Interpretation of mass spectrometry data for high-throughput proteomics

Recent developments in proteomics have revealed a bottleneck in bioinformatics: high-quality interpretation of acquired MS data. The ability to generate thousands of MS spectra per day, and the demand for this, makes manual methods inadequate for analysis and underlines the need to transfer the advanced capabilities of an expert human user into sophisticated MS interpretation algorithms. The identification rate in current high-throughput proteomics studies is not only a matter of instrumentation. We present software for high-throughput PMF identification, which enables robust and confident protein identification at higher rates. This has been achieved by automated calibration, peak rejection, and use of a meta search approach which employs various PMF search engines. The automatic calibration consists of a dynamic, spectral information-dependent algorithm, which combines various known calibration methods and iteratively establishes an optimised calibration. The peak rejection algorithm filters signals that are unrelated to the analysed protein by use of automatically generated and dataset-dependent exclusion lists. In the "meta search" several known PMF search engines are triggered and their results are merged by use of a meta score. The significance of the meta score was assessed by simulation of PMF identification with 10,000 artificial spectra resembling a data situation close to the measured dataset. By means of this simulation the meta score is linked to expectation values as a statistical measure. The presented software is part of the proteome database ProteinScape which links the information derived from MS data to other relevant proteomics data. We demonstrate the performance of the presented system with MS data from 1891 PMF spectra. As a result of automatic calibration and peak rejection the identification rate increased from 6% to 44%.Abbreviations 2-DE Two-dimensional gel electrophoresis - MALDI Matrix-assisted laser desorption ionisation - PMF Peptide mass fingerprinting - MS Mass spectrometry - TOF Time of flight     

Drug detection based on the conformational changes of calmodulin and the fluorescence of its enhanced green fluorescent protein fusion partner

A homogeneous phase protein-based assay for the high throughput screening of drugs was developed using enhanced green fluorescent protein (EGFP) as the reporter. For that, a fusion protein between calmodulin (CaM) and EGFP was constructed in order to monitor the conformational changes induced in CaM upon binding to tricyclic anti-depressant drugs. In the presence of Ca²⁺, CaM undergoes a conformational change exposing a hydrophobic pocket that interacts with CaM-binding proteins, peptides, and drugs. Further, the conformational changes induced in CaM upon binding to Ca²⁺ and the target analyte drug, leads to a change in the microenvironment of EGFP concomitant with a change in its fluorescence intensity. The observed change in fluorescence intensity of EGFP can be correlated to the concentration of the analyte present in the sample. Further, the response of CaM–EGFP fusion protein in the presence of Ca²⁺ to increasing concentrations of phenothiazines and structurally related tricyclic anti-depressants was investigated. Dose-response curves for various tricyclic anti-depressants were prepared. Moreover, this assay can serve as a model system for other homogeneous binding assays for pharmaceuticals employing genetically fused binding proteins with reporter proteins and may find applications in the high throughput screening of tricyclic anti-depressants.     

A CIEF‐LIF method for simultaneous analysis of multiple protein kinases and screening of inhibitors

Here, a CIEF‐LIF method for multiple protein kinase simultaneous analysis and inhibitors throughput screening with fast rate and low cost is presented. Comparing with CZE, CIEF‐LIF exhibited great focusing ability and high separation efficiency for substrate and phosphorylated peptides, and is applicable for multiple kinases simultaneous analysis regardless of their substrate peptides compositions and charge statuses. Thus, highly sensitive analysis for cyclic adenosine 3’, 5’‐monophosphate‐dependent protein kinase (PKA) and cyclin‐dependent kinase 1 (CDK1) was achieved in CIEF‐LIF analysis with detection sensitivity up to 1.25 mU/μL and 0.4 mU/μL, respectively, two magnitudes higher than that of CZE and comparable with that in nanomaterials or green fluorescent protein‐based kinase assay. Moreover, the inhibition effect of inhibitors on multiple kinases could be simultaneously readout in a single electrophoretic run, with half maximal inhibitory concentration of H‐89 for PKA and Ro‐3306 for CDK1 calculated as 37.0 and 35.9 nM, respectively, consistent with literatures reported. The CIEF‐LIF also exhibited strong anti‐interference ability in human breast cancer cell lysates analysis and simulators such as forskolin and 3‐isobutyl‐1‐methylxantine assessment. Therefore, CIEF‐LIF is desirable for future biological application and clinical diagnostics and drug discovery.     

