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Selective separation and enrichment of phosphoproteins possess the distinct clinical and biological importance in the diagnosis, treatment, and management of several fatal human diseases. In this study, a facile synthesis of titanium(IV) ion–immobilized arsenate-modified poly(glycidyl methacrylate) microparticles (denoted as Ti4+-arsenate-PGMA-MPs) was developed for the efficient enrichment of intact phosphoproteins found in biologically complex protein samples. By virtue of the strong interaction between the titanium ions immobilized on the surface of Ti4+-arsenate-PGMA-MPs and phosphate groups of phosphoproteins, Ti4+-arsenate-PGMA-MPs had a high saturated adsorption capacity for phosphoproteins (901 mg/g for β-casein), which was much higher than that of non-phosphoproteins (73.5 mg/g for BSA). Ti4+-arsenate-PGMA-MPs were characterized by SEM, TEM, and FT-IR, and the average particle diameter was about 2.5 μm with good dispersibility. Besides, the application of Ti4+-arsenate-PGMA-MPs in real biological samples was investigated by SDS-PAGE analysis, and the results showed that Ti4+-arsenate-PGMA-MPs were able to enrich phosphoproteins efficiently.

The repeated failures of amyloid-targeting therapies have challenged our narrow understanding of Alzheimer’s disease (AD) pathogenesis and inspired wide-ranging investigations into the underlying mechanisms of disease. Increasing evidence indicates that AD develops from an intricate web of biochemical and cellular processes that extend far beyond amyloid and tau accumulation. This growing recognition surrounding the diversity of AD pathophysiology underscores the need for holistic systems-based approaches to explore AD pathogenesis. Here we describe how network-based proteomics has emerged as a powerful tool and how its application to the AD brain has provided an informative framework for the complex protein pathophysiology underlying the disease. Furthermore, we outline how the AD brain network proteome can be leveraged to advance additional scientific and translational efforts, including the discovery of novel protein biomarkers of disease.

Two complex protein defense systems—complement and coagulation—are based on amplifying enzyme cascades triggered by specific local stimuli. Excess systemic activation of either system is pathologic and is normally prevented by a family of regulatory proteins. The 2 systems are ancient biological processes which share a common origin that predates vertebrate evolution. Recent research has uncovered multiple opportunities for cross talk between complement and coagulation including proteins traditionally viewed as coagulation factors that activate and regulate complement, and proteins traditionally seen as part of the complement system that participate in coagulation. Ten examples of cross talk between the 2 systems are described. The mutual engagement of both systems is increasingly recognized to occur in human diseases. Three conditions—paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, and the antiphospholipid syndrome—provide examples of the importance of interactions between complement and coagulation in human biology. A better understanding of the mutual engagement of these 2 ancient defense systems is expected to result in improved diagnostics and new treatments for systemic diseases.

Mimicking sensory principles encountered in animals, whereby numerous tastants and odorants are identified based on "pattern"-like sensory inputs that are generated by arrays of sensory cells, allows creating a unique technique that is distinct from conventional chemical sensing systems, as the latter usually require specific recognition of target analytes. Herein, we present a highly discriminative small fluorescent array of block copolymers that can recognize various bioanalytes in a biomimicry manner. These polyethylene glycol/poly-l-lysine block copolymers are functionalized with fluorescein as a fluorescent reporter unit and hydrophobic amino acids as cross-reactive recognition units, which provides the ability to generate fluorescent response patterns unique to proteins and cells. Multivariate analysis on the patterns obtained with an array consisting of solely 3 block copolymers allowed identifying not only 20 proteins and 10 mammalian cells individually but also complex protein mixtures with slightly different compositions. This design guideline for creating a versatile biomimicry sensing system, which is based on the bifunctionalization of polymeric materials, is expected to offer a powerful platform for simple and high-throughput sensing of a wide variety of bioanalytes.

Purpose of Review This review a highlights that to use artificial intelligence (AI) tools effectively for hypertension research, a new foundation to further understand the biology of hypertension needs to occur by leveraging genome and RNA sequencing technology and derived tools on a broad scale in hypertension. Recent Findings For the last few years, progress in research and management of essential hypertension has been stagnating while at the same time, the sequencing of the human genome has been generating many new research tools and opportunities to investigate the biology of hypertension. Cancer research has applied modern tools derived from DNA and RNA sequencing on a large scale, enabling the improved understanding of cancer biology and leading to many clinical applications. Compared with cancer, studies in hypertension, using whole genome, exome, or RNA sequencing tools, total less than 2% of the number cancer studies. While true, sequencing the genome of cancer tissue has provided cancer research an advantage, DNA and RNA sequencing derived tools can also be used in hypertension to generate new understanding how complex protein network, in non-cancer tissue, adapts and learns to be effective when for example, somatic mutations or environmental inputs change the gene expression profiles at different network nodes. The amount of data and differences in clinical condition classification at the individual sample level might be of such magnitude to overwhelm and stretch comprehension. Here is the opportunity to use AI tools for the analysis of data streams derived from DNA and RNA sequencing tools combined with clinical data to generate new hypotheses leading to the discovery of mechanisms and potential target molecules from which drugs or treatments can be developed and tested. Summary Basic and clinical research taking advantage of new gene sequencing-based tools, to uncover mechanisms how complex protein networks regulate blood pressure in health and disease, will be critical to lift hypertension research and management from its stagnation. The use of AI analytic tools will help leverage such insights. However, applying AI tools to vast amounts of data that certainly exist in hypertension, without taking advantage of new gene sequencing-based research tools, will generate questionable results and will miss many new potential molecular targets and possibly treatments. Without such approaches, the vision of precision medicine for hypertension will be hard to accomplish and most likely not occur in the near future.

