Introduction

Mass spectrometry-based phosphoproteomics is a powerful tool that aims to explore signaling network regulation and dysregulation in normal and pathological conditions. Due to the low stoichiometry of phosphorylated peptides and the high diversity of phosphorylated peptide species, the acquisition of sufficient coverage of regulatory phosphorylation sites is challenging, especially when analyzing samples with tiny amounts. In a common pipeline, a discovery method enables global profiling of proteome at first, due to many signals being missed in the profiling, a targeted method for the interested peptides has to be performed afterward. This pipeline suffers from the high cost of heavy-labeled standards, time-consuming to set up an experiment, and more sample consumption.

Methods

Here, we developed a multiplexing method using tandem mass tags with a carrier channel to amplify the signals of interested phosphorylated peptides. Sixty phosphorylated peptides were spiked in as the carrier and an SPS-MS3 with real-time searching method was applied. The proof of concept was tested using breast cancer cell lysates analyzed on the Thermo Fisher Orbitrap Eclipse. Three approaches, MS2, SPS-MS3, and SPS-MS3 with real-time search, were compared in conditions with this strategy and DDA (Data-dependent acquisition) approach. After adjustment of the parameters, the performance of identification and quantification were tested by a two-proteome interference assay. Then, the TSAP with optimized parameters was compared to a targeted method. In addition, TSAP was used to detect the phosphorylation status of breast cancer cell models treated with insulin.

Conclusions

In this project, we demonstrated a phosphoproteomics method for limited amounts of samples, which combined a multiplexing strategy and the SPS-MS3 with the real-time searching method. This approach is beneficial for applications involving limited quantities of samples. It enables targeted observation of the status of phosphoprotein peptides of interest in parallel with comprehensive global phosphoproteomics analysis in a single run. Moreover, the simple setup and high throughput of this method allow it to be used in a variety of situations.