High-throughput isotherm determination and thermodynamic modeling of protein adsorption on mixed mode adsorbents

The thermodynamic modeling of protein adsorption on mixed-mode adsorbents functionalized with ligands carrying both hydrophobic and electrostatic groups was undertaken. The developed mixed mode isotherm was fitted with protein adsorption data obtained for five different proteins on four different mixed mode adsorbents by 96-well microtitre plate high throughput batch experiments on a robotic workstation. The developed mixed mode isotherm was capable of describing the adsorption isotherms of all five proteins (having widely different molecular masses and iso-electric points) on the four mixed mode adsorbents and over a wide range of salt concentrations and solution pH, and provided a unique set of physically meaningful parameters for each resin-protein-pH combination. The model could capture the typically observed minimum in mixed mode protein adsorption and predict the precise salt concentration at which this minimum occurs. The possibility of predicting the salt concentration at which minimum protein binding occurs presents new opportunities for designing better elution strategies in mixed mode protein chromatography. Salt-protein interactions were shown to have important consequences on mixed mode protein adsorption when they occur. Finally, the mixed mode isotherm also gave very good fit with literature data of BSA adsorption on a different mixed mode adsorbent not examined in this study. Hence, the mixed mode isotherm formalism presented in this study can be used with any mixed mode adsorbent having the hydrophobic and electrostatic functional groups. It also provides the basis for detailed modeling and optimization of mixed mode chromatographic separation of proteins.     

Advances of multiplex and high throughput biomolecular detection technologies based on encoding microparticles

The rapid developments of genomics and proteomics have driven the demand for multiplex and high throughput analysis of large numbers of biomolecules in the fields of medical diagnostics, drug discovery, and environmental monitoring. Encoding the biomolecular binding events is the key technique to fulfill this demand, in which microparticles play the most important roles. This review outlines the development of multiplex and high throughput biodetections, and highlights the most recent advances in the field ...     

High throughput screening and measurements of proton conductivity of newly developed PEM materials based on proton transport visualization

An electrochemical method for proton transport visualization was developed and applied to the investigation of proton-conducting membrane materials. The method employs the change in the visual appearance of chemo-chromic tungsten oxide WO₃ in the presence of atomic hydrogen. An all-solid electrochemical cell arranged by substituting a fuel cell cathode with a thin film of WO₃-electrode was built and shown to generate both optical and electrical response to hydrogen gas exposure. The design of the cell was extended to a high throughput screening system that was utilized to characterize proton transport properties of samples, including a number of new compounds synthesized in-house by sol–gel wet chemistry. Non-destructive introduction of superacidic groups promoting proton hopping in the membrane materials was achieved by photodecomposition of a photoacid generator just after membrane casting. A model quantitatively describing current–voltage relation in the cell was developed and successfully applied to derive area-specific resistance of proton-conductive membranes from the experimental results. Area-specific resistances of membranes are derived from the slopes of optically reconstructed voltage–current curves. Sensitivity and dynamic range of the screening method are discussed.     

Utility of lab-on-a-chip technology for high-throughput nucleic acid and protein analysis

On-chip electrophoresis can provide size separations of nucleic acids and proteins similar to more traditional slab gel electrophoresis. Lab-on-a-chip (LoaC) systems utilize on-chip electrophoresis in conjunction with sizing calibration, sensitive detection schemes, and sophisticated data analysis to achieve rapid analysis times (<120 s). This work describes the utility of LoaC systems to enable and augment systems biology investigations. RNA quality, as assessed by an RNA integrity number score, is compared to existing quality control (QC) measurements. High-throughput DNA analysis of multiplex PCR samples is used to stratify gene sets for disease discovery. Finally, the applicability of a high-throughput LoaC system for assessing protein purification is demonstrated. The improvements in workflow processes, speed of analysis, data accuracy and reproducibility, and automated data analysis are illustrated.     

Design and synthesis of a novel fluorescent protein probe for easy and rapid electrophoretic gel staining by using a commonly available UV‐based fluorescent imaging system

