Variable TMT search indicates very low level of over labeled peptides during SCoPE2 analysis

We evaluated the degree of over-labeling for data generated by the Single-Cell ProtEomics by Mass Spectrometry (SCoPE2) protocol. We performed a MaxQuant search in which TMT Pro labels were specified as a variable modification on histidine, serine, threonine, and tyrosine, and as a fixed modifications on primary amines (N-terminus and lysine). We found very few peptides with TMT labeling on histidine, serine, threonine, or tyrosine. After applying standard filters used by the SCoPE2 data pipeline (keeping peptides that have PEP <= 0.02 and PIF >= 0.9 and eliminating reverse hits and potential contaminants), we find a total of 225 unique over labeled sequences, amounting to about 3% of all peptide sequences. When evaluating each SCoPE2 run individually, the amount of over-labeled and confidently identified peptides ranges from 0.1% to 2.92% of the total run.