A Rapid One-Pot Workflow for Sensitive Microscale Phosphoproteomics

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.