A new fluorescent molecular probe, methyl 3‐(3,5‐bis((bis(pyridin‐2‐ylmethyl)amino)‐methyl)‐4‐hydroxyphenyl)‐2‐(5‐(dimethylamino)naphthalene‐1‐sulfonamido) propanoate, dizinc(II) chloride salt (Dansyl‐ 1 ‐Zn(II)), which possesses Zn(II) complexes and a dansyl group, was designed and synthesized to enable the detection of proteins in solution and in high‐throughput electrophoresis by using a UV‐based detection system. Dansyl‐ 1 ‐Zn(II) exhibited weak fluorescence in the absence of proteins and strong green fluorescence at approximately 510 nm in the presence of BSA upon irradiation with light at a wavelength of 345 nm. Compared with conventional protocols for in‐gel SDS‐PAGE protein staining (e.g. silver staining, SYPRO Ruby, and Oriole), the operating times of which range from 90 min to overnight, Dansyl‐ 1 ‐Zn(II) allowed 1‐step protein staining (SDS‐PAGE →Staining →Detection) and shortened the operating time (35 min) with high sensitivity (LOD: 1 ng or less) under 312‐nm or 365‐nm light excitation with orange or red emission filters, respectively. Moreover, Dansyl‐ 1 ‐Zn(II) was successfully applied to protein identification by MS via in‐gel tryptic digestion, Western blotting, and Native‐PAGE. Accordingly, Dansyl‐ 1 ‐Zn(II) may facilitate highly sensitive and high‐throughput protein detection, and it may be widely applicable as a convenient tool in various scientific and medical fields.     

Automated protein analysis by online detection of laser-induced fluorescence in slab gels and 3-D geometry gels

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) still remains the most reliable and comprehensive analytical method for the evaluation of protein extracts. However, conventional SDS-PAGE is time-consuming and, thus, unpractical if several tens or hundreds of samples must be examined. We show that SDS-PAGE protein analysis can be automated using slab gel DNA sequencers and compare the instrument's performance with conventional SDS-PAGE in terms of resolution, sensitivity and sample capacity. Labeled protein bands are detected online by laser-induced fluorescence (LIF) and the acquired signals are electronically stored for further processing, avoiding gel staining and scanning. Appropriate software allows immediate display of recorded data and convenient evaluation. The method provides a higher sensitivity and dynamic range than conventional Coomassie-stained gels and the resolution of proteins with different masses is independent of the polyacrylamide concentration. Internal markers can also be used for direct quantification and assignment of the molecular masses. Additionally, we present a novel electrophoresis instrument for the simultaneous separation and online LIF detection of all samples of a microtiterplate in parallel lanes in a 3-D geometry gel cylinder. The specific gel thermostatting concept prevents irregular sample migration (smiling) and improves the reproducibility and comparability of individual separation patterns. In combination with the expected large capacity of 384 or 1,536 samples, this makes the instrument a valuable tool for high-throughput comparative screening applications.     

New Monofunctionalized Fluorescein Derivatives for the Efficient High‐Throughput Screening of Lipases and Esterases in Aqueous Media

Monoalkylation or acylation of fluorescein ( 1 ) with various acyloxymethyl or acyl halides afforded, respectively, a series of ether‐ ( 2 ) and ester‐functionalized ( 3 ) fluorogenic probes. The highly reactive and water‐soluble substrates release fluorescein ( 1 ) upon reaction with lipases and esterases within seconds or minutes, both under fully aqueous conditions or in the presence of DMSO (20%) as a co‐solvent. The most‐reactive substrates in the two series were the octanoic acid derivatives 2f (= 2‐{6‐[(octanoyloxy)methoxy]‐3‐oxo‐3H‐xanthen‐9‐yl}benzoic acid) and 3a (= 2‐[6‐(octanoyloxy)‐3‐oxo‐3H‐xanthen‐9‐yl]benzoic acid). Esterases were found to generally react faster under aqueous conditions, while lipases were more reactive in the presence of DMSO as a co‐solvent.     

Molecular dynamics simulations of ligand-induced backbone conformational changes in the binding site of the periplasmic lysine-, arginine-, ornithine-binding protein

The periplasmic lysine-, arginine-, ornithine-binding protein (LAOBP) traps its ligands by a large hinge bending movement between two globular domains. The overall geometry of the binding site remains largely unchanged between the open (unliganded) and closed (liganded) forms, with only a small number of residues exhibiting limited movement of their side chains. However, in the case of the ornithine-bound structure, the backbone peptide bond between Asp11 and Thr12 undergoes a large rotation. Molecular dynamics simulations have been used to investigate the origin and mechanism of this backbone movement. Simulations allowing flexibility of a limited region and of the whole binding site, with and without bound ligands, suggest that this conformational change is induced by the binding of ornithine, leading to the stabilisation of an energetically favourable alternative conformation.     

