Introduction

Thousands of proteins can be measured from different cell lines using MS based proteomics. However, to understand and characterize cell heterogeneity, these cells need to be studied individually. In recent years single cell analysis has profited from advances in LC-MS based proteomics approaches. Nevertheless, there are still challenges in this field of application. Besides sample preparation, the key challenges in looking at individual single cell proteomes are sensitivity, coverage, dynamic range, and throughput. To address some of these challenges, new technological developments, as well as improvements on existing LC-MS-based proteomics workflows are a necessity. Here we described a high-throughput workflow of TMTpro multiplexed single cells using an Orbitrap Astral mass spectrometer.

Methods

Individual HeLa cells were sorted, followed by reduction, alkylation, trypsin digestion and TMT labeling using CellenONE as per the manufacturer"s protocols. To enhance the MS1 signal of the peptides a booster channel was used.

The multiplexed single cell digests were analyzed using an Orbitrap Astral MS with FAIMS Pro Duo interface coupled to a Vanquish Neo UHPLC system. Data was acquired in DDA mode and searched with Proteome Discoverer Software 3.1.

Results

Using optimized methods for analyzing 250 Samples Per Day on average more than 3000 proteins and 13.000 peptides were quantified. 80% of those proteins had less than 10% CV.