Improved strategies for rapid identification of chemically cross-linked peptides using protein interaction reporter technology

Protein interaction reporter (PIR) technology can enable identification of in vivo protein interactions with the use of specialized chemical cross-linkers, liquid chromatography, and high-resolution mass spectrometry. PIR-cross-linkers contain labile bonds that are specifically fragmented under low energy collision or photodissociation conditions in the mass spectrometer source, thus releasing cross-linked peptides. Successful analysis of PIR-cross-linked proteins requires the use of expected mathematical relationships between cross-linked complexes and released peptides after fragmentation of the labile PIR bonds. Presented here is a next-generation software tool, BLinks, for use in the analysis and identification of PIR-cross-linked proteins. BLinks is an advancement beyond our previous efforts by incorporation of chromatographic profiles that must match between cross-linked complexes and released peptides to enable estimation of p-values to help filter true relationships from complex data sets. Additionally, BLinks was used to incorporate Mascot database searching results from subsequent MS/MS analysis of the released peptides to facilitate identification of cross-linked proteins. BLinks was used in the analysis of human serum albumin, and 46 interpeptide relationships were found spanning 30 proximal residues with a 2.2% false discovery rate. BLinks was also used to track peptides involved in multiple, coeluting relationships that make accurate identification of protein interactions difficult. An additional 10 interpeptide relationships were identified despite poor correlation using the profiling tools provided with BLinks. Additionally, BLinks can be used to globally map all interpeptide relationships from the data analysis and customize subsequent analysis to target specific peptides of interest, thus making it a useful tool for both discovery of protein interactions and mapping protein topology.     

Structural analysis of multiprotein complexes by cross-linking, mass spectrometry, and database searching

Most protein complexes are inaccessible to high resolution structural analysis. We report the results of a combined approach of cross-linking, mass spectrometry, and bioinformatics to two human complexes containing large coiled-coil segments, the NDEL1 homodimer and the NDC80 heterotetramer. An important limitation of the cross-linking approach, so far, was the identification of cross-linked peptides from fragmentation spectra. Our novel approach overcomes the data analysis bottleneck of cross-linking and mass spectrometry. We constructed a purpose-built database to match spectra with cross-linked peptides, define a score that expresses the quality of our identification, and estimate false positive rates. We show that our analysis sheds light on critical structural parameters such as the directionality of the homodimeric coiled coil of NDEL1, the register of the heterodimeric coiled coils of the NDC80 complex, and the organization of a tetramerization region in the NDC80 complex. Our approach is especially useful to address complexes that are difficult in addressing by standard structural methods.     

Protein cross-linking analysis using mass spectrometry, isotope-coded cross-linkers, and integrated computational data processing

Distance constraints in proteins and protein complexes provide invaluable information for calculation of 3D structures, identification of protein binding partners and localization of protein-protein contact sites. We have developed an integrative approach to identify and characterize such sites through the analysis of proteolytic products derived from proteins chemically cross-linked by isotopically coded cross-linkers using LC-MALDI tandem mass spectrometry and computer software. This method is specifically tailored toward the rapid analysis of low microgram amounts of proteins or multimeric protein complexes cross-linked with nonlabeled and deuterium-labeled bis-NHS ester cross-linking reagents (both commercially available and readily synthesized). Through labeling with [18O]water solvent and LC-MALDI analysis, the method further allows the possible distinction between Type 0 and Type 1 or Type 2 modified peptides (monolinks and looplinks or cross-links), although such a distinction is more readily made from analysis of tandem mass spectrometry data. When applied to the bacterial Colicin E7 DNAse/Im7 heterodimeric protein complex, 23 cross-links were identified including six intersubunit cross-links, all between residues that are close in space when examined in the context of the X-ray structure of the heterodimer. In addition, cross-links were successfully identified in five single subunit proteins, beta-lactoglobulin, cytochrome c, lysozyme, myoglobin, and ribonuclease A, establishing the generality of the approach.     

