Footprinting Techniques Research Articles

LiF-MS+, a revised technique for mapping peptide-protein interactions.

Short linear motifs are sequences of amino acids present in unstructured polypeptide regions that function as ligands for specific sites on folded protein domains. These interactions, which often occur with low to modest affinity, modulate dynamic biological processes such as signal transduction and membrane trafficking. We recently described Ligand Footprinting-Mass Spectrometry (LiF-MS), a technique that rapidly and precisely maps sites at which short peptide ligands bind their biologically relevant recognition sites on folded protein domains. This approach marks the binding location of a peptide ligand on a structured protein using a cleavable crosslinker appended to the ligand that leaves behind a stable chemical modification following cleavage. This modification serves as a mass tag detectable by mass spectrometry, pinpointing sites of peptide ligand binding. Here we present LiF-MS+, an improved version of the footprinting technique that replaces the butanol mass tag with 1-butylpyrrolidine, which is positively charged at neutral pH and thus aids in ionization of the crosslinked peptide for analysis by mass spectrometry. We show ligand-mediated butylpyrrolidine footprinting effectively maps the well characterized binding interaction of the p38α mitogen-activated protein kinase (MAPK) with a MKK6 D-motif short linear motif peptide ligand, uncovering additional binding site information not observed in our original experiment. LiF-MS+ is thus a straightforward improvement of our previously published methodology for mapping the binding of short linear motifs to folded protein domains.

Molecular Mechanism of Nucleosome Recognition by the Pioneer Transcription Factor Sox.

Pioneer transcription factors (PTFs) have the remarkable ability to directly bind to chromatin to stimulate vital cellular processes. In this work, we dissect the universal binding mode of Sox PTF by combining extensive molecular simulations and physiochemistry approaches, along with DNA footprinting techniques. As a result, we show that when Sox consensus DNA is located at the solvent-facing DNA strand, Sox binds to the compact nucleosome without imposing any significant conformational changes. We also reveal that the base-specific Sox:DNA interactions (base reading) and Sox-induced DNA changes (shape reading) are concurrently required for sequence-specific nucleosomal DNA recognition. Among three different nucleosome positions located on the positive DNA arm, a sequence-specific reading mechanism is solely satisfied at the superhelical location 2 (SHL2). While SHL2 acts transparently for solvent-facing Sox binding, among the other two positions, SHL4 permits only shape reading. The final position, SHL0 (dyad), on the other hand, allows no reading mechanism. These findings demonstrate that Sox-based nucleosome recognition is essentially guided by intrinsic nucleosome properties, permitting varying degrees of DNA recognition.

The Deuterium Calculator: An Open-Source Tool for Hydrogen-Deuterium Exchange Mass Spectrometry Analysis.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful protein footprinting technique to study protein dynamics and binding; however, HDX-MS data analysis is often challenging and time-consuming. Moreover, the HDX community is expanding to investigate multiprotein and highly complex protein systems which further complicates data analysis. Thus, a simple, open-source software package designed to analyze large and highly complex protein systems is needed. In this vein, we have developed "The Deuterium Calculator", a Python-based software package for HDX-MS data analysis. The Deuterium Calculator is capable of differential and nondifferential HDX-MS analysis, produces standardized data files according to recommendations from the International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX) to increase transparency in data analysis, and generates Woods' plots for statistical analysis and data visualization. This standard output can be used to perform time dependent deuteration studies and for the study of protein folding kinetics or differential uptake. Moreover, The Deuterium Calculator is capable of performing these analyses on large HDX-MS data sets (e.g., LC-HDX-MS from cell lysate digest). The Deuterium Calculator is freely available for download at https://github.com/OUWuLab/TheDeuteriumCalculator.git. Data are available via ProteomeXchange with identifier PXD036813.

Investigating Antimicrobial Peptide-Membrane Interactions Using Fast Photochemical Oxidation of Peptides in Nanodiscs.

