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

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

Trapping mammalian protein complexes in viral particles

Cell lysis is an inevitable step in classical mass spectrometry-based strategies to analyse protein complexes. Complementary lysis conditions, in situ cross-linking strategies and proximal labelling techniques are currently used to reduce lysis effects on the protein complex. We have developed Virotrap, a viral particle sorting approach that obviates the need for cell homogenization and preserves the protein complexes during purification. By fusing a bait protein to the HIV-1 GAG protein, we show that interaction partners become trapped within virus-like particles (VLPs) that bud from mammalian cells. Using an efficient VLP enrichment protocol, Virotrap allows the detection of known binary interactions and MS-based identification of novel protein partners as well. In addition, we show the identification of stimulus-dependent interactions and demonstrate trapping of protein partners for small molecules. Virotrap constitutes an elegant complementary approach to the arsenal of methods to study protein complexes

A COFRADIC protocol to study protein ubiquitination

Here, we apply the COmbined FRActional DIagonal Chromatography (COFRADIC) technology to enrich for ubiquitinated peptides and to identify sites of ubiquitination by mass spectrometry. Our technology bypasses the need to overexpress tagged variants of ubiquitin and the use of sequence-biased antibodies recognizing ubiquitin remnants. In brief, all protein primary amino groups are blocked by chemical acetylation, after which ubiquitin chains are proteolytically and specifically removed by the catalytic core domain of the USP2 deubiquitinase (USP2cc). Because USP2cc cleaves the isopeptidyl bond between the ubiquitin C-terminus and the epsilon-amino group of the ubiquitinated lysine, this enzyme reintroduces primary epsilon-amino groups in proteins. These amino groups are then chemically modified with a handle that allows specific isolation of ubiquitinated peptides during subsequent COFRADIC chromatographic runs. This method led to the identification of over 7500 endogenous ubiquitination sites in more than 3300 different proteins in a native human Jurkat cell lysate

Analyzing trapped protein complexes by Virotrap and SFINX

The analysis of protein interaction networks is one of the key challenges in the study of biology. It connects genotypes to phenotypes, and disruption of such networks is associated with many pathologies. Virtually all the approaches to the study of protein complexes require cell lysis, a dramatic step that obliterates cellular integrity and profoundly affects protein interactions. This protocol starts with Virotrap, a novel approach that avoids the need for cell homogenization by fusing the protein of interest to the HIV-1 Gag protein, trapping protein complexes in virus-like particles. By using the straightforward filtering index (SFINX), which is a powerful and intuitive online tool (http://sfinx.ugent.be) that enables contaminant removal from candidate lists resulting from mass-spectrometry-based analysis, we provide a complete workflow for researchers interested in mammalian protein complexes. Given direct access to mass spectrometers, researchers can process up to 24 samples in 7 d

Intelligent mixing of proteomes for elimination of false positives in affinity purification-mass spectrometry

Protein complexes are essential in all organizational and functional aspects of the cell. Different strategies currently exist for analyzing such protein complexes by mass spectrometry, including affinity purification (AP-MS) and proximal labeling-based strategies. However, the high sensitivity of current mass spectrometers typically results in extensive protein lists mainly consisting of nonspecifically copurified proteins. Finding the true positive interactors in these lists remains highly challenging. Here, we report a powerful design based on differential labeling with stable isotopes combined with nonequal mixing of control and experimental samples to discover bona fide interaction partners in AP-MS experiments. We apply this intelligent mixing of proteomes (iMixPro) concept to overexpression experiments for RAF1, RNF41, and TANK and also to engineered cell lines expressing epitope-tagged endogenous PTPN14, JIP3, and IQGAP1. For all baits, we confirmed known interactions and found a number of novel interactions. The results for RNF41 and TANK were compared to a classical affinity purification experiment, which demonstrated the efficiency and specificity of the iMixPro approach

A COFRADIC Protocol To Study Protein Ubiquitination

Here, we apply the COmbined FRActional DIagonal Chromatography (COFRADIC) technology to enrich for ubiquitinated peptides and to identify sites of ubiquitination by mass spectrometry. Our technology bypasses the need to overexpress tagged variants of ubiquitin and the use of sequence-biased antibodies recognizing ubiquitin remnants. In brief, all protein primary amino groups are blocked by chemical acetylation, after which ubiquitin chains are proteolytically and specifically removed by the catalytic core domain of the USP2 deubiquitinase (USP2cc). Because USP2cc cleaves the isopeptidyl bond between the ubiquitin C-terminus and the ε-amino group of the ubiquitinated lysine, this enzyme reintroduces primary ε-amino groups in proteins. These amino groups are then chemically modified with a handle that allows specific isolation of ubiquitinated peptides during subsequent COFRADIC chromatographic runs. This method led to the identification of over 7500 endogenous ubiquitination sites in more than 3300 different proteins in a native human Jurkat cell lysate