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Development of a Human Cell-Free Expression System to Generate Stable-Isotope-Labeled Protein Standards for Quantitative Mass Spectrometry

Reputable Mentor II
Reputable Mentor II
Bomgarden RD, Baerenwald D, Hommema E, Peterman S, Rogers JC.
ASMS 2012 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.

Thermo Fisher Scientific
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Last update:
‎10-15-2021 06:12 AM
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