Screening for small-molecule fragments that can lead to potent inhibitors of protein&ndash, protein interactions (PPIs) is often a laborious step as the fragments cannot dissociate the targeted PPI due to their low &mu, M&ndash, mM affinities. Here, we describe an NMR competition assay called w-AIDA-NMR (weak-antagonist induced dissociation assay-NMR), which is sensitive to weak &mu, mM ligand&ndash, protein interactions and which can be used in initial fragment screening campaigns. By introducing point mutations in the complex&rsquo, s protein that is not targeted by the inhibitor, we lower the effective affinity of the complex, allowing for short fragments to dissociate the complex. We illustrate the method with the compounds that block the Mdm2/X-p53 and PD-1/PD-L1 oncogenic interactions. Targeting the PD-/PD-L1 PPI has profoundly advanced the treatment of different types of cancers.

Fritz-Laylin studies the evolution of complex protein networks associated with cell movement.

Scientific reports 9(1), 18986 (2019). doi:10.1038/s41598-019-55313-5, In mitochondria, a complex protein machinery is devoted to the maturation of iron-sulfur clusterproteins. Structural information on the last steps of the machinery, which involve ISCA1, ISCA2 andIBA57 proteins, needs to be acquired in order to define how these proteins cooperate each other. Wereport here the use of an integrative approach, utilizing information from small-angle X-ray scattering(SAXS) and bioinformatics-driven docking prediction, to determine a low-resolution structural modelof the human mitochondrial [2Fe-2S]2+ ISCA2-IBA57 complex. In the applied experimental conditions,all the data converge to a structural organization of dimer of dimers for the [2Fe-2S]2+ ISCA2-IBA57complex with ISCA2 providing the homodimerization core interface. The [2Fe-2S] cluster is out of theISCA2 core while being shared with IBA57 in the dimer. The specific interaction pattern identified fromthe dimeric [2Fe-2S]2+ ISCA2-IBA57 structural model allowed us to define the molecular grounds of thepathogenic Arg146Trp mutation of IBA57. This finding suggests that the dimeric [2Fe-2S] ISCA2-IBA57hetero-complex is a physiologically relevant species playing a role in mitochondrial [4Fe-4S] proteinbiogenesis., Published by Macmillan Publishers Limited, part of Springer Nature, [London]

The study of intrinsic phosphorylation dynamics and kinetics in the context of complex protein architecture in vivo has been challenging: Method limitations have prevented significant advances in the understanding of the highly variable turnover of phosphate groups, synergy, and cooperativity between P-sites. However, over the last decade, powerful analytical technologies have been developed to determine the full catalog of the phosphoproteome for many species. The curated databases of phospho sites found by mass spectrometry analysis and the computationally predicted sites based on the linear sequence of kinase motifs are valuable tools. They allow investigation of the complexity of phosphorylation in vivo, albeit with strong discrepancies between different methods. A series of hypothetical scenarios on combinatorial processive phosphorylation is proposed that are likely unverifiable with current methodologies. These proposed a priori postulates could be considered as possible extensions of the known schemes of the activation/inhibition signaling process in vivo.

Fluorescence-based electrophoresis has been widely used for proteome analysis in which every protein species in cells is labeled with a fluorescent dye, separated by electric migration, and quantified using fluorescence detection. The ultimate limit of sensitivity for this approach could be reached by single-molecule fluorescence imaging and counting individual proteins, requiring exhaustive fluorescent labeling of proteins across molecular populations and species. However, it remains unclear how homogeneous the fluorescence labeling of individual protein molecules of each species is across the proteome. To address this question, we developed a method to measure the labeling homogeneity based on a single-molecule fluorescence counting assay. Our results reveal that the proportion of proteins labeled with at least one dye, called labeling occupancy (LO), was 35% for fluorescently labeled BSA using existing protocols. We then found that the LO could be improved to 82% under high pH and surfactant-rich conditions. Furthermore, when a proteome sample from a human cell lysate was analyzed, the total LO was 71%, whereby the values varied between 50 and 90% for low and high molecular weight proteome fractions, respectively. The results support the possibility of sensitive detection of proteins using single-molecule counting with fluorescent labeling at the proteome scale.

Chlamydia trachomatis serological assays with improved sensitivity over commercially available assays are needed to evaluate the burden of C. trachomatis infection and the effectiveness of prevention efforts. We evaluated the performance of a C. trachomatis outer membrane complex protein B (OmcB) enzyme-linked immunosorbent assay (ELISA) in the detection of anti- C. trachomatis antibody responses in C. trachomatis -infected women.

Protein immobilization and modification are widely used techniques in the fields of chemical biology and biomaterials science. While covalent strategies based on small molecules are traditionally used, supramolecular chemistry offers numerous useful opportunities for guiding the modification locations on complex protein landscapes and introducing different degrees of reversibility into the products. In this opinion, we highlight recent advances in using supramolecular interactions, particularly host-guest chemistry, for controlling protein modification and immobilization. We discuss supramolecular strategies for protein-conjugate purification and capture, as well as for protein modification via host-guest interactions and metal coordination. Lastly, we address recent advances in utilizing supramolecular interactions to direct covalent protein modification. These examples of supramolecular chemical biology present opportunities to advance a wide range of applications, including proteomics and drug delivery.

