An extra dimension in protein tagging by quantifying universal proteotypic peptides using targeted proteomics

Abstract

The use of protein tagging to facilitate detailed characterization of target proteins has not only revolutionized cell biology, but also enabled biochemical analysis through efficient recovery of the protein complexes wherein the tagged proteins reside. The endogenous use of these tags for detailed protein characterization is widespread in lower organisms that allow for efficient homologous recombination. With the recent advances in genome engineering, tagging of endogenous proteins is now within reach for most experimental systems, including mammalian cell lines cultures. In this work, we describe the selection of peptides with ideal mass spectrometry characteristics for use in quantification of tagged proteins using targeted proteomics. We mined the proteome of the hyperthermophile Pyrococcus furiosus to obtain two peptides that are unique in the proteomes of all known model organisms (proteotypic) and allow sensitive quantification of target proteins in a complex background. By combining these ’Proteotypic peptides for Quantification by SRM’ (PQS peptides) with epitope tags, we demonstrate their use in co-immunoprecipitation experiments upon transfection of protein pairs, or after introduction of these tags in the endogenous proteins through genome engineering. Endogenous protein tagging for absolute quantification provides a powerful extra dimension to protein analysis, allowing the detailed characterization of endogenous proteins.

Discovering cellular protein‐protein interactions: Technological strategies and opportunities

“…By the spike‐in of a heavier isotope‐labeled variant of this peptide after affinity purification and an even heavier variant before the LC‐MS analysis, researchers can normalize for sample losses during sample processing after the affinity purification and they can correlationally quantify the preys relative to the bait‐tag in an absolute way. Relevant in this context, the proteotypic peptides for quantification by SRM (PQS peptides) are especially selected for sensitive and reproducible tag‐based quantification of target proteins in complex backgrounds, enabled by their natural absence from the proteomes of all known model organisms.…”

Phosphorylation of the multifunctional signal transducer B-cell adaptor protein (BCAP) promotes recruitment of multiple SH2/SH3 proteins including GRB2

“…Constructs (47) for bacterial expression were generated by ligation-independent cloning (48) into pMCSG10 plasmid (DNASU) containing a His-GST-TEV cl tag (Table S2). For expression in mammalian cells, plasmids were cloned using restriction enzymes and primer sequences contained the respective tags (Table S2).…”

Targeted Proteomics Comes to the Benchside and the Bedside: Is it Ready for Us?

“…The protein (YPI) digested by trypsin in the complex sample yields tryptic peptides (Y, P, and I) . Peptides that are unique to the protein (peptides Y and P), which are ionized in a reproducible manner (peptide Y), are selected for the assay . The tryptic peptides of a complex mixture of proteins are first processed through liquid chromatography (LC).…”

“…The selection of fragment ions can again be made either by using previously acquired MS‐MS data or the available prediction tools (MRMaid; http://elvis.misc.cranfield.ac.uk/mrmaid/, SRMAtlas; http://www.srmatlas.org/). After shortlisting peptides and their fragment ions, the use of synthetic candidate peptides for optimizing the assay parameters and selection of best transitions is required …”