High-performance peptide and disulfide mapping by direct injection of intact proteins using on-line coupled UV-liquid chromatography microdroplet mass spectrometry (UVLC-MMS).
Anal Chim Acta
; 1279: 341790, 2023 Oct 23.
Article
em En
| MEDLINE
| ID: mdl-37827684
ABSTRACT
Microdroplet mass spectrometry (MMS), achieving ultra-fast enzyme digestion in the ionization source, holds great promises for innovating protein analysis. Here, in-depth protein characterization is demonstrated by direct injection of intact protein mixtures via on-line coupling MMS with capillary C4 liquid chromatography (LC) containing UV windows (UVLC-MMS) through an enzyme introduction tee. We showed complete sets of peptides of individual proteins (hemoglobin, bovine serum albumin, and ribonuclease A) in a mixture could be obtained in one injection. Such full (100%) sequence coverage, however, could not be achieved by conventional nanoLC-MS method using bottom-up approach with single enzyme. Moreover, direct injection of a chaperone α-crystalline (α-Cry) complex yielded identification of post-translational modifications including novel sites and semi-quantitative characterization including 31 stoichiometry ratio of αA- and αB-Cry sub-units and â¼1.4 phosphorylation/subunit on S45 (novel site) and S122 (main site) of αA-Cry, â¼0.7 phosphorylation/subunit on S19 (main site) and S45 of αB-Cry, as well as 100% acetylation on both N-termini of each subunits by matching the mass and retention time of the intact and its digested peptides. Furthermore, trifluoroacetic acid was able to be used in the mobile phase with UVLC-MMS to improve the separation of differentially reduced intact species and detectability of the droplet-digested products. This allowed us to completely map four disulfide linkages of ribonuclease A based on collision-induced dissociation of disulfide clusters, some of which would otherwise not be detected, preventing scrambling or shuffling errors arising from lengthy bulk solution digestion by the bottom-up approach. Integration of UVLC and MMS greatly improves droplet digestion efficiency and MS detection, enabling highly efficient workflow for in-depth and accurate protein characterization.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Ribonuclease Pancreático
/
Dissulfetos
Idioma:
En
Revista:
Anal Chim Acta
Ano de publicação:
2023
Tipo de documento:
Article