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A Rapid One-pot Workflow for Sensitive Microscale Phosphoproteomics

ABSTRACT: Rapid advancement of single-cell proteomics has revolutionized modern research to reveal cell-specific regulation and cellular phenotypes. Though phosphoproteomics is a powerful method for mapping signal transduction networks, its sensitivity has lagged behind due to significantly lower abundance, complex sample preparation and substantial sample input. Here, 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 and alkylation, direct trypsinization and phosphopeptide enrichment by TiO2 beads in a single-tube format. By reducing surface adsorptive losses via utilizing n-Dodecyl β-D-maltoside pre-coated tubes throughout the workflow, and shortening digestion time, SOP-Phos is completed within 3-4-hr with 1.4-fold higher identification coverage, especially on recovering longer and multiple phosphopeptides. SOP-Phos coupled with DIA demonstrated >90% specificity, enhanced sensitivity, lower missing values (<1%), and improved quantitative reproducibility (8%-10% CV). We established a sample size-comparable spectral library to enhance 2.6-6.4-fold more phosphopeptides compared to directDIA, offering coverage of 33,787±670 to 22,070±861 phosphopeptides from 5 to 0.5 µg (~2500 cells) cell lysate. Such sensitivity enabled mapping key lung cancer signaling sites such as autophosphorylation sites Y1197/Y1172 of EGFR and drug targets. The feasibility of SOP-Phos-DIA was demonstrated on two pairs of EGFR-Tyrosine Kinase Inhibitor (TKI)- sensitive and resistant cells, revealing the interplay of multi-pathway Hippo-EGFR-ERBB signaling cascades which not only provides the mechanistic insight to EGFR-TKI resistance but also suggest combination therapy to restore the sensitivity to EGFR-TKI therapy. Using the commonly used reagents, the SOP-Phos-DIA is an efficient and robust protocol that can easily be adapted in the community for microscale phosphoproteomic analysis with deep depth to decipher biology from low-input samples such as primary or rare immune cells.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Yu-Ju Chen

PROVIDER: PXD047646 | JPOST Repository | Tue May 06 00:00:00 BST 2025

REPOSITORIES: jPOST

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			Action	DRS
	20220928_PC9_SDC_TiO2_10000ng_1.raw	Raw
	20220928_PC9_SDC_TiO2_10000ng_2.raw	Raw
	20220928_PC9_SDC_TiO2_10000ng_3.raw	Raw
	20220928_PC9_SDC_TiO2_10000ng_DDA_1.raw	Raw
	20220928_PC9_SDC_TiO2_10000ng_DDA_2.raw	Raw

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A Rapid One-pot Workflow for Sensitive Microscale Phosphoproteomics

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