Label-Free quantitation with high accuracy and precision on a single cell scale and beyond with Orbitrap Astral mass spectrometer

Analysis of protein expression at the single-cell level, allowing to reveal subtle cellular heterogeneity, gained increasing importance over the last 5 years. Nanoflow LC coupled with high-resolution mass spectrometry is the method of choice for proteomics studies, due to its sensitivity, dynamic range, and throughput. In this study, we assessed the accuracy and precision of quantitation over the wide range of sample loads, for low-input samples, from as low as 10 pg up to 500 ng per injection, at a less than 20 min gradient length. For 10 pg-20 ng loads, FAIMS was used to decrease interference from background ions. Starting from 20ng and up MS data acquisition without FAIMS becomes more beneficial for both peptide and protein identifications. A wide (up to 40 Th) isolation window DIA methods with long (up to 100 ms) Astral ion injection times were employed to maximize the proteome coverage for low-input samples. With these settings, more data points per LC peak are acquired at MS1 level with the Orbitrap analyzer, ensuring high quantitation accuracy and precision, and MS2 information is used only for identification purposes. 100 ms maximum injection time with a 500% normalized AGC target, set at the Orbitrap analyzer, allowed delivery of a sufficient number of ions to be transferred downstream to the Astral analyzer, to ensure reliable quantitation. Linear correlation (up to R2=0.99) was observed for analyte peaks measured with the Orbitrap analyzer with FAIMS, at sample loads from 10 pg to 500 ng, demonstrating a wide dynamic range of quantitation at the scale spanning over 4 orders of magnitude. A systematic study of parameters critical for high quantitation accuracy was performed, and the best MS methods were selected. Data was analyzed using SpectronautTM 18 software (Biognosys) with directDIA, library-free processing. As a result, ~3400 protein groups, >13,000 peptides identified at 10 pg/injection, 82 to 90% of them quantified in one of the 3 ratios of proteomes; ~9300 protein groups, ~63,000 peptides identified at 250 pg/injection (single-cell level), with nearly 98% of them quantified. At 500 ng/injection, over 15.500 protein groups and >230,000 peptides were identified and quantified. Quantitation accuracy at the single-cell level loads (10-250pg) was very close to the expected values, with less than 10% relative error. Quantitation accuracy remained high throughout the entire range of sample loads, regardless of the number of data points per peak. Single-cell level LFQ was performed in three different laboratories, showing similar results, and demonstrating that high accuracy and precision of quantitation can be achieved on a routine basis.