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A Rapid One-Pot Workflow for Sensitive Microscale Phosphoproteomics.
Muneer, Gul; Chen, Ciao-Syuan; Lee, Tzu-Tsung; Chen, Bo-Yu; Chen, Yu-Ju.
Afiliação
  • Muneer G; Institute of Chemistry, Academia Sinica, Taipei 11529, Taiwan.
  • Chen CS; Institute of Biochemical Sciences, National Taiwan University, Taipei 10617, Taiwan.
  • Lee TT; Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Taipei 11529, Taiwan.
  • Chen BY; Institute of Chemistry, Academia Sinica, Taipei 11529, Taiwan.
  • Chen YJ; Institute of Chemistry, Academia Sinica, Taipei 11529, Taiwan.
J Proteome Res ; 23(8): 3294-3309, 2024 Aug 02.
Article em En | MEDLINE | ID: mdl-39038167
ABSTRACT
Compared to advancements in single-cell proteomics, phosphoproteomics sensitivity has lagged behind due to low abundance, complex sample preparation, and substantial sample input requirements. We present a simple and rapid one-pot phosphoproteomics workflow (SOP-Phos) integrated with data-independent acquisition mass spectrometry (DIA-MS) for microscale phosphoproteomic analysis. SOP-Phos adapts sodium deoxycholate based one-step lysis, reduction/alkylation, direct trypsinization, and phosphopeptide enrichment by TiO2 beads in a single-tube format. By reducing surface adsorptive losses via utilizing n-dodecyl ß-d-maltoside precoated tubes and shortening the digestion time, SOP-Phos is completed within 3-4 h with a 1.4-fold higher identification coverage. SOP-Phos coupled with DIA demonstrated >90% specificity, enhanced sensitivity, lower missing values (<1%), and improved reproducibility (8%-10% CV). With a sample size-comparable spectral library, SOP-Phos-DIA identified 33,787 ± 670 to 22,070 ± 861 phosphopeptides from 5 to 0.5 µg cell lysate and 30,433 ± 284 to 6,548 ± 21 phosphopeptides from 50,000 to 2,500 cells. Such sensitivity enabled mapping key lung cancer signaling sites, such as EGFR autophosphorylation sites Y1197/Y1172 and drug targets. The feasibility of SOP-Phos-DIA was demonstrated on EGFR-TKI sensitive and resistant cells, revealing the interplay of multipathway Hippo-EGFR-ERBB signaling cascades underlying the mechanistic insight into EGFR-TKI resistance. Overall, SOP-Phos-DIA is an efficient and robust protocol that can be easily adapted in the community for microscale phosphoproteomic analysis.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Fosfopeptídeos / Fosfoproteínas / Proteômica / Fluxo de Trabalho Limite: Humans Idioma: En Revista: J Proteome Res Assunto da revista: BIOQUIMICA Ano de publicação: 2024 Tipo de documento: Article País de afiliação: Taiwan

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Fosfopeptídeos / Fosfoproteínas / Proteômica / Fluxo de Trabalho Limite: Humans Idioma: En Revista: J Proteome Res Assunto da revista: BIOQUIMICA Ano de publicação: 2024 Tipo de documento: Article País de afiliação: Taiwan