Affinity mass spectrometry-based approaches for the analysis of protein-protein interaction and complex mixtures of peptide-ligands.

Combined applications of affinity purification procedures and mass-spectrometric analyses (affinity mass spectrometry or affinity-directed mass spectrometry) have gained broad interest in various fields of biological sciences. We have extended these techniques to the purification and analysis of closely related peptides from complex mixtures and to the characterization of binding motifs and relative affinities in protein-protein interactions. The posttranslational modifications in the carboxy-terminal region of porcine brain tubulin are used as an example for the applicability of affinity mass spectrometry in the characterization of complex patterns of related peptides. We also show that affinity mass spectrometry allows the mapping of sequential binding motifs of two interacting proteins. Using the ActA/Mena protein-protein complex as a model system, we show that we can selectively purify Mena-binding peptides from a tryptic digest of ActA. The results from this assay are compared to data sets obtained earlier by classical methods using synthetic peptides and molecular genetic experiments. As a further expansion of affinity mass spectrometry, we have established an internally standardized system that allows comparison of the affinities of related ligands for a given protein. Here the affinities of two peptide ligands for the monoclonal tubulin-specific antibody YL1/2 are determined in terms of half-maximal competition.     

PUB-MS: a mass spectrometry-based method to monitor protein-protein proximity in vivo

The common techniques to study protein-protein proximity in vivo are not well adapted to the capabilities and the expertise of a standard proteomics laboratory, typically based on the use of mass spectrometry. With the aim of closing this gap, we have developed PUB-MS (for proximity utilizing biotinylation and mass spectrometry), an approach to monitor protein-protein proximity, based on biotinylation of a protein fused to a biotin-acceptor peptide (BAP) by a biotin-ligase, BirA, fused to its interaction partner. The biotinylation status of the BAP can be further detected by either Western analysis or mass spectrometry. The BAP sequence was redesigned for easy monitoring of the biotinylation status by LC-MS/MS. In several experimental models, we demonstrate that the biotinylation in vivo is specifically enhanced when the BAP- and BirA-fused proteins are in proximity to each other. The advantage of mass spectrometry is demonstrated by using BAPs with different sequences in a single experiment (allowing multiplex analysis) and by the use of stable isotopes. Finally, we show that our methodology can be also used to study a specific subfraction of a protein of interest that was in proximity with another protein at a predefined time before the analysis.     

Computational approaches to protein-protein interaction

The interactions between proteins allow the cell's life. A number of experimental, genome-wide, high-throughput studies have been devoted to the determination of protein-protein interactions and the consequent interaction networks. Here, the bioinformatics methods dealing with protein-protein interactions and interaction network are overviewed. 1. Interaction databases developed to collect and annotate this immense amount of data; 2. Automated data mining techniques developed to extract information about interactions from the published literature; 3. Computational methods to assess the experimental results developed as a consequence of the finding that the results of high-throughput methods are rather inaccurate; 4. Exploitation of the information provided by protein interaction networks in order to predict functional features of the proteins; and 5. Prediction of protein-protein interactions.     

Recent developments in mass spectrometry-based quantitative phosphoproteomics.

Protein phosphorylation is a reversible post-translational modification that is involved in virtually all eukaryotic cellular processes and has been studied in great detail in recent years. Many developments in mass spectrometry (MS)-based proteomics have been successfully applied to study protein phosphorylation in highly complicated samples. Furthermore, the emergence of a variety of enrichment strategies has allowed some of the challenges associated with low phosphorylation stoichiometry and phosphopeptide copy number to be overcome. The dynamic nature of protein phosphorylation complicates its analysis; however, a number of methods have been developed to successfully quantitate phosphorylation changes in a variety of cellular systems. The following review details some of the most recent breakthroughs in the study of protein phosphorylation, or phosphoproteomics, using MS-based approaches. The majority of the focus is placed on detailing strategies that are currently used to conduct MS-based quantitative phosphoproteomics.     

