While the sensitivity of mass spectrometry (MS) is often cited as a major limitation for single-cell proteomics, current methods can detect over 60 000 peptide-like features from individual cells analyzed by label-free MS. Yet, because of time constraints, shotgun methods can perform MS2 scans on only a fraction of all detected peptide-like features and identify the amino acid sequences of even a smaller fraction. Thus, proteome coverage can be increased by allocating the limited MS analysis time only to peptide features that result in confidently identified and well quantified peptides.
This can be acheived by sending for MS2 scans only identifiable precursors from an inclusion list. This may be performed efficiently using real-time retention time alignment. This approach also provides the opportunity to select peptides of biological interest and to tailor their analysis based on biological considerations. For example, one may increase the ion accumulation times and thus the sampled peptide copies for peptides of relevance to the investigated biological question. To acheived these objectives, we developed prioritized single-cell proteomics. It ensures duty-cycle time for analyzing prioritized peptides across all single cells (thus increasing data consistency) while analyzing identifiable peptides at full duty-cycle, thus increasing proteome depth. These strategies increased the quantified data points for challenging peptides and the overall proteome coverage about 2-fold.
Huffman RG, Leduc A, Wichmann C, et al. (2022)
Prioritized single-cell proteomics reveals molecular and functional polarization across primary macrophages
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