Serial and multi-level proteome analysis for microscale protein samples

Post-translational modifications (PTMs), such as phosphorylation and ubiquitination, play key roles in signal transduction and protein homeostasis. The crosstalk of PTMs greatly expands the components of proteome and protein functions. Multi-level proteome analysis, which involves proteome investigations of total lysate and PTMs in this context, provides a comprehensive approach to explore the PTM crosstalk of a biological system under diverse disturbances. However, multi-level proteome practice remains technically challenging. Here we intended to build a strategy for multi-level proteome analysis, in which we focus on the serial profiling the total proteome, ubiquitinome and phosphoproteome from the microscale of starting material. We started by evaluating five common lysis buffers and found that the sodium deoxycholate buffer provided the best overall performance. We then developed an approach for serial enrichment and profiling of the multi-level proteome. To expand the depth of identification, we customized the variable windows to perform data-independent acquisition (DIA) sequencing for each proteome. In total, we identified 6465 proteins, ∼20,000 GlyGly sites (class 1), and ∼ 19,000 phosphosites (class 1) sequentially using 1 mg of HeLa digest by three DIA measurements. We applied this strategy to analyze MG132-treated HeLa cells and observed the crosstalk between ubiquitination and phosphorylation. Our method can be referenced for other multi-level proteome studies with microscale samples. SIGNIFICANCE: Lysis buffer containing sodium deoxycholate provided the best overall performance in multi-level proteome analysis. One step of ubiquitination enrichment before phosphorylation enrichment does not reduce the reproducibility of phosphoproteome. Customized isolation windows were established for DIA analysis on each level of proteome. Combined the serial enrichment approach and the customized single-shot DIA method enabled the multi-level proteome of microscale protein samples.