Phosphotyrosine-based phosphoproteomics scaled down to sub-mg needle biopsy level for analysis of individual tumor biology and treatment selection

Introduction

Mass spectrometry-based phosphoproteomics of cancer cell and tumor tissue lysates provides insight in aberrantly activated signaling pathways and potential drug targets. For a comprehensive understanding of individual patient's tumor biology that includes phosphotyrosine signaling and to allow selection of tyrosine kinase inhibitors in individual patients, pTyr-based phosphopeptide enrichment coupled to mass spectrometry is required. However, it needs to be feasible and reproducible with the low protein yields obtained from tumor needle biopsies. Previously, we down-scaled the phosphotyrosine immunoprecipitation to 1 mg, yet most needle biopsies have protein yields that are lower. Here we explore whether further down-scaling of the tyrosine immunoprecipitation is possible and yields reproducible profiles.

Methods

To this end, phosphopeptide immunoprecipitation using anti-phosphotyrosine beads was performed using 5, 2, 1 and 0.5 mg protein input from lysates of a mix of colorectal cancer (CRC) tissues that yields similar phosphosite numbers as our previously published CRC cell line reference HCT116. All volumina were linearly scaled or left at 1 mg protein input level. In addition, for low protein input samples, smaller vials and glassware was used.

Results

The total number of phosphopeptides captured and detected by LC-MS/MS (Timstof-HT) searched using MQ 2.3.1.0 ranged from 2387 at 5 mg input to 457-604 at 0.5 mg CRC mix input, depending on the condition used. These numbers may be further boosted by match-between-run ID propagation. Reproducibility of 6 replicates at the 0.5 mg protein level was high. Detailed results will be reported along with optimization of MS acquisition and exploration of phosphoproteomics in DIA mode.

Conclusions

This study demonstrates the feasibility of label-free pTyr-phosphoproteomics at sub-mg protein level, a protein yield that in our experience may be obtained from ~50% of tumor needle biopsies collected in clinical trials.