To increase the throughput and quantitative accuracy of single-cell protein analysis by SCoPE-MS, we introduced many technical improvements in both the sample preparation and in the mass-spectrometry analysis. The synergistic effect is to increase quantitative accuracy by 4-fold and the throughput of data acquisition about 8-fold. Below, we outline controlled experiments that illustrate the benefits of individual improvements. To comprehensively compare the mass-spec data at all levels (including chromatographic separation, precursor abundance, ion isolation, spectral purity, and peptide sequence identification), we include the full Data-driven Optimization of MS (DO-MS) reports for each set of experiments.
Decreasing co-isolation with narrow isolation windows
The degree of coisolation (or purity of MS2 spectra) was evluated as a function of the width of the isolation window. MaxQuant uses the precursor intensity fractions (PIF) to estimate the fraction of the ion intensity originating from the precursor assigned to a peptide spectral match for each MS2 spectrum. The distributions of PIF values for two controlled experiments indicate that the isolation window width used by SCoPE2 (0.7 Th) results in purer spectra compared to the isolation with used with SCoPE-MS. The associated DO-MS report includes dozens of additional plots demonstrating that these experiments were well controlled. It alos shows that the wider isolation wiwndow of 1 Th results in slighly more identified peptides.
Preparing single cells for mass-spec analysis
Improving apex targetting