Antimicrobial peptides (AMPs) are an important part of the innate immune system and demonstrate promising applications in the fight against antibiotic-resistant infections due to their unique mechanism of targeting bacterial membranes. However, it is challenging to study the interactions of these peptides within lipid bilayers, making it difficult to understand their mechanisms of toxicity and selectivity. Here, we used fast photochemical oxidation of peptides, an irreversible footprinting technique that labels solvent accessible residues, and native charge detection-mass spectrometry to study AMP-lipid interactions with different lipid bilayer nanodiscs. We observed differences in the oxidation of two peptides, indolicidin and LL-37, in three distinct lipid environments, which reveal their affinity for lipid bilayers. Our findings suggest that indolicidin interacts with lipid head groups via a simple charge-driven mechanism, but LL-37 is more specific for Escherichia coli nanodiscs. These results provide complementary information on the potential modes of action and lipid selectivity of AMPs.

A heterotrinuclear bioinspired coordination complex capable of binding to DNA and emulation of nuclease activity

The investigation of compounds capable of strongly and selectively interacting with DNA comprises a field of research in constant development. In this work, we demonstrate that a trinuclear coordination complex based on a dinuclear Fe(III)Zn(II) core designed for biomimicry of the hydrolytic enzyme kidney bean purple acid phosphatase, containing an additional pendant arm coordinating a Pd(II) ion, has the ability to interact with DNA and to promote its hydrolytic cleavage. These results were found through analysis of plasmid DNA interaction and cleavage by the trinuclear complex 1 and its derivatives 2 and 3, in addition to the analysis of alteration in the DNA structure in the presence of the complexes through circular dichroism and DNA footprinting techniques. The suggested covalent interaction of the palladium-containing complex with DNA was analysed using an electrophoretic mobility assay, circular dichroism, high resolution gel separation techniques and kinetic analysis. This is a new and promising metal complex targeted to nucleic acids and acting in two separate ways: strong DNA interaction and hydrolytic cleavage.

Structurally distinct external solvent-exposed domains drive replication of major human prions.

There is a limited understanding of structural attributes that encode the iatrogenic transmissibility and various phenotypes of prions causing the most common human prion disease, sporadic Creutzfeldt-Jakob disease (sCJD). Here we report the detailed structural differences between major sCJD MM1, MM2, and VV2 prions determined with two complementary synchrotron hydroxyl radical footprinting techniques—mass spectrometry (MS) and conformation dependent immunoassay (CDI) with a panel of Europium-labeled antibodies. Both approaches clearly demonstrate that the phenotypically distant prions differ in a major way with regard to their structural organization, and synchrotron-generated hydroxyl radicals progressively inhibit their seeding potency in a strain and structure-specific manner. Moreover, the seeding rate of sCJD prions is primarily determined by strain-specific structural organization of solvent-exposed external domains of human prion particles that control the seeding activity. Structural characteristics of human prion strains suggest that subtle changes in the organization of surface domains play a critical role as a determinant of human prion infectivity, propagation rate, and targeting of specific brain structures.

Mapping the interaction between eukaryotic initiation factor 4A (eIF4A) and the inhibitor hippuristanol using carbene footprinting and mass spectrometry.

Protein-ligand interactions are central to protein activity and cell functionality. Improved knowledge of these relationships greatly benefits our understanding of key biological processes and aids in rational drug design towards the treatment of clinically relevant diseases. Carbene footprinting is a recently developed mass spectrometry-based chemical labelling technique that provides valuable information relating to protein-ligand interactions, such as the mapping of binding sites and associated conformational change. Here, we show the application of carbene footprinting to the interaction between eIF4A helicase and a natural product inhibitor, hippuristanol, found in the coral Isis hippuris. Upon addition of hippuristanol we identified reduced carbene labelling (masking) in regions of eIF4A previously implicated in ligand binding. Additionally, we detected hippuristanol-associated increased carbene labelling (unmasking) around the flexible hinge region of eIF4A, indicating ligand-induced conformational change. This work represents further development of the carbene footprinting technique and demonstrates its potential in characterising medicinally relevant protein-ligand interactions.

Mapping the Interactions of PKNB with Small Molecule Inhibitors using Plasma Induced Modifications of Biomolecules (PLIMB)

Newly advanced PLIMB (Plasma Induced Modifications of Biomolecules) technology provides a uniquely powerful method for mapping protein-protein and protein-ligand interactions. PLIMB is a mass spectrometry (MS)-based protein footprinting technique that generates μs bursts of hydroxyl radicals from water, to measure changes in protein structure via altered solvent accessibility of amino acid side chains. Here, we utilized PLIMB to characterize the interactions of PknB, a mycobacterial Ser/Thr kinase with two small molecule inhibitors, GSK690693 and GW779439X.