Experimental analysis of co‐evolution within protein complexes: The yeast exosome as a model

Extensive bioinformatics analysis suggests that the stability and function of protein complexes are maintained throughout evolution by coordinated changes (co‐evolution) of complex subunits. Yet, relatively little is known regarding the actual dynamics of such processes and the functional implications of co‐evolution within protein complexes, since most of the bioinformatics predictions were not analyzed experimentally. Here, we describe a systematic experimental approach that allows a step‐by‐step observation of the co‐evolution process in protein complexes. The exosome complex, an essential complex exhibiting a 3′→5′ RNA degradation activity, served as a model system. In this study, we show that exosome subunits diverged very early during fungal evolution. Interestingly, we found that despite significant differences in conservation between Rrp41 and Mtr3 both subunits exhibit similar divergence pattern and co‐evolutionary behavior through fungi evolution. Activity analysis of mutated exosomes exposes another layer of co‐evolution between the core subunits and RNA substrates. Overall, our approach allows the experimental analysis of co‐evolution within protein complexes and together with bioinformatics analysis can significantly deepen our understanding of the evolution of these complexes. Proteins 2013; 81:1997–2006. © 2013 Wiley Periodicals, Inc.     

Tandem repeats in proteins: from sequence to structure

The bioinformatics analysis of proteins containing tandem repeats requires special computer programs and databases, since the conventional approaches predominantly developed for globular domains have limited success. Here, I survey bioinformatics tools which have been developed recently for identification and proteome-wide analysis of protein repeats. The last few years have also been marked by an emergence of new 3D structures of these proteins. Appraisal of the known structures and their classification uncovers a straightforward relationship between their architecture and the length of the repetitive units. This relationship and the repetitive character of structural folds suggest rules for better prediction of the 3D structures of such proteins. Furthermore, bioinformatics approaches combined with low resolution structural data, from biophysical techniques, especially, the recently emerged cryo-electron microscopy, lead to reliable prediction of the protein repeat structures and their mode of binding with partners within molecular complexes. This hybrid approach can actively be used for structural and functional annotations of proteomes.     

Proteome informatics I: bioinformatics tools for processing experimental data

Bioinformatics tools for proteomics, also called proteome informatics tools, span today a large panel of very diverse applications ranging from simple tools to compare protein amino acid compositions to sophisticated software for large-scale protein structure determination. This review considers the available and ready to use tools that can help end-users to interpret, validate and generate biological information from their experimental data. It concentrates on bioinformatics tools for 2-DE analysis, for LC followed by MS analysis, for protein identification by PMF, by peptide fragment fingerprinting and by de novo sequencing and for data quantitation with MS data. It also discloses initiatives that propose to automate the processes of MS analysis and enhance the quality of the obtained results.     

Protein topography of the 40 S ribosomal subunit from Saccharomyces cerevisiae as shown by chemical cross-linking

Protein-protein cross-linking was used to examine the spatial arrangement of proteins within the 40 S ribosomal subunits of Saccharomyces cerevisiae. Purified ribosomal subunits were treated with either 2-iminothiolane or dimethyl 3,3'-dithiobispropionimidate under conditions such that the ribosomal particle was intact and that formation of 40 S subunit dimers was minimized. Proteins were extracted from the treated subunits and fractionated on Sephadex G-150 or by acid-urea-polyacrylamide gel electrophoresis. Cross-linked proteins in these fractions were analyzed by two-dimensional diagonal sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Constituent members of cross-linked pairs were radiolabeled with 125I and identified by two-dimensional gel electrophoresis and comparison with nonradioactive ribosomal protein markers. Forty-two pairs involving 25 of the 32 40 S subunit proteins were identified. Many proteins were detected in several cross-linked dimers. These proteins with multiple cross-links form foci for the construction of a schematic model of the spatial arrangement of proteins within the 40 S subunit.     

Mapping the interaction of the [2Fe-2S] Clostridium pasteurianum ferredoxin with nitrogenase MoFe protein

The [2Fe-2S] ferredoxin from Clostridium pasteurianum had previously been shown to interact specifically with the nitrogenase MoFe protein, and electrostatic forces were found to be important contributors to the interaction. This phenomenon has now been analyzed in detail by using ferredoxin variants in which charge inversions or cancellations were introduced on all charged residues. The mutated forms of the ferredoxin were covalently cross-linked to the MoFe protein. The reaction products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and their nitrogenase activity was measured. The latter displayed a consistent inverse correlation with the amount of cross-linked MoFe protein. The data allowed an unambiguous identification of the ferredoxin residues (glutamates 31, 34, 38, 39, 84, 85) that are involved in the interaction with the MoFe protein. Furthermore, whereas the wild-type ferredoxin yielded approximately equal amounts of cross-linked products with the alpha and beta subunits of the MoFe protein, some of its molecular variants displayed a differential decrease of reactivity towards one or the other of these subunits. The positions on the ferredoxin molecule of the residues interacting with the MoFe protein were determined using the recently elucidated crystal structure of the homologous [2Fe-2S] ferredoxin from Aquifex aeolicus.     

