AUTO-SP: Automated Sample Preparation for Analyzing Proteins and Protein Modifications

作者：T. Mamie Lih, Liyuan Jiao, Lijun Chen, Jongmin Jacob Woo, Yuefan Wang, Hui Zhang · 发表于：Analytical Chemistry · 年份：2025 · DOI：10.1021/acs.analchem.5c00886 · 被引用次数：6 · 研究领域：Advanced Proteomics Techniques and Applications、Mass Spectrometry Techniques and Applications、Peptidase Inhibition and Analysis

Liquid chromatography (LC) tandem mass spectrometry (MS/MS) is one of the widely used proteomic techniques to study the alterations occurring at the protein level as well as post-translational modifications (PTMs) of proteins that are relevant to different physiological or pathological statuses. The mass spectrometric analysis of peptides digested from proteins (bottom-up proteomics) has emerged as one of the major approaches for proteomics. In this approach, proteins are first cleaved by one or more proteases into peptides for MS 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 AUTO-SP, a sample preparation platform providing automated protocols for BCA analysis, protein digestion, and PTM enrichment for protein and PTM analyses. We utilized patient-derived xenograft (PDX) breast cancer tumor tissues (basal-like and luminal subtypes) to demonstrate the efficacy of AUTO-SP. 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 that samples of the same breast cancer subtypes were highly correlated (≥0.98). Additionally, >25,000 phosphopeptides and >14,000 ubiquitinated peptides were identified in the PDX samples when using AUTO-SP fo...