Increasing the Throughput and Reproducibility of Activity-Based Proteome Profiling Studies with Hyperplexing and Intelligent Data Acquisition.
J Proteome Res
; 23(8): 2934-2947, 2024 Aug 02.
Article
em En
| MEDLINE
| ID: mdl-38251652
ABSTRACT
Intelligent data acquisition (IDA) strategies, such as a real-time database search (RTS), have improved the depth of proteome coverage for experiments that utilize isobaric labels and gas phase purification techniques (i.e., SPS-MS3). In this work, we introduce inSeqAPI, an instrument application programing interface (iAPI) program that enables construction of novel data acquisition algorithms. First, we analyze biotinylated cysteine peptides from ABPP experiments to demonstrate that a real-time search method within inSeqAPI performs similarly to an equivalent vendor method. Then, we describe PairQuant, a method within inSeqAPI designed for the hyperplexing approach that utilizes protein-level isotopic labeling and peptide-level TMT labeling. PairQuant allows for TMT analysis of 36 conditions in a single sample and achieves â¼98% coverage of both peptide pair partners in a hyperplexed experiment as well as a 40% improvement in the number of quantified cysteine sites compared with non-RTS acquisition. We applied this method in the ABPP study of ligandable cysteine sites in the nucleus leading to an identification of additional druggable sites on protein- and DNA-interaction domains of transcription regulators and on nuclear ubiquitin ligases.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Temas:
Geral
Base de dados:
MEDLINE
Assunto principal:
Proteoma
/
Cisteína
/
Proteômica
Limite:
Humans
Idioma:
En
Revista:
J Proteome Res
Assunto da revista:
BIOQUIMICA
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
Estados Unidos