The analysis of protein expression at the single-cell level has gained increased importance over recent years. Nanoflow HPLC coupled with high-resolution mass spectrometry is the method of choice for studies at the single-cell level, due to its sensitivity, dynamic range, and throughput. The work presented here demonstrates accurate and precise quantitation from 100 pg to 10 ng loads across an international interlaboratory study.
Digests of Human, Yeast and E. coli proteomes were mixed at different ratios and analyzed at total protein loads from 100 pg to 10 ng/injection. Peptides were separated under nanoflow conditions using a 50 samples per day throughput and MS analysis was performed using DIA methods on a Thermo ScientificTM OrbitrapTM AstralTM mass spectrometer with FAIMS –Pro Duo. Data were analyzed using SpectronautTM 18 software (Biognosys) with directDIA, library-free processing.
We assessed the accuracy and precision of quantitation for low-input samples using wide isolation windows for DIA with longer Astral analyzer ion injection times to balance proteome coverage with quantitative accuracy and precision for low-input samples. With these settings, more data points per LC peak are acquired at the MS1 level with the Orbitrap analyzer, ensuring quantitative accuracy and precision, while MS2 identification is performed. A strong linear correlation was observed for analyte peaks at sample loads from 100 pg to 10 ng. A systematic study of parameters critical for high quantitative accuracy was performed to optimize MS methods. As a result, more than 7,000 protein groups and 40,000 unique peptides were quantified with FDR<1% for a 100 pg sample load per run, with a median CV of less than 7%, and accurate quantitation at the single-cell level loads. The interlab study demonstrated that high accuracy and precision of quantitation can be achieved on a routine basis globally with optimized methods.