Biology subjects

Hartlmayr, D.

Publications and source records attributed to Hartlmayr, D..

3 recordsLinked to original sources

bioRxiv2023

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

Spatial tissue proteomics integrating whole-slide imaging, laser microdissection and ultrasensitive mass spectrometry is a powerful approach to link cellular phenotypes to functional proteome states in (patho)physiology. To be applicable to large patient cohorts and low sample input amounts, including single-cell applications, loss-minimized and streamlined end-to-end workflows are key. We here introduce an automated sample preparation protocol for laser microdissected samples utilizing the cellenONE(R) robotic system, which has the capacity to process 192 samples in three hours. Following laser microdissection collection directly into the proteoCHIP LF 48 or EVO 96 chip, our 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, particularly pertinent for laser microdissection workflows. We validate our method in human tonsil archival tissue, where we profile proteomes of spatially-defined B-cell, T-cell and epithelial microregions of 4,000 {micro}m2 to a depth of [~]2,000 proteins and with high cell type specificity. We finally provide detailed equipment templates and experimental guidelines for broad accessibility.

systems biology↗

bioRxiv2023

High-throughput and scalable single cell proteomics identifies over 5000 proteins per cell

The emergence of mass spectrometry (MS)-based single-cell proteomics (SCP) promise to revolutionize the study of cellular biology and biomedicine by providing an unparalleled view of the proteome in individual cells. Despite its groundbreaking potential, SCP is nascent and faces challenges including limited sequence depth, throughput, and reproducibility, which have constrained its broader utility. This study introduces key methodological advances, which considerably improve the sensitivity, coverage and dependability of protein identification from single cells. We developed an almost lossless SCP workflow encompassing sample preparation to MS analysis, doubling the number of identified proteins from roughly 2000 to over 5000 in individual HeLa cells. A comprehensive evaluation of analytical software tools, alongside strict false discovery rate (FDR) controls solidified the reliability of our results. These enhancements also facilitated the direct detection of post-translational modifications (PTMs) in single cells, negating the need for enrichment and thereby simplifying the analytical process. Although throughput in MS remains a challenge, our study demonstrates the feasibility of processing up to 80 label-free SCP samples per day. Moreover, an optimized tissue dissociation buffer enabled effective single cell disaggregation of drug-treated cancer cell spheroids, refining the overall proteomic analysis. Our workflow sets a new benchmark in SCP for sensitivity and throughput, with broad applications ranging from the study of cellular development to disease progression and the identification of cell type-specific markers and therapeutic targets.

systems biology↗

bioRxiv2021

An automated workflow for label-free and multiplexed single cell proteomics sample preparation at unprecedented sensitivity.

The analysis of single-cell proteomes has recently become a viable complement to transcriptomics and genomics studies. Proteins are the primary driver of cellular functionality and mRNA levels are often an unreliable proxy of such. Therefore, the global analysis of the proteome is essential to study cellular identities. Multiplexed and label-free mass spectrometry-based approaches with single-cell resolution have lately attributed surprising heterogeneity to presumed homogenous cell populations. Even though specialized experimental designs and instrumentation have demonstrated remarkable advances, the efficient sample preparation of single cells still lag. Here, we introduce the proteoCHIP, a universal option for single-cell proteomics sample preparation at surprising sensitivity and throughput. The automated processing using a commercial system combining single-cell isolation and picoliter dispensing, the cellenONE(R), reduces final sample volumes to low nanoliters submerged in a hexadecane layer simultaneously eliminating error-prone manual sample handling and overcoming evaporation. The specialized proteoCHIP design allows direct injection of single cells via a standard autosampler resulting in around 1,500 protein groups per analytical run at remarkable reporter ion signal to noise while reducing or eliminating the carrier proteome. We identified close to 2,600 proteins across 170 multiplexed single cells from two highly similar human cell types. This dedicated loss-less workflow allows distinguishing in vitro co-differentiated cell types of self-organizing cardiac organoids based on indicative markers across 150 single cells. In-depth characterization revealed enhanced cellular motility of cardiac endothelial cells and sarcomere organization in cardiomyocytes. Our versatile and automated sample preparation has not only proven to be easily adaptable but is also sufficiently sensitive to drive biological applications of single-cell proteomics.

biochemistry↗