Article:Monitoring both extended and tryptic forms of stable isotope-labeled standard peptides provides an internal quality control of proteolytic digestion in targeted mass spectrometry-based assays

The article provides an easy-to-implement approach to determining the extent of tryptic or other enzymatic digestion of a sample prior to the analysis of a surrogate peptide by LC-MS/MS.  Using an extended length (e.g., winged peptide) stable labeled internal standard, they showed that the efficiency of the digestion process could be monitored by analyzing for both the undigested extended length SIL IS and the target SIL IS after digestion.  They looked at a total of 524 assays - 398 using a direct measure of the digest, and 126 using an immunoaffintity extraction).  Of these, measurable amounts of the extended length SIL were found in 54% of the direct and 62% of the immunoaffinity extraction assays. Time course of digestion and using stressors on the digestion aided in characterizing the ability of the undigested/digested SIL to be an indicator of digestion efficiency.

Their work shows that 1) The ratio can be used to optimize the digestion conditions and 2) it is possible to use the measurement of the undigested extended length SIL IS and the SIL IS to assess the extent of enzymatic digestion.  The latter, when performed as part of routine sample analysis, provides a within-sample quality control (QC) check of the digestion. This may lead to the ability to normalize results based on the extent of digestion in each sample. 

Lundeen RA, Kennedy JJ, Murillo OD, Ivey RG, Zhao L, Schoenherr RM,
Hoofnagle AN, Wang P, Whiteaker JR, Paulovich AG, Monitoring both extended and tryptic forms of
stable isotope-labeled standard peptides provides an internal quality control of proteolytic digestion
in targeted mass spectrometry-based assays, Molecular & Cellular Proteomics (2023), doi: https://
doi.org/10.1016/j.mcpro.2023.100621.