Analisis Keamanan Website Menggunakan Teknik Footprinting dan Vulnerability Scanning


 
 
 Website security is an effort to protect websites from hacker attacks connected through a network. Website security needs to be a concern in the midst of many cases of website hacking from irresponsible people, including websites at Indonesian tertiary institutions that provide information and services to the public, students, and alumni of tertiary institutions.
 Higher education institution websites that can be accessed widely online can create vulnerabilities against threats from hacker attacks. To minimize this vulnerability, it is necessary to test the website of the higher education institution to assess and evaluate the security system of the university's website. The research method used in this study is the Ethical Hacking method which focuses on footprinting techniques and vulnerability scanning by only testing passive attacks. The results of this study have found information related to the target website (the website of an educational institution in one of the cities in Indonesia) and several vulnerability alerts after testing vulnerability scanning with high to low risk levels so that researchers recommend improving vulnerability to minimize security holes exploited by hackers .
 
 

Parallel Chemoselective Profiling for Mapping Protein Structure

Solution-based structural techniques complement high-resolution structural data by providing insight into the oft-missed links between protein structure and dynamics. Here, we present Parallel Chemoselective Profiling, a solution-based structural method for characterizing protein structure and dynamics. Our method utilizes deep mutational scanning saturation mutagenesis data to install amino acid residues with specific chemistries at defined positions on the solvent-exposed surface of a protein. Differences in the extent of labeling of installed mutant residues are quantified using targeted mass spectrometry, reporting on each residue's local environment and structural dynamics. Using our method, we studied how conformation-selective, ATP-competitive inhibitors affect the local and global structure and dynamics of full-length Src kinase. Our results highlight how parallel chemoselective profiling can be used to study a dynamic multi-domain protein, and suggest that our method will be a useful addition to the relatively small toolkit of existing protein footprinting techniques.

Analysis of the In Vivo Translation Process in Trypanosoma cruzi Using Ribosome Profiling.

The technique of ribosome profiling is based on the isolation of sequences around 30 nucleotides in size protected by mRNA-associated ribosomes, following digestion with specific nucleases, generating a footprint. After isolation and purification, these 30-nucleotide sequences are converted to a cDNA library and analyzed by deep sequencing, providing a high-precision picture of the translation process in vivo. In addition, this powerful technique allows for the study of several biological phenomena such as alternative splicing, alternative codon usage and initiation of translation by non-AUG codons. Furthermore, the ribosome footprinting technique has proved to be very efficient for studies of ribosome pause sites on mRNAs, which could act as key regulators in the translation process. Here we describe a modified protocol of the ribosome footprinting technique for translation efficiency analysis in Trypanosoma cruzi.

Implementing In-Cell Fast Photochemical Oxidation of Proteins in a Platform Incubator with a Movable XY Stage.

Fast photochemical oxidation of proteins (FPOP) is a protein footprinting technique that is being increasingly used in MS-based proteomics. FPOP is utilized to study protein–protein interactions, protein–ligand interactions, and protein conformational dynamics. This method has recently been extended to protein labeling in live cells (IC-FPOP), allowing the study of protein conformations in the complex cellular environment. Traditionally, IC-FPOP has been executed using a single cell flow system, in which hydrodynamic focusing drives cells along in a single file line, keeping the cells from clumping and thus ensuring equal exposure to the laser irradiation required for photochemical oxidation. Here, we introduce a novel platform that allows IC-FPOP to occur in a sterile incubation system complete with a mobile stage for XY movement, peristaltic pumps equipped with perfusion lines for chemical transport, and mirrors for laser beam guidance. This new system, called Platform Incubator with movable XY stage (PIXY), also utilizes software enabling automated communication between equipment and execution of the entire system. Further, comparison with a standard IC-FPOP flow system results reveal that this platform can successfully be used in lieu of the flow system while also decreasing the time to complete analysis of a single sample.