Creating hierarchical models of protein families based on Expressed Sequence Tags: the "Sprockets" analysis pipeline

We have created an analysis pipeline called Sprockets, which can be used to classify proteins into various hierarchical “families”, and build searchable models of these families. The construction of these families is based on data from Expressed Sequence Tags (ESTs) and Coding DNA Sequences (CDSs), making Sprockets clusters especially suitable for studying gene families in organisms for which the completely sequenced genome does not (yet) exist. The pipeline consists of two main parts: pair-wise analysis and grouping of sequences with Z-score statistics, followed by hierarchical splitting of clusters into alignable protein families. Various computational and statistical techniques applied in Sprockets allow it to act like a massive and selective multiple sequence alignment engine for combining individual sequence collections and related public sequences. The end result is a database of gene Hidden Markov Models, each related to the other by three levels of similarity: secondary structure, function and evolutionary origin. For a sample 20,000 EST set from Lactuca spp., Sprockets provided a 9% improvement in mapping of function to unknown sequences over traditional pair-wise search methods and InterPro mapping.     

A high throughput metabolic stability screening workflow with automated assessment of data quality in pharmaceutical industry

One of the most commonly performed in vitro ADME assays during the lead generation and lead optimization stage of drug discovery is metabolic stability evaluation. Metabolic stability is typically assessed in liver microsomes, which contain Phase I metabolizing enzymes, mainly cytochrome P450 enzymes (CYPs). The amount of parent drug metabolized by these CYPs is determined by LC/MS/MS. The metabolic stability data are typically used to rank order compounds for in vivo evaluation. We describe a streamlined and intelligent workflow for the metabolic stability assay that permits high throughput analyses to be carried out while maintaining the standard of high quality. This is accomplished in the following ways: a novel post-incubation pooling strategy based on c Log D_3.0 values, coupled with ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS), enables sample analysis times to be reduced significantly while ensuring adequate chromatographic separation of compounds within a group, so as to reduce the likelihood of compound interference. Assay quality and fast turnaround of data reports is ensured by performing automated real-time intelligent re-analysis of discrete samples for compounds that do not pass user-definable criteria during the pooling analysis. Intelligent, user-independent data acquisition and data evaluation are accomplished via a custom visual basic program that ties together every step in the workflow, including cassette compound selection, compound incubation, compound optimization, sample analysis and re-analysis (when appropriate), data processing, data quality evaluation, and database upload. The workflow greatly reduces labor and improves data turnaround time while maintaining high data quality.     

The effects of aggregation, protein binding and cellular incorporation on the photophysical properties of benzoporphyrin derivative monoacid ring A (BPDMA)

Absorption, fluorescence and laser flash photolysis spectroscopies were used to investigate the effects of self-aggregation, binding to human serum albumin and incorporation in cancer cells on the photophysics of benzoporphyrin derivative monoacid ring A (BPDMA). Aggregation of BPDMA has been studied in mixtures of methanol and phosphate-buffered saline (PBS). The extent of aggregation was dependent on dye concentration and solvent composition, becoming particularly marked in mixtures containing less than 30% methanol. A dimerization constant K_d or 9 × 10⁶ M⁻¹ was determined by fluorescence experiments for BPDMA in pure PBS. In addition to spectral modifications, aggregation induces a lowering of the fluorescence and intersystem crossing quantum yields. Human serum albumin binds BPDMA with an association constant K_b of 5.2 × 10⁵ M⁻¹ in PBS. When bound to HSA, BPDMA displays photophysical properties very similar to the monomer in organic solvents. The molar ratio [HSA]/[BPDMA] corresponding to complete binding of the dye was determined to be approximately 10. Efficient generation of the triplet state of BPDMA was also observed from aqueous cellular suspensions containing incorporated photosensitizer.     

Fabrication of hexagonal boron nitride based 2D nanopore sensor for the assessment of electro-chemical responsiveness of human serum transferrin protein

In this work, we present a step-by-step workflow for the fabrication of 2D hexagonal boron nitride (h-BN) nanopores which are then used to sense holo-human serum transferrin (hSTf) protein at pH ∼8 under applied voltages ranging from +100 mV to +800 mV. 2D nanopores are often used for DNA, however, there is a great void in the literature for single-molecule protein sensing and this, to the best of our knowledge, is the first time where h-BN—a material with large band-gap, low dielectric constant, reduced parasitic capacitance and minimal charge transfer induced noise—is used for protein profiling. The corresponding ΔG (change in pore conductance due to analyte translocation) profiles showed a bimodal Gaussian distribution where the lower and higher ΔG distributions were attributed to (pseudo-) folded and unfolded conformations respectively. With increasing voltage, the voltage induced unfolding increased (evident by decrease in ΔG) and plateaued after ∼400 mV of applied voltage. From the ΔG versus voltage profile corresponding to the pseudo-folded state, we calculated the molecular radius of hSTf, and was found to be ∼3.1 nm which is in close concordance with the literature reported value of ∼3.25 nm.     

