Large-scale mapping of human protein–protein interactions by mass spectrometry

Mapping protein–protein interactions is an invaluable tool for understanding protein function. Here, we report the first large-scale study of protein–protein interactions in human cells using a mass spectrometry-based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large-scale immunoprecipitation of Flag-tagged versions of these proteins followed by LC-ESI-MS/MS analysis resulted in the identification of 24 540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross-validated using previously published and predicted human protein interactions. In-depth mining of the data set shows that it represents a valuable source of novel protein–protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations

Development of a proteomic approach for identifying protein-protein interactions

The purification of protein complexes can be accomplished by different types of affinity chromatography. In a typical immunoaffinity experiment, protein complexes are captured from a cell lysate by an immobilized antibody that recognizes an epitope on one of the known components of the complex. After extensive washing to remove unspecifically bound proteins, the complexes are eluted and analyzed by mass spectrometry. Transient protein complexes, which are characterized by high dissociation constants, are typically lost by this approach. The primary objective of this study is to describe a novel method for identifying transient protein-protein interactions using a combination of in vivo cross-linking, immunoaffinity purification, and mass spectrometry-based protein identification. Live cells expressing an epitope-tagged protein of interest are treated with formaldehyde, which rapidly permeates the cell membrane and generates protein-protein cross-links. Proteins cross-linked to the protein of interest are co-purified by immunoaffinity chromatography and subjected to a procedure which dissociates the cross-linked complexes. After separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis, interacting proteins are identified by mass spectrometry. Application of this method enabled the identification of numerous proteins that co-purified with a constitutively active form of a Ras GTPase known as M-Ras (MRasQ71L), which was stably expressed in the murine mast cell line R6X. Among the proteins that were co-purified, the RasGAP-related protein IQGAP1 was shown to be a novel interaction partner of M-RasQ71L. We believe that this method is applicable to many proteins and will prove to be a valuable tool for the study of protein-protein interactions.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat

Tryptic digestion of ubiquitin standards reveals an improved strategy for identifying ubiquitinated proteins by mass spectrometry

Ubiquitination plays an essential role in maintaining cellular homeostasis by regulating a multitude of essential processes. The ability to identify ubiquitinated proteins by MS currently relies on a strategy in which ubiquitinated peptides are identified by a 114.1 Da diglycine (GG) tag on lysine residues, which is derived from the C-terminus of ubiquitin, following trypsin digestion. In the following study, we report a more comprehensive approach for mapping ubiquitination sites by trypsin digestion and MS/MS analysis. We demonstrate that ubiquitination sites can be identified by signature peptides containing a GG-tag (114.1 Da) and an LRGG-tag (383.2 Da) on internal lysine residues as well as a GG-tag found on the C-terminus of ubiquitinated peptides. Application of this MS-based approach enabled the identification of 96 ubiquitination sites from proteins purified from human MCF-7 breast cancer cells, representing a 2.4-fold increase in the number of ubiquitination sites that could be identified over standard methods. Our improved MS-based strategy will aid future studies which aim to identify and/or characterize ubiquitinated proteins in human cells

Differential proteomic screen to evidence proteins ubiquitinated upon mitotic exit in cell-free extract of Xenopus laevis embryos.

International audiencePost-translational modification of proteins via ubiquitination plays a crucial role in numerous vital functions of the cell. Polyubiquitination is one of the key regulatory processes involved in regulation of mitotic progression. Here we describe a differential proteomic screen dedicated to identification of novel proteins ubiquitinated upon mitotic exit in cell-free extract of Xenopus laevis embryo. Mutated recombinant His6-tagged ubiquitin (Ubi (K48R)) was added to mitotic extract from which we purified conjugated proteins, as well as associated proteins in nondenaturing conditions by cobalt affinity chromatography. Proteins eluted from Ubi (K48R) supplemented and control extracts were compared by LC-MS/MS analysis after monodimensional SDS-PAGE. A total of 144 proteins potentially ubiquitinated or associated with them were identified. Forty-one percent of these proteins were shown to be involved in ubiquitination and/or proteasomal degradation pathway confirming the specificity of the screen. Twelve proteins, among them ubiquitin itself, were shown to carry a "GG" or "LRGG" remnant tag indicating their direct ubiquitination. Interestingly, sequence analysis of ubiquitinated substrates carrying these tags indicated that in Xenopus cell-free embryo extract supplemented with Ubi (K48R) the majority of polyubiquitination occurred through lysine-11 specific ubiquitin chain polymerization. The potential interest in this atypical form of ubiquitination as well as usefulness of our method in analyzing atypical polyubiquitin species is discussed

Systematic determination of ion score cutoffs based on calculated false positive rates: Application for identifying ubiquitinated proteins by tandem mass spectrometry

We report a simple approach for determining ion score cutoffs that permit the confident identification of ubiquitinated proteins by tandem mass spectrometry (MS/MS). Initial experiments involving the analysis of gel bands containing multi-Ubiquitin chains with quadrupole time-of-flight and quadrupole ion trap mass spectrometers revealed that standard ion score cutoffs used for database searching were not sufficiently stringent. We also found that false positive and false negative rates (FPR and FNR) varied significantly depending on the cutoff scores used and that appropriate cutoffs could only be determined following a systematic evaluation of false positive rates. When standard cutoff scores were used for the analysis of complex mixtures of ubiquitinated proteins, unacceptably high FPR were observed. Finally, we found that FPR for ub