Bomgarden RD, Baerenwald D, Hommema E, Peterman S, Rogers JC.
ASMS 2012 Video Poster
Stable-isotope-labeled peptides are routinely used as internal standards for the
quantification of enzymatically-digested protein samples. Sable-isotope-labeled
proteins are ideal for MS sample preparation standardization. Traditional in vivo
expression systems, such as 15N-labeled E. coli or SILAC, have been used to express
recombinant heavy proteins. However, these systems are limited in their expression of
toxic or insoluble proteins, require two to three days for protein expression, and may
have low yield of functional (i.e. properly folded) proteins. In addition, because in vivo
systems use stable-isotope-labeled cell lines, all proteins in the cell are isotopically
labeled leading to significantly higher waste and cost.
An alternative method to in vivo protein expression, in vitro translation (IVT), uses a
cellular-extract system to transcribe DNA into mRNA, which is subsequently translated
into protein. Most IVT systems utilize prokaryotic (e.g. bacteria) or non-human
eukaryotic (e.g. rabbit reticulocyte) cell extracts. However, these systems lack the
components needed for proper folding and modification of human proteins, have lower
expression yields, and inefficiently incorporate stable-isotope-labeled amino acids. In
this study, we describe a novel human cell-free system3,4 to express stable-isotopelabeledprotein standards as controls for sample-preparation loss, for
digestion-efficiency determination, and as quantification standards.
http://www.youtube.com/watch?v=j7XLDOZc9AkThermo Fisher Scientific
United States