Advances in the analysis of dynamic protein complexes by proteomics and data processing

Signal transduction governs virtually every cellular function of multicellular organisms, and its deregulation leads to a variety of diseases. This intricate network of molecular interactions is mediated by proteins that are assembled into complexes within individual signaling pathways, and their composition and function is often regulated by different post-translational modifications. Proteomic approaches are commonly used to analyze biological complexes and networks, but often lack the specificity to address the dynamic and hence transient nature of the interactions and the influence of the multiple post-translational modifications that govern these processes. Here we review recent developments in proteomic research to address these limitations, and discuss several technologies that have been developed for this purpose. The synergy between these proteomic and computational tools, when applied together with global methods to the analysis of individual proteins, complexes and pathways, may allow researchers to unravel the underlying mechanisms of signaling networks in greater detail than previously possible.     

Direct injection of HILIC fractions on the reversed‐phase trap column improves protein identification rates for salivary proteins

Online combination of hydrophilic interaction chromatography (HILIC) and RP chromatography for separation of tryptic peptides is a challenging approach due to the incompatibility of direct loading HILIC fractions on the RP trapping column. High amounts of organic modifiers in loading solvents decrease the binding efficiency of tryptic peptides on C18 phases and lower the number of identifications. A 500 μL loop upfront of the trapping column filled with aqueous mobile phase was employed as a mixing chamber and enabled direct injections and improved saliva protein identification rates of HILIC fractions.     

A High‐Throughput Assay for Arylamine Halogenation Based on a Peroxidase‐Mediated Quinone–Amine Coupling with Applications in the Screening of Enzymatic Halogenations

Arylhalides are important building blocks in many fine chemicals, pharmaceuticals and agrochemicals, and there has been increasing interest in the development of more “green” halogenation methods based on enzyme catalysis. However, the screening and development of new enzymes for biohalogenation has been hampered by a lack of high‐throughput screening methods. Described herein is the development of a colorimetric assay for detecting both chemical and enzymatic arylamine halogenation reactions in an aqueous environment. The assay is based on the unique UV/Vis spectrum created by the formation of an ortho‐benzoquinone‐amine adduct, which is produced by the peroxidase‐catalysed benzoquinone generation, followed by Michael addition of either a halogenated or non‐halogenated arylamine. This assay is sensitive, rapid and amenable to high‐throughput screening platforms. We have also shown this assay to be easily coupled to a flavin‐dependent halogenase, which currently lacks any convenient colorimetric assay, in a “one‐pot” workflow.     

Effect of protein solution components in the adsorption of Herbaspirillum seropedicae GlnB protein on mica

The adsorption of proteins and its buffer solution on mica surfaces was investigated by atomic force microscopy (AFM). Different salt concentration of the Herbaspirillum seropedicae GlnB protein (GlnB-Hs) solution deposited on mica was investigated. This protein is a globular, soluble homotrimer (36 kDa), member of PII-like proteins family involved in signal transducing in prokaryote. Supramolecular structures were formed when this protein was deposited onto bare mica surface. The topographic AFM images of the GlnB-Hs films showed that at high salt concentration the supramolecular structures are spherical-like, instead of the typical doughnut-like shape for low salt concentration. AFM images of NaCl and Tris from the buffer solution showed structures with the same pattern as those observed for high salt protein solution, misleading the image interpretation. XPS experiments showed that GlnB protein film covers the mica surface without chemical reaction.     

Cluster-C, an algorithm for the large-scale clustering of protein sequences based on the extraction of maximal cliques

Although the characterization of proteins cannot solely rely upon sequence similarity, it has been widely proved that all-vs-all massive sequence comparisons may be an effective approach and a good basis for the prediction of biochemical functions or for the delineation of common shared properties. The program Cluster-C presented here enables a stand-alone and efficient construction of protein families within whole proteomes. The algorithm, which is based on the detection of cliques, ensures a high level of connectivity within the clusters. As opposed to the single transitive linkage method, Cluster-C allows a large number of sequences to be classified in such a way that the multidomain proteins do not produce a chain-grouping effect resulting in meaningless clusters. Moreover, some proteins can be present in several different but relevant clusters, which is of help in the determination of their functional domains. In the present analysis we used the Z-value, an evaluation of the significance of the similarity score, as the criterion for connecting sequences (the user can freely define the threshold of the similarity criterion). The clusters built with a rather low threshold (Z= 14) include more than 97% of the sequences and are consistent with known protein families and PROSITE patterns.