High-throughput workflow for in-depth proteome coverage and quantitative analysis of low sample amounts and single-cells

In MS based proteomics, millions of cells are analyzed at the same time and protein changes are presented as a sum to the total number of cells analyzed. However, it is known that each cell carries out its own biological processes. Therefore, a better understanding of this diversity can be achieved by studying as many individual cells as possible. Improvements in MS instrumentation have enhanced sensitivity, dynamic range, and large-scale profiling with improved throughput. However, maintaining the proteome depth at throughput is still challenging. Thus, we optimized and benchmarked the performance of a low nanoflow LC-MS methods in data-independent acquisition (DIA), on the Orbitrap Astral MS, to achieve deep proteome profiling at highest throughput for single cell.

For the dilution, Pierce™ HeLa digest (20 ug) was reconstituted by adding 200 μL of 0.015% DDM (Dodecyl β D maltosid) solution, sonicated for 5 min, then diluted to 5 ng/μL in 0.015% DDM. Individual HeLa cells were isolated using CellenONE® system from Cellenion, followed by reduction, alkylation, and trypsin digestion as per manufacturer"s instructions. The data was acquired on a Thermo Scientific™ OrbitrapTM Astral™ mass spectrometer with a Thermo Scientific™ FAIMS Pro™ interface. Samples were separated using Thermo Scientific™ Vanquish™ Neo UHPLC system on an IonOpticks Aurora 25 cm TS C18 column. Data was acquired in DIA mode. Raw files were processed with Spectronaut 18, with and without library, using a human database with 20k sequences.

Though throughput is determined by sample preparation, LC separation and MS acquisition, we focused more on the LC optimization since the MS part has been previously optimized [Santosh et al.]. During the separation, the flow rate is ramped multiple times, starting from 450nl\min at the beginning of the gradient. During peptide elution, the flow rate dropped to 200nl\min and later ramped up to 300nl\min during the washing step. Spectronaut analysis, with no library, from 250pg HeLa digest on column, separated using a 19.5min active gradient, we could identify more than 5600 protein groups from individual files. When triplicated files are combined and processed together, the number of protein groups at 1 % FDR goes up to over 6100, with median CVs below 10%. With a library generated from 10ng the protein groups increased to over 7400. To evaluate the reproducibility and reliability of the optimized method, it was transferred to multiple Thermo Fisher Scientific sites across the globe to be executed on different instruments and users. All results from the different sites were within ± 10%, demonstrating the reproducibility and reliability of this method. Analyzing real HeLa single cells, we identified on average of over 6,500 protein groups without using a library.