Getting a handle on a cross-linking motif Although protein backbones consist exclusively of amino acids, various other molecules in the cell often get latched on afterward in a process termed posttranslational modification. In one such motif, called glucosepane, the side chains of lysine and arginine form a condensed cross-link through a reaction sequence with glucose. Formation of this cross-link is of interest in diabetes research. Draghici et al. now report a chemical synthesis of glucosepane outside the broader environment of a surrounding protein (see the Perspective by Boger). This synthesis should facilitate more precise characterization of the structure and function of the motif in vivo. Science , this issue p. 294 ; see also p. 275

Measurement of protein phosphorylation plays an essential role in delineating cell signaling pathways. Although the detection of a specific phosphoprotein has been largely accomplished by immunological methods, a specific and sensitive assay to measure total protein phosphorylation level in complex samples such as whole cell extracts has yet to be established. Here, we present a sensitive phosphorylation assay on a microwell plate to determine total protein phosphorylation level calibrated to a phosphoprotein standard. The core of the assay is a reagent termed pIMAGO that is multi-functionalized with titanium ions for its superior selectivity towards phosphorylated proteins and with fluorophores for quantification. The specificity, sensitivity, and quantitative nature of the assay were demonstrated with standard proteins and whole cell lysates. The method was then employed to measure the overall protein phosphorylation level of human cells under different treatments. At last, we investigated the practicability of the assay to serve as a sensitive tool to estimate the amount of phosphorylated samples prior to a mass spectrometry-based phosphoproteomic analysis.

Analysis of proteomic composition of human follicular fluid (hFF) has been previously proposed as a potential tool of oocyte quality evaluation. In order to develop an efficient method to investigate the hFF proteome and peptidome components, we applied and tested a few prefractionation schemes of hFF material consisting of ultrafiltration, optional immunodepletion, and high pH RP-HPLC separation by building spectral libraries and comparing their quantification capabilities of unfractionated samples. Low Molecular-Weight Fraction peptides (LMWF,10 kDa) and High Molecular-Weight Fraction proteins (HMWF,10 kDa) resulting from ultrafiltration were analyzed separately. We identified 302 proteins in HMWF and 161 proteins in LMWF in all qualitative experiments. All LMWF peptidomic libraries turned out to be of poor quantification quality, however they enabled measurement of higher numbers of peptides with increasing input of experiment data, in contrast to HMWF proteomic libraries. We were able to quantify a total of 108 HMWF proteins and 250 LMWF peptides (from 84 proteins) in all experiments. Employment of high RP-HPLC fractionation allowed for identification of a much broader set of proteins, however did not significantly improve the quantification capabilities of the applied method. Data are available via ProteomeXchange with identifier PXD008073. SIGNIFICANCE: In the search of biomarkers for assessment of oocyte quality in assisted reproductive technology, many studies are devoted to analysis of follicular fluid composition. Candidates for such biomarkers can be located in both the proteome and the recently investigated peptidome of hFF. Reliable qualitative and especially quantitative analysis of complex mixtures such as hFF, requires development of a fast and preferably inexpensive analytical procedure. The powerful SWATH-MS technique is well suited for quantitative label-free analysis of complex protein and peptide mixtures. However, for efficient usage it needs well designed and constructed MS-spectral libraries as well as a proper protocol for sample preparation. We investigated the influence of the size and quality of MS-spectral libraries (different spectral libraries are constructed using various sample prefractionation protocols) on SWATH experiments on hFF proteome and peptidome. In the case of peptidome investigation, increasing the size of spectral libraries led to quantification of more peptides in a single experiment. For the proteome, increasing the size of spectral libraries improved quantification only to a limited extend, and further extension of spectral libraries even worsened results. Nevertheless, using the best selected prefractionation schemes and spectral libraries we were able to quantify as many as 79 proteins of hFF proteome and 106 peptides (from 53 proteins) of hFF peptidome in single experiments. The spectral libraries and prefractionation protocols we developed allow for a large scale fast scan of hundreds of clinical hFF samples in the search for biomarkers for evaluation of oocyte quality.

The synthesis of complex protein-based bioconjugates has been facilitated greatly by recent developments in chemoselective methods for biomolecular modification. The oxidative coupling of o-aminophenols or catechols with aniline functional groups is chemoselective, mild, and rapid; however, the oxidatively sensitive nature of the electron-rich aromatics and the paucity of commercial sources pose some obstacles to the general use of these reactive strategies. Herein, we identify o-methoxyphenols as air-stable, commercially available derivatives that undergo efficient oxidative couplings with anilines in the presence of periodate as oxidant. Mechanistic considerations informed the development of a preoxidation protocol that can greatly reduce the amount of periodate necessary for effective coupling. The stability and versatility of these reagents was demonstrated through the synthesis of complex protein-protein bioconjugates using a site-selective heterobifunctional cross-linker comprising both o-methoxyphenol and 2-pyridinecarboxaldehyde moieties. This compound was used to link epidermal growth factor to genome-free MS2 viral capsids, affording nanoscale delivery vectors that can target a variety of cancer cell types.

The idea of using hybrid proteins containing transcription factor domains to analyze protein-protein interactions was described in 1989. Over the past 25 years, this method has begun to reveal the complex protein networks that underlie cellular behavior.

