In BriefWe developed a lauryl maltose neopentyl glycol (LMNG)-assisted sample preparation (LASP) method for low-input proteomics. LMNG surfactant is added for reducing protein and peptide loss into th...

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 β-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.

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 pres...

In BriefWe developed a lauryl maltose neopentyl glycol (LMNG)-assisted sample preparation (LASP) method for low-input proteomics. LMNG surfactant is added for reducing protein and peptide loss into th...

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Orbitrap Exploris 480 MS with FAIMS Pro provides fast, sensitive and robust profiling of proteomes when combined with Evosep One. The combination of Data Independent Acquisition (DIA) and FAIMS with s...