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Background  Malaria control in Panama is problematic due to the high diversity of morphologically similar  Anopheles  mosquito species, which makes identification of vectors of human  Plasmodium  challenging. Strategies by Panamanian health authorities to bring malaria under control targeting  Anopheles  vectors could be ineffective if they tackle a misidentified species.    Methods  A rapid mass spectrometry identification procedure was developed to accurately and timely sort out field-collected Neotropical  Anopheles  mosquitoes into vector and non-vector species. Matrix-assisted laser desorption/ionization (MALDI) mass spectra of highly-abundant proteins were generated from laboratory-reared mosquitoes using different extraction protocols, body parts, and sexes to minimize the amount of material from specimen vouchers needed and optimize the protocol for taxonomic identification. Subsequently, the mass spectra of field-collected Neotropical  Anopheles  mosquito species were classified using a combination of custom-made unsupervised (i.e., Principal component analysis—PCA) and supervised (i.e., Linear discriminant analysis—LDA) classification algorithms.    Results  Regardless of the protocol used or the mosquito species and sex, the legs contained the least intra-specific variability with enough well-preserved proteins to differentiate among distinct biological species, consistent with previous literature. After minimizing the amount of material needed from the voucher, one leg was enough to produce reliable spectra between specimens. Further, both PCA and LDA were able to classify up to 12 mosquito species, from different subgenera and seven geographically spread localities across Panama using mass spectra from one leg pair. LDA demonstrated high discriminatory power and consistency, with validation and cross-validation positive identification rates above 93% at the species level.    Conclusion  The selected sample processing procedure can be used to identify field-collected  Anopheles  species, including vectors of  Plasmodium , in a short period of time, with a minimal amount of tissue and without the need of an expert mosquito taxonomist. This strategy to analyse protein spectra overcomes the drawbacks of working without a reference library to classify unknown samples. Finally, this MALDI approach can aid ongoing malaria eradication efforts in Panama and other countries with large number of mosquito’s species by improving vector surveillance in epidemic-prone sites such as indigenous  Comarcas .    Electronic supplementary material  The online version of this article (10.1186/s12936-019-2723-0) contains supplementary material, which is available to authorized users.

Application of matrix-assisted laser desorption/ionization mass spectrometry to identify species of Neotropical Anopheles vectors of malaria