Hierarchical assemblies of biomolecular subunits can carry out versatile tasks at the cellular level with remarkable spatial and temporal precision. As an example, the collective motion and mutual cooperation between complex protein machines mediate essential functions for life, such as replication, synthesis, degradation, repair and transport. Nucleic acid molecules are far less dynamic than proteins and need to bind to specific proteins to form hierarchical structures. The simplest example of these nucleic acid-based structures is provided by a rod-shaped tobacco mosaic virus, which consists of genetic material surrounded by coat proteins. Inspired by the complexity and hierarchical assembly of viruses, a great deal of effort has been devoted to design similarly constructed artificial viruses. However, such a wrapping approach makes nucleic acid dynamics insensitive to environmental changes. This limitation generally restricts, for example, the amplification of the conformational dynamics between the right-handed B form to the left-handed Z form of double-stranded deoxyribonucleic acid (DNA). Here we report a virus-like hierarchical assembly in which the native DNA and a synthetic coat undergo repeated collective helicity switching triggered by pH change under physiological conditions. We also show that this collective helicity inversion occurs during translocation of the DNA-coat assembly into intracellular compartments. Translating DNA conformational dynamics into a higher level of hierarchical dynamics may provide an approach to create DNA-based nanomachines.

Nanoelectromechanical systems (NEMS) resonators can detect mass with exceptional sensitivity. Previously, mass spectra from several hundred adsorption events were assembled in NEMS-based mass spectrometry using statistical analysis. Here, we report the first realization of single-molecule NEMS-based mass spectrometry in real time. As each molecule in the sample adsorbs on the resonator, its mass and position of adsorption are determined by continuously tracking two driven vibrational modes of the device. We demonstrate the potential of multimode NEMS-based mass spectrometry by analysing IgM antibody complexes in real time. NEMS-based mass spectrometry is a unique and promising new form of mass spectrometry: it can resolve neutral species, provide a resolving power that increases markedly for very large masses, and allow the acquisition of spectra, molecule-by-molecule, in real time.

The implementation of high-throughput technologies based on qualitative and quantitative methodologies for the characterization of complex protein mixtures is increasingly required in clinical laboratories. MALDI profiling is a robust and sensitive technology although the serum high dynamic range imposes a major limitation hampering the identification of less abundant species decreasing the quality of MALDI profiling. A setup to improve these parameters has been performed for recombinant human erythropoietin (rhEPO) monitoring in serum, analyzing the effects of two commercially available columns (MARS Hu7 and Hu14) for immunodepletion, and two matrices (α-cyano-4-hydroxycinnamic acid and 2',4'-dihydroxyacetophenone) for peak quality improvement. The immunodepletion capability of both columns was determined by 2-D DIGE, which precisely revealed the efficacy of Hu14 in protein removal and the serum dynamic range decrement. In addition, the type of matrix, the sample dilution, and the efficacy of optimized parameters were used for serum profiling of ten healthy subjects before and after rhEPO treatment. The principal component analysis indicates that a combination of Hu14 column and 2',4'-dihydroxyacetophenone matrix increases data quality allowing the discrimination between treated and untreated samples, making serum MALDI profiling suitable for clinical monitoring of rhEPO.

FeS cluster biogenesis is an essential process in virtually all forms of life. Complex protein machineries that are conserved from bacteria through higher eukaryotes facilitate assembly of the FeS cofactor in proteins. In the last several years, significant strides have been made in our understanding of FeS cluster assembly and the functional overlap of this process with cellular iron homeostasis. This minireview summarizes the present understanding of the cytosolic iron-sulfur cluster assembly (CIA) system in eukaryotes, with a focus on information gained from studies in budding yeast and mammalian systems.

One major limitation in proteomics is the detection and analysis of low-abundant proteins, i.e. in plasma. Several years ago, a technique to selectively enrich the relative concentration of low-abundant proteins was introduced by Boschetti and co-workers. It is based on a specific and saturable interaction of proteins to a high diversity of binding sites, realized by a hexapeptide library coupled to beads. This technology was commercialized as Equalizer beads or ProteoMiner. However, during application of ProteoMiner beads to plasma samples unexpected results questioned the proposed mode of action. Therefore, ProteoMiner beads were compared with chromatographic beads exhibiting completely different surface chemistry. Sepabeads FP-OD400 octadecyl, FP-DA400 diethylamine, FP-BU400 butyl, FP-HG400 hydroxyl and EXE056 epoxy were used. The results show that ProteoMiner or the different Sepabeads behave surprisingly similarly in the separation of complex protein mixtures. ProteoMiner beads interact with protein mixtures according to a general hydrophobic binding mechanism, where diversity in surface ligands plays only a negligible role.

Purpose of Review: To describe progress in the application of proteomic approaches to advance our understanding of the biology of pancreatic cancer as well as contribute potential protein biomarkers for this disease. Recent Findings: Here we review proteomic studies relating to pancreatic cancer that have been published in the past 12 months. We describe novel techniques for the simplification of complex protein samples, focusing particularly on emerging methods for reducing the complexity of blood. We provide examples, where possible, of the application of these novel technologies to pancreatic cancer research. Summary: Both the range of proteomic-based approaches and their sensitivities for the detection of low-abundance proteins has increased. This provides promise that further research will yield insight into pancreatic cancer, including valuable information on proteins that may ultimately serve as biomarkers for pancreatic cancer.

Considerable interest, speculation and controversy have been generated utilising surface-enhanced laser desorption/ionization in conjunction with mass spectrometry (SELDI-MS) for the diagnosis, prognosis and therapeutic monitoring of cancer and offers an attractive approach to cancer biomarker discovery from tissues and biological fluids. This technology utilises a combination of mass spectrometry and chromatography to facilitate protein profiling of complex biological mixtures. Compared to some other more traditional proteomic platforms, such as 2D polyacrylamide gel electrophoresis, it has a high-throughput capability and can resolve low-mass proteins. However, a considerable number of challenging issues related to the design of studies, including reproducibility, sensitivity, specificity, variation in sample collection, processing and storage, have been reported as problematic with this technology; albeit some of these concerns could perhaps also be lauded against other proteomic approaches that have attempted to address complex protein mixtures, such as plasma. Applications, successes and limitations of SELDI-MS in both clinical and basic science arenas will be reviewed in this article.

