Fig. 2.

Experimental outline for temporal comparison of SIL peptide introduction into a FASP workflow. Twenty 200 μl aliquots of SDX protein extract were reduced and alkylated then pooled to create a uniform, bulk sample that was aliquoted into 20, 10-kDa MWCO-filters for FASP-digestions. Two sets of digests were performed in parallel. In the first set, the SIL mixture was added immediately following the digest quenching step (postdigest). In the second set, the SIL mixture was added concurrently with the trypsin (concurrent). During both sets of digests, the double-labeled peptide for HCT1.338–354 (indicated as SIL**) was added at the opposite time of the SIL mixture containing the corresponding single-labeled peptide. Note, predigest introduction of the SIL mixture could not be tested within this FASP workflow as the SIL peptides would be removed by the various rinse steps.