AUTO-SP: automated sample preparation for analyzing proteins and protein modifications

ABSTRACT Liquid chromatography (LC) tandem mass spectrometry (MS/MS) is one of the widely used proteomic techniques to study the alterations occurred at protein expression level as well as post-translation modifications (PTMs) of proteins that are relevant to different physiological or pathological statuses. The mass spectrometric analysis of peptides fragmented from proteins (bottom-up proteomics) has emerged as one of the major approaches for proteomics. In this approach, proteins are first cleaved into peptides for mass spectrometric analysis and peptides with PTMs are further enriched followed by the LC-MS/MS analysis. To achieve a reproducible and quantitative proteomic characterization, a well-established protease digestion and PTM peptide enrichment protocol is critical. In this study, we developed an automated sample preparation (AUTO-SP) for analyzing proteins and protein modifications utilizing Patient-Derived Xenograft (PDX) breast cancer tumors (basal-like and luminal subtypes). The protein amount was quantified and proteins were further digested by using AUTO-SP for each PDX sample. Based on the data-independent acquisition (DIA)-MS data, we observed samples of the same breast cancer subtypes were highly correlated (>0.98). Additionally, >14,000 ubiquitinated peptides were identified in the PDX samples when using AUTO-SP for ubiquitin enrichment, while unique pathways were enriched from the basal-like and luminal subtypes. AUTO-SP demonstrated its efficacy to provide reliable and reproducible sample preparation procedure for MS-based proteomic and PTM analyses.