Previously, we reported the analysis of human plasma proteins by 2-DE under nondenaturing conditions (Type-I 2-DE) followed by the assignment of stained spots using MALDI-MS and PMF [1]. Here, we employ 2-DE conditions modified only in the second-dimensional separation; SDS was added in the gradient slab gel aiming to dissociate noncovalently bound proteins/polypeptides (Type-II 2-DE). Totally 169 CBB-stained spots on a micro-2-DE gel were numbered and subjected to polypeptide assignment using MALDI-Ms.PMF. One hundred sixty spots out of the 169 provided significant match (p

Tandem mass spectrometry (MS/MS) has become a common and useful tool for analyzing complex protein mixtures. Database search programs are the most popular means for peptide identification from MS/MS spectra. However, estimations of charge states of peptide MS/MS spectra obtained from low-resolution mass spectrometers have not been reliable. They require repetitive database searches and additional analyses of the search results. We propose here an algorithm designed to reliably differentiate doubly charged spectra from triply charged ones. We conducted a rigorous analysis of various spectral features and their effects. We employed the distinguishing features found in our analysis and developed a classifier for multiply charged spectra using a machine learning approach. The test on various data sets showed that our method could be successfully applied independent of experimental setup and mass instrument. This algorithm can be used to prefilter spectra so that only reasonably good spectra are submitted to database search programs, thereby saving considerable time. The software for MS/MS charge-state determination, which we named "CIFTER", is available at a website http://prix.uos.ac.kr/sifter/cifter.

Previously, we have reported on the analysis of human plasma proteins on a nondenaturing micro-2-DE (mu2-DE) gel, using in-gel digestion followed by MALDI-MS and PMF [1]. Many of the spots on the mu2-DE gel showed apparent masses much larger than the calculated masses of their assigned polypeptides, suggesting noncovalent or covalent interactions between the polypeptides. In the present study, we aimed to further analyze the plasma protein spots on a nondenaturing mu2-DE gel, on which protein/polypeptide interactions have been suggested. The proteins in the spots were extracted under alkaline conditions and subjected to 3-D separation using SDS-PAGE in microslab gel format (muSDS gel) with or without the sample treatment of reduction-alkylation. The clear bands in each lane of the muSDS gels demonstrated the successful extraction of proteins from the relevant gel spot and visualized the relative contents of the polypeptides in the spot. Most of the bands were assigned by in-gel digestion followed by MALDI-MS and PMF (MASCOT/Swiss-Prot). The large discrepancy between the apparent mass value of a protein spot and the estimated mass values of the polypeptide bands on a nonreducing muSDS gel strongly suggested noncovalent polypeptide interactions. The differences in the polypeptide separation patterns on the muSDS gels, between with and without the treatment of reduction-alkylation, confirmed polypeptide disulfide bonding. The method employed here, aiming to integrate information on the proteins separated on nondenaturing 2-DE gels with that on the interactions between polypeptides, would help the comprehensive understanding of complex protein systems.

A myriad of co- and post-translational modifications occur at protein N- and C-termini, resulting in an extra layer of proteome complexity and an additional source of protein regulation. Here, we review N- and C-terminal modifications and the contemporary positional proteomics techniques used to isolate protein terminal peptides from complex protein mixtures and characterize their diversity and occurrence in biological systems. Furthermore, these degradomics strategies--often referred to as N- and C-terminomics--represent dedicated high-throughput techniques to study proteolysis in dynamic living systems. Over the past decade, terminomics studies have provided indispensable information on the functional states of individual proteins, cell types, tissues, and biological processes and delivered fundamental new data for the Human Proteome Project, including high confidence identifications of many so-called "missing proteins", which had not been identified by traditional proteomics analyses.

Nontuberculous mycobacteria (NTM) have been isolated from water, soil, air, food, protozoa, plants, animals, and humans. Although most NTM are saprophytes, approximately one-third of NTM have been associated with human diseases. In this study, we did a comparative proteomic analysis among five NTM strains isolated from several sources. There were different numbers of protein spots fromM. gordonae(1,264),M. nonchromogenicumtype I (894),M. nonchromogenicumtype II (935),M. peregrinum(806), andM. scrofulaceum/Mycobacterium mantenii(1,486) strains, respectively. We identified 141 proteins common to all strains and specific proteins to each NTM strain. A total of 23 proteins were selected for its identification. Two of the common proteins identified (short-chain dehydrogenase/reductase SDR and diguanylate cyclase) did not align withM. tuberculosiscomplex protein sequences, which suggest that these proteins are found only in the NTM strains. Some of the proteins identified as common to all strains can be used as markers of NTM exposure and for the development of new diagnostic tools. Additionally, the specific proteins to NTM strains identified may represent potential candidates for the diagnosis of diseases caused by these mycobacteria.

Since the formulation of the concept of "proteomics" in 1995, a plethora of proteomic technologies have been developed in order to study proteomes of tissues, cells and organelles. The powerful new technologies enabled by proteomic approaches have lead to the application of these methods to an exponentially increasing variety of biological questions for highly complex protein mixtures. Continuous technical optimization allows for an ever-increasing sensitivity of proteomic techniques. In this review, a brief overview of currently available proteomic techniques and their applications is given, followed by a more detailed description of advantages and technical challenges of two-dimensional electrophoresis (2-DE). Some solutions to circumvent currently encountered technical difficulties for 2-DE analyses are proposed.