Insights on the Conformational Ensemble of Cyt C Reveal a Compact State during Peroxidase Activity

Cytochrome c (cyt c) is known for its role in the electron transport chain but transitions to a peroxidase-active state upon exposure to oxidative species. The peroxidase activity ultimately results in the release of cyt c into the cytosol for the engagement of apoptosis. The accumulation of oxidative modifications that accompany the onset of the peroxidase function are well-characterized. However, the concurrent structural and conformational transitions of cyt c remain undercharacterized. Fast photochemical oxidation of proteins (FPOP) coupled with mass spectrometry is a protein footprinting technique used to structurally characterize proteins. FPOP coupled with native ion mobility separation shows that exposure to H2O2 results in the accumulation of a compact state of cyt c. Subsequent top-down fragmentation to localize FPOP modifications reveals changes in heme coordination between conformers. A time-resolved functional assay suggests that this compact conformer is peroxidase active. Altogether, combining FPOP, ion mobility separation, and top-down and bottom-up mass spectrometry allows us to discern individual conformations in solution and obtain a better understanding of the conformational ensemble and structural transitions of cyt c as it transitions from a respiratory role to a proapoptotic role.

Metabolic pathway analysis and dynamic macroscopic model development for lovastatin production by Monascus purpureus using metabolic footprinting concept

Understanding and modeling of the metabolic pathway of a cell by probing the fraction of extracellular metabolites is gaining much interest in biological reactions. This communication deals with metabolic footprinting technique to understand the effect of static- and shake-culture conditions on the production of lovastatin by Monascus purpureus by probing the predominant exometabolites. A strong glycolytic flux pattern was observed in the shake culture of M. purpureus that resulted in the accumulation of tricarboxylic acids such as, citric acid, succinic acid, and oxalic acid, apart from glycerol and ethanol. Interestingly, those exometabolites were most probably utilized for enhanced production of lovastatin in shake culture after 10 days of fermentation. This suggests a correlation with the higher production of lovastatin in shake culture compared to that of in static-culture. The predominant metabolic network was developed from metabolic footprinting technique. Metabolic flux analysis was performed to check the consistency of the proposed metabolic cascade. A reduced dynamic metabolic model was developed from eight elementary macro-reactions. Predicted specific substrate utilization and product excretion rates have also been correlated well with the experimental observations, which validate the proposed metabolic pathway developed from metabolic footprinting data.

A simple MS method to characterize the higher order structures of antibody therapeutics

A scheme with enhanced throughput using the hydroxyl radical foot-printing technique to map the higher order structures (HOSs) of antibody therapeutics was developed. This method takes advantage of the simplicity of multiple reaction monitoring (MRM) by monitoring the remaining unmodified peptides, thus overcoming the complexity of the data analysis of the conventional free radical foot-printing technique, wherein various oxidized forms of the peptides are measured. In this study, a preliminary method validation was carried out to show that the technique has good specificity, sensitivity, and repeatability. Finally, we were able to use the method to differentiate the HOSs of the disulfide isoforms of the IgG2 antibody, provide site-specific structural differences between IgG1 and its deglycosylated counterpart, and determine similarities between a biosimilar candidate and the originator product.

What does “green quality” really mean?

PurposeThe purpose of this paper is to identify various dimensions of green quality. It integrates the existing carbon footprinting technique with the eight dimensions of quality proposed by Garvin (1984, 1987). Apart from extending these concepts, it also proposes two new dimensions – traceability and standardization which are not explicitly considered by the above two.Design/methodology/approachConceptual theory building is used to develop a framework consisting of three interrelated propositions which explain the underlying dimensions of green quality and provide a better understanding of the same.FindingsSimilar to the eight dimensions of quality proposed by Garvin, the authors propose various dimensions of green quality and develop three propositions around these dimensions. This conceptual framework is developed by integrating the works of traditional quality (specifically Garvin’s eight dimensions), emergent literature on green products and their attributes, carbon footprinting from environmental economics discipline by summarizing their common elements and contrasting their differences.Originality/valueThis research is one of the first studies that explore the dimensions of green quality of a product. Apart from discovering and exploring inherent greenness in Garvin’s eight dimensions of quality, the authors also discuss about two new dimensions – traceability and standardization.

Implementing fast photochemical oxidation of proteins (FPOP) as a footprinting approach to solve diverse problems in structural biology.

