Mapping of polyglutamylation in tubulins using nanoLC-ESI-MS/MS.
Anal Biochem
; 612: 113761, 2021 01 01.
Article
en En
| MEDLINE
| ID: mdl-32502490
ABSTRACT
Tubulin polyglutamylation is a polymeric modification that extends from the carboxyl-terminus of tubulins. Molecular description of amino acids and their branching polyglutamyls is a hallmark of tubulin in microtubules. There are different chemical approaches for detecting these polymeric structures, mostly reported prior to development of nESI peptide analysis. Here we demonstrate a novel and simple approach to detect shared regions of amino acid ions from tubulin polyglutamylated peptides in nanoLC-MS/MS. This involves two parallel in gel digestions with trypsin and subtilisin followed by mapping of di- and triglutamyl modifications of α- and ß-tubulins using a routine proteomics assay. We present three levels of information i) identification of proteomics MS/MS data, ii) description of internal fragment ion series common across digests, and iii) extracted ion chromatograms mapped relative to retention time standards for confirmation of relative hydrophobicity values. Our nanoLC assay positive ion ESI detects up to 3 conjugated glutamates in tubulins. We implemented an analytical column only bottom up approach that characterizes molecular features of polyglutamylated tubulins.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Ácido Poliglutámico
/
Tubulina (Proteína)
/
Cromatografía Líquida de Alta Presión
/
Espectrometría de Masa por Ionización de Electrospray
/
Nanotecnología
/
Espectrometría de Masas en Tándem
Límite:
Animals
Idioma:
En
Revista:
Anal Biochem
Año:
2021
Tipo del documento:
Article
País de afiliación:
Estados Unidos