Our ability to alter and control the structure and function of biomolecules, and of proteins in particular, will be of utmost importance in order to understand their respective biological roles in complex systems such as living organisms. This challenge has prompted the development of powerful modern techniques in the fields of molecular biology, physical biochemistry and chemical biology. These fields complement each other and their successful combination has provided unique insights into protein structure and function at the level of isolated molecules, cells and organisms. Chemistry is without doubt most suited for introducing subtle changes into biomolecules down to the atomic level, but often struggles when it comes to large targets, such as proteins. In this review, we attempt to give an overview of modern and broadly applicable techniques that permit chemical synthesis to be applied to complex protein targets in order to gain control over their structure and function. As will be demonstrated, these approaches offer unique possibilities in our efforts to understand the molecular basis of protein functioning in vitro and in vivo. We will discuss modern synthetic reactions that can be applied to proteins and give examples of recent highlights. Another focus of this review will be the application of inteins as versatile protein engineering tools.

The objective of this presentation is to review the major proteomic technologies available to developmental toxicologists and, when possible, to provide examples of how various proteomic technologies have been used in developmental toxicology or toxicology in general. The field of proteomics is too broad for us to go into great depth about each technology, so we have attempted to provide brief overviews supplemented with many references that cover the subjects in more detail. Proteomics tools produce a global view of complex biological systems by examining complex protein mixtures using large-scale, high-throughput technologies. These technologies speed up the process of protein separation, quantification, and identification. As an important complement to genomics, proteomics allows for the examination of the entire complement of proteins in an organism, tissue, or cell-type. Current proteomics technologies not only identify protein expression, but also post-translational modifications and protein interactions. The field of proteomics is expanding rapidly to provide greater volume and quality of protein information to help understand the multifaceted nature of biological systems.

Tandem mass spectrometry has been used increasingly for high-throughput analysis of complex protein samples. A major challenge lies in the consistent, objective and transparent analysis of the large amounts of data generated by such experiments and in their dissemination and publication. Here, we review currently available computational tools and discuss the need for statistical criteria in the analysis of large proteomics datasets.

Advances in molecular biology over the past decade have helped to enhance our understanding of the complex interplay between genetic, transcriptional and translational alterations in human cancers. These molecular changes are the basis for an evolving field of high-throughput cancer discovery techniques using microscopic amounts of patient-based materials. Laser capture microdissection allows pure populations of cells to be isolated from both the tumor and stroma in order to identify subtle differences in RNA and protein expression. Comparative analysis of these alterations between normal, pre-invasive, and invasive tissue using powerful bioinformatics programs has allowed us to identify novel tumor markers, profile complex protein pathways, and develop new molecular-based therapies. Continued refinement of such high-throughput microtechnologies will enable us to rapidly query patient specimens to identify novel methods for early detection, treatment, and follow-up of a wide array of human cancers.

The use of data-independent acquisition (DIA) approaches for the reproducible and precise quantification of complex protein samples has increased in the last years. The protein information arising from DIA analysis is stored in digital protein maps (DIA maps) that can be interrogated in a targeted way by using ad hoc or publically available peptide spectral libraries generated on the same sample species as for the generation of the DIA maps. The restricted availability of certain difficult-to-obtain human tissues (i.e., brain) together with the caveats of using spectral libraries generated under variable experimental conditions limits the potential of DIA. Therefore, DIA workflows would benefit from high-quality and extended spectral libraries that could be generated without the need of using valuable samples for library production. We describe here two new targeted approaches, using either classical data-dependent acquisition repositories (not specifically built for DIA) or ad hoc mouse spectral libraries, which enable the profiling of human brain DIA data set. The comparison of our results to both the most extended publically available human spectral library and to a state-of-the-art untargeted method supports the use of these new strategies to improve future DIA profiling efforts.

Absolute quantification of protein expression and post-translational modifications by mass spectrometry has been challenging due to a variety of factors, including the potentially large dynamic range of phosphorylation response. To address these issues, we have developed MARQUIS — Multiplex Absolute Regressed Quantification with Internal Standards — a novel mass spectrometry-based approach using a combination of isobaric tags and heavy-labeled standard peptides to construct internal standard curves for peptides derived from key nodes in signal transduction networks. We applied MARQUIS to quantify phosphorylation dynamics within the EGFR network at multiple time points following stimulation with several ligands, enabling a quantitative comparison of EGFR phosphorylation sites and demonstrating that receptor phosphorylation is qualitatively similar but quantitatively distinct for each EGFR ligand tested. MARQUIS was also applied to quantify the effect of EGFR kinase inhibition on glioblastoma patient derived xenografts. MARQUIS is a versatile method, broadly applicable and extendable to multiple mass spectrometric platforms.

