom into two peaks. The two peaks are separated by only a few Daltons. While the left peak is more common in A. arabiensis, the right peak is more common in A. gambiae s.s. In this representation the peak intensities were normalised against the highest intensity measured in each spectrum.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585343&req=5

pone-0057486-g001: Examples of MALDI-TOF MS spectra for Anopheles gambiae sensu stricto and A. arabiensis.Examples of representative MALDI-TOF MS spectra measured from 3 A. arabiensis (blue) and 3 A. gambiae s.s. (red) colonies. The spectra were taken from crude suspensions of heads and thoraces in SA solution. The vertical, dashed lines indicate peaks that are characteristic (but not exclusive) for one or the other species. The left panels show the whole spectra between 2 and 14 kDa, while the right panels zoom into two peaks. The two peaks are separated by only a few Daltons. While the left peak is more common in A. arabiensis, the right peak is more common in A. gambiae s.s. In this representation the peak intensities were normalised against the highest intensity measured in each spectrum.
Mentions: Raw spectra were acquired with an Axima™ Confidence MALDI-TOF mass spectrometer (Shimadzu-Biotech Corp., Kyoto, Japan) in the linear, positive mode over a m/z range of 2–30 kDa for a total of 1,000 laser shots per spotted sample. The machine’s parameters were chosen by setting the ion source at 20 kV and the extraction delay time at 200 ns. The spectra obtained by the spectrometer were then loaded into Launchpad™ 2.8 software (Shimadzu-Biotech Corp., Kyoto, Japan) to create a peak list for each sample spotted on the plate (i.e. four lists for each mosquito specimen). The peak lists with size (m/z value) and intensity values (arbitrary units) were then saved as ASCII text files for further data analysis. The peak lists and additional information are provided in Dataset S1 and Table S1. The software parameters were set to the following values: parent peak cleanup = “advanced scenario”; peak width = “80 chans”; smoothing filter width = “50 chans”; baseline filter width = “500 chans”; peak detection method = “threshold apex”. For the “threshold apex” peak detection, the threshold type was set as dynamic and the threshold offset to 0.02 mV with a response factor of 1.2. For external calibration purposes the software also used the spectra of the Escherichia coli DH5 alpha strain which was spotted alongside the mosquito samples on each 48-well plate. In addition, internal reference peaks that appear to be highly conserved in mosquitoes (i.e. m/z 2670.5, 4554.4, 5115.2, 5217.4, 5328.7, 5345.6, 5371.2, 5551.8, 5591.3, 6560.1, 7683.7, 8560.0, 9109.6, 9234.2, 9453.3, 10255.6 and 16724.8, see also results Figure 1) were used for internal calibration within a range of ±700 ppm.

Bottom Line: The approach also classifies specimens from different laboratory colonies; hence proving also very promising for its use in colony authentication as part of quality assurance in laboratory studies.While being exceptionally accurate and robust, MALDI-TOF MS has several advantages over other typing methods, including simple sample preparation and short processing time.As the method does not require DNA sequence information, data can also be reviewed at any later stage for diagnostic or functional patterns without the need for re-designing and re-processing biological material.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Services and Diagnostic, Swiss Tropical and Public Health Institute, Basel, Switzerland. pie.mueller@unibas.ch

ABSTRACT
Vector control is the mainstay of malaria control programmes. Successful vector control profoundly relies on accurate information on the target mosquito populations in order to choose the most appropriate intervention for a given mosquito species and to monitor its impact. An impediment to identify mosquito species is the existence of morphologically identical sibling species that play different roles in the transmission of pathogens and parasites. Currently PCR diagnostics are used to distinguish between sibling species. PCR based methods are, however, expensive, time-consuming and their development requires a priori DNA sequence information. Here, we evaluated an inexpensive molecular proteomics approach for Anopheles species: matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). MALDI-TOF MS is a well developed protein profiling tool for the identification of microorganisms but so far has received little attention as a diagnostic tool in entomology. We measured MS spectra from specimens of 32 laboratory colonies and 2 field populations representing 12 Anopheles species including the A. gambiae species complex. An important step in the study was the advancement and implementation of a bioinformatics approach improving the resolution over previously applied cluster analysis. Borrowing tools for linear discriminant analysis from genomics, MALDI-TOF MS accurately identified taxonomically closely related mosquito species, including the separation between the M and S molecular forms of A. gambiae sensu stricto. The approach also classifies specimens from different laboratory colonies; hence proving also very promising for its use in colony authentication as part of quality assurance in laboratory studies. While being exceptionally accurate and robust, MALDI-TOF MS has several advantages over other typing methods, including simple sample preparation and short processing time. As the method does not require DNA sequence information, data can also be reviewed at any later stage for diagnostic or functional patterns without the need for re-designing and re-processing biological material.

Show MeSH
Related in: MedlinePlus