Parallel, High-Quality Proteomic and Targeted Metabolomic Quantification Using Laser Capture Microdissected Tissues

• Published in: Analytical chemistry (Washington) 2021-06, Vol.93 (25), p.8711-8718
• Main Authors: Shen, Shichen, Li, Jun, Huo, Shihan, Ma, Min, Zhu, Xiaoyu, Rasam, Sailee, Duan, Xiaotao, Qu, Miao, Titus, Mark A, Qu, Jun
• Format: Article
• Language: English
• Subjects: Androgens; Chemistry; Crosstalk; Humans; Laser Capture Microdissection; Lasers; Male; Metabolomics; Molecular weight; Parameter robustness; Prostate cancer; Prostatic Neoplasms; Proteins; Proteome; Proteomes; Proteomics; Robustness; Sample preparation; Stromal cells; Surveying; Tandem Mass Spectrometry
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.1c01026

Quantitative proteomics/metabolomics investigation of laser-capture-microdissection (LCM) cell populations from clinical cohorts affords precise insights into disease/therapeutic mechanisms, nonetheless high-quality quantification remains a prominent challenge. Here, we devised an LC/MS-based approach allowing parallel, robust global-proteomics and targeted-metabolomics quantification from the same LCM samples, using biopsies from prostate cancer (PCa) patients as the model system. The strategy features: (i) an optimized molecular weight cutoff (MWCO) filter-based separation of proteins and small-molecule fractions with high and consistent recoveries; (ii) microscale derivatization and charge-based enrichment for ultrasensitive quantification of key androgens (LOQ = 5 fg/1k cells) with excellent accuracy/precision; (iii) reproducible/precise proteomics quantification with low-missing-data using a detergent-cocktail-based sample preparation and an IonStar pipeline for reproducible and precise protein quantification with excellent data quality. Key parameters enabling robust/reproducible quantification have been meticulously evaluated and optimized, and the results underscored the importance of surveying quantitative performances against key parameters to facilitate fit-for-purpose method development. As a proof-of-concept, high-quality quantification of the proteome and androgens in LCM samples of PCa patient-matched cancerous and benign epithelial/stromal cells was achieved (N = 16), which suggested distinct androgen distribution patterns across cell types and regions, as well as the dysregulated pathways involved in tumor–stroma crosstalk in PCa pathology. This strategy markedly leverages the scope of quantitative-omics investigations using LCM samples, and combining with IonStar, can be readily adapted to larger-cohort clinical analysis. Moreover, the capacity of parallel proteomics/metabolomics quantification permits precise corroboration of regulatory processes on both protein and small-molecule levels, with decreased batch effect and enhanced utilization of samples.