Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 2 de 2
Filtrar
Más filtros




Base de datos
Intervalo de año de publicación
1.
J Am Soc Mass Spectrom ; 34(8): 1753-1760, 2023 Aug 02.
Artículo en Inglés | MEDLINE | ID: mdl-37463113

RESUMEN

Electron capture dissociation (ECD) is now a well-established method for sequencing peptides and performing top-down analysis on proteins of less than 30 kDa, and there is growing interest in using this approach for studies of larger proteins and protein complexes. Although much progress on ECD has been made over the past few decades, establishing methods for obtaining informative spectra still poses a significant challenge. Here we describe how digital quadrupole (DigiQ) ion isolation can be used for the mass selection of single charge states of proteins and protein complexes prior to undergoing ECD and/or charge reduction. First, we demonstrate that the DigiQ can isolate single charge states of monomeric proteins such as ubiquitin (8.6 kDa) and charge states of large protein complexes such as pyruvate kinase (234 kDa) using a hybrid quadrupole-TOF-MS (Agilent extended m/z range 6545XT). Next, we demonstrate that fragment ions resulting from ECD can be utilized to provide information about the sequence and structure of the cytochrome c/heme complex and the ubiquitin monomer. Lastly, an especially interesting result for DigiQ isolation and electron capture (EC) was noted; namely, the 16+ charge state of the streptavidin/biotin complex reveals different electron capture patterns for the biotinylated proteoforms of streptavidin. This result is consistent with previous reports that apo streptavidin exists in multiple conformations and that biotin binding shifts the conformational dynamics of the complex (Quintyn, R. Chem. Biol. 2015, 22 (55), 583-592).


Asunto(s)
Biotina , Electrones , Estreptavidina , Proteínas/química , Ubiquitina/química
2.
J Am Chem Soc ; 142(16): 7245-7249, 2020 04 22.
Artículo en Inglés | MEDLINE | ID: mdl-32251579

RESUMEN

Labeling of biomolecules in live eukaryotic cells has been limited by low component stability and slow reaction rates. We show that genetically encoded tetrazine amino acids in proteins reach reaction rates of 8 × 104 M-1 s-1 with sTCO reagents, making them the fastest site-specific bioorthogonal labels in eukaryotic systems. We demonstrate that tetrazine amino acids are stable on proteins and are capable of quantitative labeling with sTCO reagents. The exceptionally high reaction rate of this ligation minimizes label concentration, allowing for substoichiometric in vivo eukaryotic protein labeling where the concentration of the label is less than the concentration of the protein. This approach offers unprecedented control over the composition and stability of the protein tag. We anticipate that this system will have a broad impact on labeling and imaging studies because it can be used where all generally orthogonal PylRS/tRNA pairs are employed.


Asunto(s)
Aminoácidos/metabolismo , Células Eucariotas/metabolismo
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA