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. 2021 Jul 22;2(3):100682. doi: 10.1016/j.xpro.2021.100682

Figure 1.

Figure 1

Schematic workflow

Conditioned media from three 15 cm dishes are pooled to make a replicate. For N-terminomics, samples are digested with LysargiNase, subjected to N-terminal peptide enrichment, labeled with TMT, and then mixed. The multiplexed sample is divided into three parts and analyzed by triplicate LC/MS/MS runs. Note that TMT labeling should be performed after enrichment of N-terminal peptides, since the TMT label affects the enrichment efficiency. For C-terminomics, samples are digested with trypsin and LysC, labeled with TMT and mixed, divided into three parts and subjected to C-terminal peptide enrichment using three SCX-StageTips. Analyzing two fractions, the flow-through and the 0.5% TFA-eluted fraction, allows identification and quantification of C-terminal peptides on a comparable scale to the N-terminal peptide counterpart with the same amount of loaded peptides. The peptide purification process (steps 36–43) appears four times in this workflow (N1, N2, C1, and C2).