Recent advances in RNA-protein interaction studies

Conclusions The RNA binding sites for several small proteins have been characterised. These sites include double helical regions with hairpins, bulged bases and internal loops. As seen in Flock House virus structure, some proteins may recognise phosphate backbone of the canonical A-form helix not in a sequence-specific manner. If sequence-specific base contacts are to be made, then the A-helic major groove must be widened. This can be accomplished by introducing bulges, internal loops and hairpin loops into double helical regions. In these cases proteins may recognise both distorted backbone conformations and read out base sequences in a widened major groove. Crystallographic studies on complexes of aminoacyl-tRNA synthetase and tRNA showed that even RNAs with stable tertiary fold undergo substantial structural changes upon binding to the synthetases. The structural variability of RNA as well as the ability of RNA to distort upon protein binding may be crucial in RNA-protein interactions.     

Correction: Recent advances in biomedical applications of accelerator mass spectrometry

After publication of our article, it was noted that we inadvertently failed to include the complete list of authors. The full list, including co-authors, has now been added and the Authors' contributions and Competing interests sections modified accordingly.     

Recent advances in clustering methods for protein interaction networks

The increasing availability of large-scale protein-protein interaction data has made it possible to understand the basic components and organization of cell machinery from the network level. The arising challenge is how to analyze such complex interacting data to reveal the principles of cellular organization, processes and functions. Many studies have shown that clustering protein interaction network is an effective approach for identifying protein complexes or functional modules, which has become a major research topic in systems biology. In this review, recent advances in clustering methods for protein interaction networks will be presented in detail. The predictions of protein functions and interactions based on modules will be covered. Finally, the performance of different clustering methods will be compared and the directions for future research will be discussed.     

Recent advances in biomedical applications of accelerator mass spectrometry

The use of radioisotopes has a long history in biomedical science, and the technique of accelerator mass spectrometry (AMS), an extremely sensitive nuclear physics technique for detection of very low-abundant, stable and long-lived isotopes, has now revolutionized high-sensitivity isotope detection in biomedical research, because it allows the direct determination of the amount of isotope in a sample rather than measuring its decay, and thus the quantitative analysis of the fate of the radiolabeled probes under the given conditions. Since AMS was first used in the early 90's for the analysis of biological samples containing enriched ¹⁴C for toxicology and cancer research, the biomedical applications of AMS to date range from in vitro to in vivo studies, including the studies of 1) toxicant and drug metabolism, 2) neuroscience, 3) pharmacokinetics, and 4) nutrition and metabolism of endogenous molecules such as vitamins. In addition, a new drug development concept that relies on the ultrasensitivity of AMS, known as human microdosing, is being used to obtain early human metabolism information of candidate drugs. These various aspects of AMS are reviewed and a perspective on future applications of AMS to biomedical research is provided.     

Recent advances in mass spectrometry: data independent analysis and hyper reaction monitoring

New mass spectrometry (MS) methods, collectively known as data independent analysis and hyper reaction monitoring, have recently emerged. These methods hold promises to address the shortcomings of data-dependent analysis and selected reaction monitoring (SRM) employed in shotgun and targeted proteomics, respectively. They allow MS analyses of all species in a complex sample indiscriminately, or permit SRM-like experiments conducted with full high-resolution product ion spectra, potentially leading to higher sequence coverage or analytical selectivity. These methods include MS^E, all-ion fragmentation, Fourier transform-all reaction monitoring, SWATH Acquisition, multiplexed MS/MS, pseudo-SRM (pSRM) and parallel reaction monitoring (PRM). In this review, the strengths and pitfalls of these methods are discussed and illustrated with examples. In essence, the suitability of the use of each method is contingent on the biological questions posed. Although these methods do not fundamentally change the shape of proteomics, they are useful additional tools that should expedite biological discoveries.     

Target class strategies in mass spectrometry-based proteomics

The cornerstone of proteomics resides in using traditional methods of protein chemistry, to extract and resolve complex mixtures, in concert with the powerful engines of mass spectrometry to decipher peptide and protein identities. The broad utility of proteomics technologies to map protein interactions, understand regulatory mechanisms and identify biomarkers associated with disease states and drug treatments necessitates a targeted biochemical approach tailored to the characteristics of the tissue, fluid or cellular extract being studied. The application of affinity methods in proteomic studies to focus on particular classes of molecules is being used with increasing frequency and comprises the subject of this review. An overview of successfully applied affinity methods is provided, along with speculation on the use of innovative approaches. Sample preparation and processing are critical for proteomics with affinity reagents, as only functional and active proteins can be isolated in most cases. Considerations for methods of sample preparation to optimize affinity capture and release are also discussed.