Isotopically coded cleavable cross-linker for studying protein-protein interaction and protein complexes

An emerging approach for studying protein-protein interaction in complexes is the combination of chemical cross-linking and mass spectrometric analysis of the cross-linked peptides (cross-links) obtained after proteolysis of the complex. This approach, however, has several challenges and limitations, including the difficulty of detecting the cross-links, the potential interference from non-informative "cross-linked peptides" (dead end and intrapeptide cross-links), and unambiguous identification of the cross-links by mass spectrometry. Thus, we have synthesized an isotopically coded ethylene glycol bis(succinimidylsuccinate) derivate (D12-EGS), which contains 12 deuterium atoms for easy detection of cross-links when applied in a 1:1 mixture with its H12 counterpart and is also cleavable for releasing the cross-linked peptides allowing unambiguous identification by MS sequencing. Moreover, hydrolytic cleavage permits rapid distinguishing between different types of cross-links. Cleavage of a dead end cross-link produces a doublet with peaks 4.03 Da apart, with the lower peak appearing at a molecular mass 162 Da lower than the mass of the H12 form of the original cross-linked peptide. Cleavage of an intrapeptide cross-link leads to a doublet 8.05 Da apart and 62 Da lower than the molecular mass of the H12 form of the original cross-linked peptide. Cleavage of an interpeptide cross-link forms a pair of 4.03-Da doublets, with the lower mass member of each pair each shifted up from its unmodified molecular weight by 82 Da because of the attached portion of the cross-linker. All of this information has been incorporated into a software algorithm allowing automatic screening and detection of cross-links and cross-link types in matrix-assisted laser desorption/ionization mass spectra. In summary, the ease of detection of these species through the use of an isotopically coded cleavable cross-linker and our software algorithm, followed by mass spectrometric sequencing of the cross-linked peptides after cleavage, has been shown to be a powerful tool for studies of multi-component protein complexes.     

Characterization of an antagonist interleukin-6 dimer by stable isotope labeling,cross-linking,and mass spectrometry

The homodimeric form of a recombinant cytokine interleukin-6 (IL-6(D)) is known to antagonize IL-6 signaling. In this study, spatially proximal residues between IL-6 chains in IL-6(D) were identified using a method for specific recognition of intermolecular cross-linked peptides. Our strategy involved mixing 1:1 (15)N-labeled and unlabeled ((14)N) protein to form a mixture of isotopically labeled and unlabeled homodimers, which was chemically cross-linked. This cross-linked IL-6(D) was subjected to proteolysis by trypsin and the generated peptides were analyzed by electrospray ionization time-of-flight mass spectrometry (MS). Molecular ions from cross-linked peptides of intermolecular origin are labeled with [(15)N/(15)N] + [(15)N/(14)N] + [(14)N/(15)N] + [(14)N/(14)N] yielding readily identified triplet/quadruplet MS peaks. All other peptide species are labeled with [(15)N] + [(14)N] yielding doublet peaks. Intermolecular cross-linked peptides were identified by MS, and cross-linked residues were identified. This intermolecular cross-link detection method, which we have designated "mixed isotope cross-linking" MIX may have more general application to protein-protein interaction studies. The pattern of proximal residues found was consistent with IL-6(D) having a domain-swapped fold similar to IL-10 and interferon-gamma. This fold implies that IL-6(D)-mediated antagonism of IL-6 signaling is caused by obstruction of cooperative gp130 binding on IL-6(D), rather than direct blocking of gp-130-binding sites on IL-6(D).     

A novel strategy for defining critical amino acid residues involved in protein/glycosaminoglycan interactions

The binding of proteins to glycosaminoglycans (GAGs) is the prerequisite for a large number of cellular processes and regulatory events and is associated to many pathologies. However, progress in the understanding of these mechanisms has been hampered by the lack of simple and comprehensive analytical tools for the identification of the structural attributes involved in protein/saccharide interaction. Characterization of GAG binding motifs on proteins has so far relied on site-directed mutagenesis studies, protein sequence mapping using synthetic peptides, molecular modeling, or structural analysis. Here, we report the development of a novel approach for identifying protein residues involved in the binding to heparin, the archetypal member of the GAG family. This method, which uses native proteins, is based on the formation of cross-linked complexes of the protein of interest with heparin beads, the proteolytic digestion of these complexes, and the subsequent identification of the heparin binding containing peptides by N terminus sequencing. Analysis of the CC chemokine regulated on activation, normal T-cell expressed, and secreted (RANTES), the envelope glycoprotein gC from pseudorabies virus and the laminin-5 alpha 3LG4/5 domain validated the techniques and provided novel information on the heparin binding motifs present within these proteins. Our results highlighted this method as a fast and valuable alternative to existing approaches. Application of this technique should greatly contribute to facilitate the structural study of protein/GAG interactions and the understanding of their biological functions.     

