An exciting new development in the field of spacial tissue proteomics by Makhmut et. al., from the Coscia lab is presented in Molecular and Cellular Proteomics. Spatial tissue proteomics, which combines whole-slide imaging, laser microdissection, and ultrasensitive mass spectrometry, is a powerful technique for linking cellular phenotypes to functional proteome states in (patho)physiology. However, to be applicable to large patient cohorts, low sample input amounts (e.g single-cell applications), loss-minimized and streamlined end-to-end workflows are crucial.

This study introduces an automated sample preparation protocol for laser microdissected samples utilizing the cellenONE® robotic system, which has the capacity to process 192 samples in three hours. After laser microdissection collection directly into the proteoCHIP LF 48 or EVO 96 chip, the optimized protocol facilitates lysis, formalin de-crosslinking, and tryptic digest of low-input archival tissue samples. The seamless integration with the Evosep ONE LC system by centrifugation allows ‘on-the-fly’ sample clean-up, which is particularly pertinent for laser microdissection workflows.

The method is validated in human tonsil archival tissue, where proteomes of spatially-defined B-cell, T-cell, and epithelial microregions of 4,000 μm2 are profiled to a depth of approximately 2,000 proteins with high cell type specificity. Detailed equipment templates and experimental guidelines are provided for broad accessibility. This study is important as it enables high-throughput spatial proteomics analysis of low-input samples, facilitating insights into cellular phenotypes and proteome states in various biological contexts, and the Aurora Elite column (15cm x 75um, 1.7um, IonOpticks) was used for peptide separation.

Publication
Molecular & Cellular Proteomics – March 2024

Authors

A. Makhmut; D. Qin; D. Hartlmayr; A. Seth; F. Coscia

Title

An automated and fast sample preparation workflow for laser microdissection guided ultrasensitive proteomics