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Description
  • Apidaecins represent an important group of antimicrobial peptides occurring in honey bee hemolymph, where they play an important role as key components of humoral immunity. The present study demonstrates the development of a highly sensitive assay for apidaecin 1 isoforms quantification in the hemolymph or body parts from honey bee individuals. The analytical protocol comprises apidaecins 1 purification and enrichment steps by weak cation-exchange chromatography (WCX) in laboratory-made WCX-Tip microcolumns combined with a desalting step on a reversed-phase sorbent (C8) carried in StageTips. Apidaecin-enriched fraction was analyzed by a reversed-phase based nanoliquid chromatography (C4) separation coupled with high-resolution mass spectrometry. The method performance was validated in its specificity, linearity (0-5 pmol), recovery (?45%), precision ({10% at 0.1 pmol), limit of detection (?50 fmol), limit of quantification (0.1 pmol) and sample stability. The method was successfully applied to analyze the content of apidaecin 1 isoforms in the following samples: hemolymph - 13.0 ng/mýL (95% confidence interval of 7.5-18.6 ng/mýL), thoraxes - 36.2 ng/unit (95% CI of 18.9-53.6 ng/unit) and heads - 12.9 ng/unit (95% CI of 9.1-16.7 ng/unit). Freshly emerged bees had apidaecin 1 isoforms levels below the limit of detection. Thus it was possible to use them as a competitive matrix for calibration standards to prevent losses of highly basic apidaecins. This new protocol for apidaecin 1 isoforms quantification represents a promising tool to study the role of apidaecins in honey bee immunity and can be considered as a proof-of-concept for the development of sensitive quantification methods for basic antimicrobial peptides in various organisms.
  • Apidaecins represent an important group of antimicrobial peptides occurring in honey bee hemolymph, where they play an important role as key components of humoral immunity. The present study demonstrates the development of a highly sensitive assay for apidaecin 1 isoforms quantification in the hemolymph or body parts from honey bee individuals. The analytical protocol comprises apidaecins 1 purification and enrichment steps by weak cation-exchange chromatography (WCX) in laboratory-made WCX-Tip microcolumns combined with a desalting step on a reversed-phase sorbent (C8) carried in StageTips. Apidaecin-enriched fraction was analyzed by a reversed-phase based nanoliquid chromatography (C4) separation coupled with high-resolution mass spectrometry. The method performance was validated in its specificity, linearity (0-5 pmol), recovery (?45%), precision ({10% at 0.1 pmol), limit of detection (?50 fmol), limit of quantification (0.1 pmol) and sample stability. The method was successfully applied to analyze the content of apidaecin 1 isoforms in the following samples: hemolymph - 13.0 ng/mýL (95% confidence interval of 7.5-18.6 ng/mýL), thoraxes - 36.2 ng/unit (95% CI of 18.9-53.6 ng/unit) and heads - 12.9 ng/unit (95% CI of 9.1-16.7 ng/unit). Freshly emerged bees had apidaecin 1 isoforms levels below the limit of detection. Thus it was possible to use them as a competitive matrix for calibration standards to prevent losses of highly basic apidaecins. This new protocol for apidaecin 1 isoforms quantification represents a promising tool to study the role of apidaecins in honey bee immunity and can be considered as a proof-of-concept for the development of sensitive quantification methods for basic antimicrobial peptides in various organisms. (en)
Title
  • A sensitive quantification of the peptide apidaecin 1 isoforms in single bee tissues using a weak cation exchange pre-separation and nanocapillary liquid chromatography coupled with mass spectrometry
  • A sensitive quantification of the peptide apidaecin 1 isoforms in single bee tissues using a weak cation exchange pre-separation and nanocapillary liquid chromatography coupled with mass spectrometry (en)
skos:prefLabel
  • A sensitive quantification of the peptide apidaecin 1 isoforms in single bee tissues using a weak cation exchange pre-separation and nanocapillary liquid chromatography coupled with mass spectrometry
  • A sensitive quantification of the peptide apidaecin 1 isoforms in single bee tissues using a weak cation exchange pre-separation and nanocapillary liquid chromatography coupled with mass spectrometry (en)
skos:notation
  • RIV/61989592:15310/14:33151657!RIV15-MSM-15310___
http://linked.open...avai/riv/aktivita
http://linked.open...avai/riv/aktivity
  • P(LO1204)
http://linked.open...iv/cisloPeriodika
  • DEC
http://linked.open...vai/riv/dodaniDat
http://linked.open...aciTvurceVysledku
http://linked.open.../riv/druhVysledku
http://linked.open...iv/duvernostUdaju
http://linked.open...titaPredkladatele
http://linked.open...dnocenehoVysledku
  • 1159
http://linked.open...ai/riv/idVysledku
  • RIV/61989592:15310/14:33151657
http://linked.open...riv/jazykVysledku
http://linked.open.../riv/klicovaSlova
  • Quantification; Mass spectrometry; Nanoflow liquid chromatography; Honey bee; Apidaecin; Antimicrobial peptide (en)
http://linked.open.../riv/klicoveSlovo
http://linked.open...odStatuVydavatele
  • NL - Nizozemsko
http://linked.open...ontrolniKodProRIV
  • [3669AA2159A1]
http://linked.open...i/riv/nazevZdroje
  • Journal of Chromatography A
http://linked.open...in/vavai/riv/obor
http://linked.open...ichTvurcuVysledku
http://linked.open...cetTvurcuVysledku
http://linked.open...vavai/riv/projekt
http://linked.open...UplatneniVysledku
http://linked.open...v/svazekPeriodika
  • 1374
http://linked.open...iv/tvurceVysledku
  • Petřivalský, Marek
  • Šebela, Marek
  • Lenobel, René
  • Danihlík, Jiří
http://linked.open...ain/vavai/riv/wos
  • 000347605000015
issn
  • 0021-9673
number of pages
http://bibframe.org/vocab/doi
  • 10.1016/j.chroma.2014.11.041
http://localhost/t...ganizacniJednotka
  • 15310
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