Isolation of thylakoid membrane complexes from rice by a new double-strips BN/SDS-PAGE and bioinformatics prediction of stromal ridge subunits interaction

Thylakoid membrane complexes of rice (Oryza sativa L.) play crucial roles in growth and crop production. Understanding of protein interactions within the complex would provide new insights into photosynthesis. Here, a new "Double-Strips BN/SDS-PAGE" method was employed to separate thylakoid membrane complexes in order to increase the protein abundance on 2D-gels and to facilitate the identification of hydrophobic transmembrane proteins. A total of 58 protein spots could be observed and subunit constitution of these complexes exhibited on 2D-gels. The generality of this new approach was confirmed using thylakoid membrane from spinach (Spinacia oleracea) and pumpkin (Cucurita spp). Furthermore, the proteins separated from rice thylakoid membrane were identified by the mass spectrometry (MS). The stromal ridge proteins PsaD and PsaE were identified both in the holo- and core- PSI complexes of rice. Using molecular dynamics simulation to explore the recognition mechanism of these subunits, we showed that salt bridge interactions between residues R19 of PsaC and E168 of PasD as well as R75 of PsaC and E91 of PsaD played important roles in the stability of the complex. This stromal ridge subunits interaction was also supported by the subsequent analysis of the binding free energy, the intramolecular distances and the intramolecular energy.     

Computational Analysis of C-Reactive Protein for Assessment of Molecular Dynamics and Interaction Properties

Serum C-reactive protein (CRP) is used as a marker of inflammation in several diseases including autoimmune disease and cardiovascular disease. CRP, a member of the pentraxin family, is comprised of five identical subunits. CRP has diverse ligand-binding properties which depend upon different structural states of CRP. However, little is known about the molecular dynamics and interaction properties of CRP. In this study, we used SAPS, SCRATCH protein predictor, PDBsum, ConSurf, ProtScale, Drawhca, ASAView, SCide and SRide server and performed comprehensive analyses of molecular dynamics, protein–protein and residue–residue interactions of CRP. We used 1GNH.pdb file for the crystal structure of human CRP which generated two pentamers (ABCDE and FGHIJ). The number of residues involved in residue–residue interactions between A–B, B–C, C–D, D–E, F–G, G–H, H–I, I–J, A–E and F–J subunits were 12, 11, 10, 11, 12, 11, 10, 11, 10 and 10, respectively. Fifteen antiparallel β sheets were involved in β-sheet topology, and five β hairpins were involved in forming the secondary structure. Analysis of hydrophobic segment distribution revealed deviations in surface hydrophobicity at different cavities present in CRP. Approximately 33 % of all residues were involved in the stabilization centers. We show that the bioinformatics tools can provide a rapid method to predict molecular dynamics and interaction properties of CRP. Our prediction of molecular dynamics and interaction properties of CRP combined with the modeling data based on the known 3D structure of CRP is helpful in designing stable forms of CRP mutants for structure–function studies of CRP and may facilitate in silico drug design for therapeutic targeting of CRP.     

Resistivity to denaturation of the apoprotein of aequorin and reconstitution of the luminescent photoprotein from the partially denatured apoprotein.

A dimeric glycoprotein, glucose oxidase, was allowed to react with lysine-specific cross-linkers, both when immobilized on a succinoylated lectin matrix at a critically low density and also at a high density in solution. Analysis of the cross-linked complexes thus obtained led to the following inferences with regard to the structure of this protein. (1) Of the 15 lysine residues on each glucose oxidase protomer, none is available on the non-interfacial surfaces. (2) Assuming that this protein possesses C2 symmetry with isologous bonding between subunits, it may be inferred that on each promoter there are at least two lysine clusters along or close to the interprotomeric interface. (3) These "interfacial' lysine residues on each protomer are so oriented that the epsilon-amino groups of lysine residues a and b on protomer 1 "face', and are very close to, the epsilon-amino groups of lysine residues b' and a' respectively on protomer 2. General inferences on the geometry of dimeric proteins derivable from an analysis of the cross-linked complexes obtained (as well as those not seen) by using this low-density matrix cross-linking approach were enumerated. Modified lectin matrices may prove useful in studying the three-dimensional structure of glycoproteins, particularly non-crystallizable oligomers.     