Fast photochemical oxidation of proteins (FPOP) is a footprinting technique used in mass spectrometry-based structural proteomics. It has been applied to solve a variety of problems in different areas of biology. A FPOP platform requires a laser, optics, and sample flow path properly assembled to enable fast footprinting. Sample preparation, buffer conditions, and reagent concentrations are essential to obtain reasonable oxidations on proteins. FPOP samples can be analyzed by LC-MS methods to measure the modification extent, which is a function of the solvent-accessible surface area of the protein. The platform can be expanded to accommodate several new approaches, including dose-response studies, new footprinting reagents, and two-laser pump-probe experiments. Here, we briefly review FPOP applications and in a detailed manner describe the procedures to set up an FPOP protein footprinting platform.

The Molecular Basis for Binding of an Electron Transfer Protein to a Metal Oxide Surface.

Achieving fast electron transfer between a material and protein is a long-standing challenge confronting applications in bioelectronics, bioelectrocatalysis, and optobioelectronics. Interestingly, naturally occurring extracellular electron transfer proteins bind to and reduce metal oxides fast enough to enable cell growth, and thus could offer insight into solving this coupling problem. While structures of several extracellular electron transfer proteins are known, an understanding of how these proteins bind to their metal oxide substrates has remained elusive because this abiotic-biotic interface is inaccessible to traditional structural methods. Here, we use advanced footprinting techniques to investigate binding between the Shewanella oneidensis MR-1 extracellular electron transfer protein MtrF and one of its substrates, α-Fe2O3 nanoparticles, at the molecular level. We find that MtrF binds α-Fe2O3 specifically, but not tightly. Nanoparticle binding does not induce significant conformational changes in MtrF, but instead protects specific residues on the face of MtrF likely to be involved in electron transfer. Surprisingly, these residues are separated in primary sequence, but cluster into a small 3D putative binding site. This binding site is located near a local pocket of positive charge that is complementary to the negatively charged α-Fe2O3 surface, and mutational analysis indicates that electrostatic interactions in this 3D pocket modulate MtrF-nanoparticle binding. Strikingly, these results show that binding of MtrF to α-Fe2O3 follows a strategy to connect proteins to materials that resembles the binding between donor-acceptor electron transfer proteins. Thus, by developing a new methodology to probe protein-nanoparticle binding at the molecular level, this work reveals one of nature's strategies for achieving fast, efficient electron transfer between proteins and materials.

Credit Alpha and CO2 Reduction: A Portfolio Manager Perspective

This paper discusses the challenges of carbon-dioxide emission measurement on corporate credit portfolios. We illustrate how it can be difficult to translate traditional CO2 reductive strategies into incentives for portfolio managers. As an alternative approach to the footprinting techniques commonplace in equities, we introduce the ECOBAR model which looks at CO2 missions from an ordinal standpoint and takes a risk-based approach to measuring this in credit portfolios. We build out the model to encompass important credit alpha factors such as short positions, leverage and derivatives as well as explicit green investments such as green bonds. We apply the model on two sets of data, where the first is a historical real traded investment-grade credit portfolio and the second is a systematic CDS trading strategy. In the traded portfolio, we find that it has been possible to own a clearly CO2 efficient portfolio whilst still generating average alpha of 4.5 percentage points per annum. In the CDS-based strategy, alpha loss turns out to be insignificant with reasonable investment constraints on high-CO2 emitting issuers. We conclude that there is a good potential for low-CO2 strategies in a variety of operational, mainstream credit trading settings.

Understanding the impact of water management on people for investment in sustainable infrastructure in the UK

This study explored whether digital footprinting through the use of mobile data could be used to improve the understanding of water and environmental events on people, to enhance the evidence base for investment in sustainable infrastructure. The study focused on Margate, UK, where an integrated stakeholder surface water management plan was being developed to support the town’s regeneration and urban growth aspirations. The objectives were to assess (a) the usefulness of mobile data in infrastructure studies and (b) the impacts of a recent water pollution and beach closure event on visitor footfall to and from Margate. It was found that digital footprinting techniques provided substantial new insight into human dynamics (spatially, temporally, and demographically) with visitor footfall to and from Margate strongly driven by weather, predominately from the local area and over 65 years of age, but with 16% of visitors from London. With further work (longer time periods, further population data, and statistical analysis) digital footprinting techniques could give Margate’s regeneration strategy an area to focus on in diversifying its economy and reliance on tourism, as well as moreover, a basis for future infrastructure appraisal and evaluation in the UK (potentially in real time). The development of the latter will pose many of the real privacy challenges that previous study has highlighted and is likely to be pursued due to the current financial climate needing to deliver better services for lower cost.