Chapter 5
Shotgun Proteomics for L3 and L4 Anisakis simplex
Development Stages
Robert Stryin ´ ski, Jesu ´ s Mateos, Elz ˙ bieta Łopien ´ ska-Biernat,
and Mo ´ nica Carrera
Abstract
Anisakis simplex s.s. is a parasitic nematode that causes anisakiasis in humans. L3 stage larvae, which are
present in many fish species and cephalopods all over the globe, might be consumed and develop
occasionally into the L4 stage but cannot reproduce. Anisakiasis is an emerging health problem and
economic concern. In recent years, proteomic methods have gained greater acceptance among scientists
involved in parasitology and food science. According to that, here, we present tandem mass tag (TMT)based shotgun proteomics to define differences in proteomic composition between L3 and L4 development
stages of A. simplex s.s.
Key words Shotgun proteomics, LC–MS/MS, Tandem mass tag (TMT), Anisakis simplex,
Anisakiasis
1 Introduction
The tandem mass tag (TMT)-based shotgun proteomics enables
multiplex relative quantification and identification of proteins by
liquid chromatography coupled with tandem mass spectrometry
(LC–MS/MS). Each mass-tagging reagent has the same nominal
mass (isobaric) and chemical structure composed of an aminereactive NHS-ester group, a spacer arm, a mass reporter, and a
mass normalizer (Fig. 1). The chemical structures of all the tags
are identical but each contains isotopes substituted at various positions, such that the mass reporter and mass normalization regions
have different molecular masses in each tag. Upon fragmentation in
MS/MS mode, sequence information is obtained from the fragmentation of the peptide backbone and quantification data are
simultaneously obtained from the fragmentation of the tags, giving
rise to mass reporter ions [1–3].
Mo ´ nica Carrera and Jesu ´ s Mateos (eds.), Shotgun Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 2259,
https://doi.org/10.1007/978-1-0716-1178-4_5, © Springer Science+Business Media, LLC, part of Springer Nature 2021
59
Shotgun Proteomics for L3 and L4 Anisakis simplex
Development Stages
Robert Stryin ´ ski, Jesu ´ s Mateos, Elz ˙ bieta Łopien ´ ska-Biernat,
and Mo ´ nica Carrera
Abstract
Anisakis simplex s.s. is a parasitic nematode that causes anisakiasis in humans. L3 stage larvae, which are
present in many fish species and cephalopods all over the globe, might be consumed and develop
occasionally into the L4 stage but cannot reproduce. Anisakiasis is an emerging health problem and
economic concern. In recent years, proteomic methods have gained greater acceptance among scientists
involved in parasitology and food science. According to that, here, we present tandem mass tag (TMT)based shotgun proteomics to define differences in proteomic composition between L3 and L4 development
stages of A. simplex s.s.
Key words Shotgun proteomics, LC–MS/MS, Tandem mass tag (TMT), Anisakis simplex,
Anisakiasis
1 Introduction
The tandem mass tag (TMT)-based shotgun proteomics enables
multiplex relative quantification and identification of proteins by
liquid chromatography coupled with tandem mass spectrometry
(LC–MS/MS). Each mass-tagging reagent has the same nominal
mass (isobaric) and chemical structure composed of an aminereactive NHS-ester group, a spacer arm, a mass reporter, and a
mass normalizer (Fig. 1). The chemical structures of all the tags
are identical but each contains isotopes substituted at various positions, such that the mass reporter and mass normalization regions
have different molecular masses in each tag. Upon fragmentation in
MS/MS mode, sequence information is obtained from the fragmentation of the peptide backbone and quantification data are
simultaneously obtained from the fragmentation of the tags, giving
rise to mass reporter ions [1–3].
Mo ´ nica Carrera and Jesu ´ s Mateos (eds.), Shotgun Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 2259,
https://doi.org/10.1007/978-1-0716-1178-4_5, © Springer Science+Business Media, LLC, part of Springer Nature 2021
59