Size distribution of native cytosolic proteins of Thermoplasma acidophilum

We used molecular sieve chromatography in combination with LC‐MS/MS to identify protein complexes that can serve as templates in the template matching procedures of visual proteomics approaches. By this method the sample complexity was lowered sufficiently to identify 464 proteins and – on the basis of size distribution and bioinformatics analysis – 189 of them could be assigned as subunits of macromolecular complexes over the size of 300 kDa. From these we purified six stable complexes of Thermoplasma acidophilum whose size and subunit composition – analyzed by electron microscopy and MALDI‐TOF‐MS, respectively – verified the accuracy of our method.     

Unraveling tobacco BY-2 protein complexes with BN PAGE/LC-MS/MS and clustering methods

To understand physiological processes, insight into protein complexes is very important. Through a combination of blue native gel electrophoresis and LC-MS/MS, we were able to isolate protein complexes and identify their potential subunits from Nicotiana tabacum cv. Bright Yellow-2. For this purpose, a bioanalytical approach was used that works without a priori knowledge of the interacting proteins. Different clustering methods (e.g., k-means and hierarchical clustering) and a biclustering approach were evaluated according to their ability to group proteins by their migration profile and to correlate the proteins to a specific complex. The biclustering approach was identified as a very powerful tool for the exploration of protein complexes of whole cell lysates since it allows for the promiscuous nature of proteins. Furthermore, it searches for associations between proteins that co-occur frequently throughout the BN gel, which increases the confidence of the putative associations between co-migrating proteins. The statistical significance and biological relevance of the profile clusters were verified using functional gene ontology annotation. The proof of concept for identifying protein complexes by our BN PAGE/LC-MS/MS approach is provided through the analysis of known protein complexes. Both well characterized long-lived protein complexes as well as potential temporary sequential multi-enzyme complexes were characterized.     

The pitfalls of proteomics experiments without the correct use of bioinformatics tools

The elucidation of the entire genomic sequence of various organisms, from viruses to complex metazoans, most recently man, is undoubtedly the greatest triumph of molecular biology since the discovery of the DNA double helix. Over the past two decades, the focus of molecular biology has gradually moved from genomes to proteomes, the intention being to discover the functions of the genes themselves. The postgenomic era stimulated the development of new techniques (e.g. 2-DE and MS) and bioinformatics tools to identify the functions, reactions, interactions and location of the gene products in tissues and/or cells of living organisms. Both 2-DE and MS have been very successfully employed to identify proteins involved in biological phenomena (e.g. immunity, cancer, host-parasite interactions, etc.), although recently, several papers have emphasised the pitfalls of 2-DE experiments, especially in relation to experimental design, poor statistical treatment and the high rate of 'false positive' results with regard to protein identification. In the light of these perceived problems, we review the advantages and misuses of bioinformatics tools - from realisation of 2-DE gels to the identification of candidate protein spots - and suggest some useful avenues to improve the quality of 2-DE experiments. In addition, we present key steps which, in our view, need to be to taken into consideration during such analyses. Lastly, we present novel biological entities named 'interactomes', and the bioinformatics tools developed to analyse the large protein-protein interaction networks they form, along with several new perspectives of the field.     

Properly reading the histone code by MS‐based proteomics

Histone proteins are essential elements for DNA packaging. Their PTMs contribute in modeling chromatin structure and recruiting enzymes involved in gene regulation, DNA repair, and chromosome condensation. This fundamental aspect, together with the fact that histone PTMs can be epigenetically inherited through cell generations, enlightens their importance in chromatin biology, and the consequent necessity of having biochemical techniques for their characterization. Nanoflow LC coupled to MS (nanoLC‐MS) is the strategy of choice for protein PTM accurate quantification. However, histones require adjustments to the digestion protocol such as lysine derivatization to obtain suitable peptides for the analysis. nanoLC‐MS has numerous advantages, spanning from high confidence identification to possibility of high throughput analyses, but the peculiarity of the histone preparation protocol requires continuous monitoring with the most modern available technologies to question its reliability. The work of Meert et al. (Proteomics 2015, 15, 2966–2971) establishes which protocols lead to either incomplete derivatization or derivatization of undesired amino acid residues using a combination of high resolution MS and bioinformatics tools for the alignment and the characterization of nanoLC‐MS runs. As well, they identify a number of side reactions that could be potentially misinterpreted as biological PTMs.