The modular architecture of protein-protein interaction (PPI) networks is evident in diverse species with a wide range of complexity. However, the molecular components that lead to the evolution of modularity in PPI networks have not been clearly identified. Here, we show that weak domain-linear motif interactions (DLIs) are more likely to connect different biological modules than strong domain-domain interactions (DDIs). This molecular division of labor is essential for the evolution of modularity in the complex PPI networks of diverse eukaryotic species. In particular, DLIs may compensate for the reduction in module boundaries that originate from increased connections between different modules in complex PPI networks. In addition, we show that the identification of biological modules can be greatly improved by including molecular characteristics of protein interactions. Our findings suggest that transient interactions have played a unique role in shaping the architecture and modularity of biological networks over the course of evolution., Author Summary Modular architecture is important for the evolution of cellular systems. Modular rearrangements facilitate functional innovations and modular insulations provide robustness to perturbations. However, molecular-level understanding of the mechanisms underlying modular network evolution is currently not well understood. Here we show that strong domain-domain interactions (DDIs) and weak domain-linear motif interactions (DLIs) made different contributions to the evolution of the modular architecture of PPI networks. Especially, DLIs mediate between-module interactions, and that their relative abundance has dramatically increased in metazoan species. Linear motifs have been identified as evolutionary interaction switches since subtle amino acid changes can cause the short sequences in linear motifs to appear and disappear. Our results suggest that subtle changes in linear motifs have contributed to the rewiring of functional modules and, consequently, to functional innovations in metazoan species.

With whole genomes being sequenced almost routinely, the next logical step towards a better understanding of cellular mechanisms lies in studying the functional units of gene expression-proteins. One of the fundamental approaches in proteomics is the use of two-dimensional gel electrophoresis as a mode of separation and visualization of complex protein mixtures. Despite several limitations of the method, its ability to separate large numbers of proteins, including their post-translationally modified forms, ensures that it will continue to be popular in several well-defined areas of proteomics. In this article, we discuss the merits and drawbacks of two-dimensional gels and compare them with alternative systems such as one-dimensional gels and liquid chromatography-based separation methods. In the wake of recent advances in mass spectrometry and related areas, we outline areas where two-dimensional gels can best be utilized as the preferred separation method in proteomic strategies.

Immunology has contributed to biomedical education in many important ways since the creation of scientific medicine in the last quarter of the 19th century. Today, immunology is a major area of biomedical research. Nevertheless, there are many basic problems unresolved in immunological activities and phenomena. Solving these problems is probably necessary to devise predictable and safe ways to produce new vaccines, treat allergy and autoimmune diseases and perform safe transplants. This challenge involves not only technical developments but also changes in attitude, of which the most fundamental is to abandon the traditional stimulus-response perspective in favor of more "systemic" views. Describing immunological activities as the operation of a complex multi connected network, raises biological and epistemological issues not usually dealt with in biomedical education. Here we point to one example of systemic approaches. A new form of immunoblot (Panama blot), by which the reaction of natural immunoglobulins with complex protein mixtures may be analyzed by a special software and multivariate statistics, has been recently used to characterize human autoimmune diseases. Our preliminary data show that Panama blots can also be used to characterize global (systemic) immunological changes in chronic human parasitic diseases, such as malaria and schistosomiasis mansoni, that correlate with the clinical status.

Three early proteins expressed by adenovirus type 5, E1b 55K, E4 Orf3 and E4 Orf6, are involved in regulating late viral gene expression. It has previously been shown that 55K associates with Orf6. Here we show that 55K also associates with Orf3 and that this interaction is necessary for 55K to localize to the nuclear matrix fraction of the cell. From our data, we infer that the Orf3 and Orf6 interactions with 55K may be mutually exclusive. The Orf3 protein is also known to associate with and cause the reorganization of cell nucleus structures known as ND10 or PODs. Consistent with the observed increase in the biochemical interaction between 55K and Orf3 in the absence of Orf6, the 55K association with Orf3 in ND10 was also found to increase in the absence of Orf6. The most studied cellular component of ND10 is PML, a complex protein present in a range of isoforms, some of which are modified by conjugation to the small ubiquitin-like protein PIC-1. The pattern of PML isoforms was altered in adenovirus-infected cells, in that a number of additional isoform bands appeared in an Orf3-dependent manner, one of which became predominant later in infection. As for ND10 reorganization, neither Orf6 nor 55K was required for this effect. Therefore it is likely that these changes in PML are related to the changes in ND10 structure that occur during infection.

Proteins immobilized on insoluble carriers or immunoadsorbents are now widely used in many areas of biological research. They are especially efficient in studies of complex protein mixtures because immunoadsorbents are very useful for isolation of individual components for further investigation. Antibodies immobilized on paper retain their specific activity and can be applied as very sensitive measurement tool.

Eradication of meningitis and septicemia caused by Neisseria meningitidis is approaching reality. Capsule polysaccharide (CPS)-conjugate vaccines are expected to virtually eliminate meningococcal d...

The widespread use of biologics has paved way for newer options in therapeutics for once incurable illnesses. Their large and complex protein structure, post-translational modifications, elaborate manufacturing/production process and risk for immunogenicity adds to the uniqueness of a biologic product. Patent expiration of innovator biologics has led to the development of biosimilars; biologics similar /comparable to the reference product in terms of quality, safety and efficacy. We discuss the clinical safety and regulatory requirements for biosimilars in various countries across the world. Future holds promise for biosimilars to provide affordable, efficacious and safe treatment to a vast majority of patients with significant cost savings to the nation.

Application of nuclear magnetic resonance (NMR) methods for the structural characterization to larger and more complex protein systems can be facilitated through the development of new methods for resonance assignment. Here, a novel approach that relies on integration of NMR and mass spectrometry (MS) methods is explored. The approach relies on the fact that both NMR and MS are able to monitor rates of exchange of amide protons for water deuterons. Correlating the rates can connect cross-peak positions from NMR data with fragment masses from MS data to support sequential assignment. The example provided is for a small model protein, ubiquitin, but the potential for application to large, more difficult to express proteins is clear.

