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

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.