Background

Proteomics is the large-scale study of proteins; those proteins are often contained within cells, tissues, or an entire organism. Several technologies exist for the absolute quantitation of peptides within a complex mixture, namely isotope labeled peptides, and artificial proteins that are concatenations of multiple different peptides (known as "QconCATs"). QconCATS genes are normally expressed in E. coli in the presence of media supplemented with isotope labeled amino acids and added to a biological sample prior to digestion. Once digested, the QconCAT peptides are all at a 1:1 ratio and correspond to naturally occurring peptides in the biological sample; therefore, each isotopically labeled peptide can be used as a standard for absolute quantitation of peptides of interest. Although QconCATs have tremendous utility for quantitation of known peptides in a mixture, they are not helpful for scientists who need a proteomics standard that can be spiked into a protein mixture at an extremely low level, that co-purifies during sample fractionation, and be unobtrusive by mass spectrometry. The invention described here is focused on using the concatemeric protein as a non-intrusive process control that is carried along for the protein purification steps, but when it is digested, it behaves as single peptide species by mass spectrometry.

Description

LLNL researchers have designed a synthetic, concatemeric bacterial expression vector that expresses a protein sequence that can be digested into a single peptide. The synthetic protein is designed to be secreted outside E. coli cells, and therefore can be purified using a His-tag from the cell supernatant (thereby reducing the need to lyse the cells for purification). The protein sequence is optimized for protein solubility, and the concatemeric peptide sequence is optimized for ionization using electrospray ionization (ESI), which is the most common ion source for proteomics. Taken together, the protein can be spiked into a complex mixture at very low levels as an internal control, and then digested and detected as single peptide species.

Advantages
  • Provides a non-intrusive internal process control; when digested in a mixture it gives rise to a single peptide species by mass spectroscopy
  • Can be added to protein mixtures at a virtually “invisible” amounts (non-disruptive, non-intrusive, presence only known if looking for a signature peptide sequence)
  • Inexpensive (not a custom QconCAT or isotopically labelled peptide) and is only a single species recombinant protein
  • Can be easily produced at a larger scale, but used in small amounts/aliquots (only small amounts needed for mass spec detection)
Potential Applications
  • Proteomics- superior internal control for absolute quantitation of peptides within a complex mixture
Development Status

LLNL has filed a patent application (US Patent Application 16/908521) covering this technology (LLNL Internal Case # IL-13366).

Reference Number
492/20