Current ideas about the composition of the focal adhesion complexes in cultured cells are based mainly on indirect immunocytochemical data. We here report a two-dimensional (2-D) gel electrophoresis analysis of the focal adhesion associated-structures that remain in the growth substrate after removal of cells by mechanical shearing. Many proteins additional to the known adhesion proteins, and in higher abundance, could be identified. Using selective extraction procedures, employing detergent or gelsolin, these could be classified as either membrane-associated, actin-associated or both. Cross correlation of these polypeptode patterns with a 2-D gel database allowed identification of some proteins, not previously considered as resident of focal adhesions. The data point to a more complex make up of focal adhesions than formerly supposed.

The compatibility of superficially porous (SP) resin for label-free intact protein analysis with online capillary LC/MS is demonstrated to give improved chromatographic resolution, sensitivity, and reproducibility. The robustness of the platform was measured against several samples of varying complexity and sample loading amount. The results indicate that capillary SP columns provide high loading capacities and that ∼6 s chromatographic peak widths are typical for standard proteins in simple mixtures and proteins isolated from cell and tissue lysates. Subfemtomole detection limits for standard proteins were consistently observed, with the lowest levels at 12 amol for ubiquitin. The analysis of total heart homogenates shows that capillary SP columns provide theoretical peak capacity of 106 protein forms with 30 min total analysis time and enabled detection of proteins from complex mixtures with a single high-resolution scan. The SPLC/MS platform also detected 343 protein forms from two HeLa acid extract replicate analyses that consumed 5 × 10(4) cells and 30 min analysis time, each. Comparison of all the species observed in each HeLa replicate showed 90% overlap (309 forms) with a Pearson correlation coefficient of 89.9% for the common forms observed in the replicates. Efficient acid extract of 1 × 10(4) HeLa cells allowed reproducible detection of common modification states and members from all five of the histone families and demonstrated that capillary SPLC/MS supports reproducible label-free profiling of histones in15 min total analysis time. The data presented demonstrate that a capillary LC/MS platform utilizing superficially porous stationary phase and a LTQ-Orbitrap FT-MS is fast, sensitive, and reproducible for intact protein profiling from small tissue and cell amounts.

Collagen contains the unique imino acid hydroxyproline (HyPro), which is involved in the stabilization of this triple helical molecule. The concentration of HyPro is customarily used to calculate the total collagen content in a cell culture environment and in acid hydrolysates of normal and pathophysiological tissues. Radiolabelling, chromatographic and calorimetric assays have been developed over the years for the accurate determination of collagen content through HyPro estimation. Recently, the Sircol Collagen Assay (SCA) has been almost exclusively adopted as the fastest and simplest colorimetric method for the determination of collagen concentration in complex protein solutions. We show here that the colorimetric SCA, which is based on the binding of Sirius red (SR) to collagen, is flawed by interference of non-collagenous proteins (e.g. serum). In fact, we demonstrate that SCA in cell culture systems and tissue hydrolysates results in a dramatic overestimation of collagen content ranging from 3- to 24-fold. In order to rescue this otherwise very practical assay, we introduce a simple purification procedure that allows the removal of interfering non-collagenous proteins from culture media and tissue samples so that accurate measurements with SCA are now possible.

Protein X-ray crystallography recently celebrated its 50th anniversary. The structures of myoglobin and hemoglobin determined by Kendrew and Perutz provided the first glimpses into the complex protein architecture and chemistry. Since then, the field of structural molecular biology has experienced extraordinary progress and now more than 55 000 protein structures have been deposited into the Protein Data Bank. In the past decade many advances in macromolecular crystallography have been driven by world-wide structural genomics efforts. This was made possible because of third-generation synchrotron sources, structure phasing approaches using anomalous signal, and cryo-crystallography. Complementary progress in molecular biology, proteomics, hardware and software for crystallographic data collection, structure determination and refinement, computer science, databases, robotics and automation improved and accelerated many processes. These advancements provide the robust foundation for structural molecular biology and assure strong contribution to science in the future. In this report we focus mainly on reviewing structural genomics high-throughput X-ray crystallography technologies and their impact.

Due to physical and chemical phenomena, a simple sample can give rise to a complex mass spectrum with many more peaks than the number of molecular species present in the sample. We link peaks within and between different spectra, and come up with an advanced analysis approach to produce reliable estimates of the molecule masses and abundances. By linking peaks, we can locate multiple-charge peaks at the correct position in the spectrum, we can deconvolute complex regions with many overlapping peaks by including information from related regions with lower complexity and higher resolution, and we reduce the total number of observed peaks in a spectrum to a much smaller number of underlying molecular species. In this paper we properly model 29 952 peaks in 64 spectra, using only 39 location parameters and one shape parameter. This major reduction from many different molecules to a limited set of molecular species reduces the statistical test multiplicity for biomarker discovery and therefore we imply that the reduction should eventually increase the biomarker discovery power significantly, too.

At first, a short history of the beginning of 2-DE is provided. Based on the present state of the art at the time I developed a 2-DE technique in 1975 that was able to resolve complex protein extracts from mouse tissues in hundreds of protein spots. My intention was to study proteins from a global point of view. Questions of interest were, how do proteins change during embryonic development, and what is the effect of induced mutations on the protein level. At that time protein chemistry was a matter of analyzing single proteins in detail. Therefore, my approach was frequently criticized as inappropriate because it would be impossible to identify and characterize the hundreds of proteins resolved. But soon it was realized that studying total proteins gives opportunities to answer many interesting questions. This led to a research field nowadays called "proteomics". Already in the beginning of the 1980s the idea to analyze the total human proteins had come up. By entering the post-genome era it became obvious that a human proteome project is needed in order to explain the human genome in terms of its functions. The problems in realizing such a project are considered.