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A quantitative bioanalytical liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for GSK2126458, a dual PI3K/mTOR inhibitor, was developed and validated. Plasma and tumor homogenate samples were pre-treated using protein precipitation with acetonitrile containing dabrafenib as internal standard. After dilution with water, the extract was directly injected into the reversed-phase liquid chromatographic system. The eluate was transferred into the electrospray interface with positive ionization and compounds were detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was completely validated for plasma in a 4–4000 ng/ml calibration range with r 2 = 0.9996 ± 0.0003 using double logarithmic calibration ( n = 5). Within-run precisions ( n = 6) were 2.0–5.3% and between-run (3 runs; n = 18) precisions 2.7–5.8%. Accuracies were between 101 and 105% for the whole calibration range. The drug was sufficiently stable under all relevant analytical conditions. Finally, the assay was successfully applied to determine plasma and tumor drug levels after oral administration of GSK2126458 to mice with AMC711T neuroblastoma xenografts.

Protein-protein interactions are important in providing compartmentalization and specificity in cellular signal transduction. Many studies have hallmarked the well designed compartmentalization of the cAMP-dependent protein kinase (PKA) through its anchoring proteins. Much less data are available on the compartmentalization of its closest homolog, cGMP-dependent protein kinase (PKG), via its own PKG anchoring proteins (GKAPs). For the enrichment, screening, and discovery of (novel) PKA anchoring proteins, a plethora of methodologies is available, including our previously described chemical proteomics approach based on immobilized cAMP or cGMP. Although this method was demonstrated to be effective, each immobilized cyclic nucleotide did not discriminate in the enrichment for either PKA or PKG and their secondary interactors. Hence, with PKG signaling components being less abundant in most tissues, it turned out to be challenging to enrich and identify GKAPs. Here we extend this cAMP-based chemical proteomics approach using competitive concentrations of free cyclic nucleotides to isolate each kinase and its secondary interactors. Using this approach, we identified Huntingtin-associated protein 1 (HAP1) as a putative novel GKAP. Through sequence alignment with known GKAPs and secondary structure prediction analysis, we defined a small sequence domain mediating the interaction with PKG Iβ but not PKG Iα. In vitro binding studies and site-directed mutagenesis further confirmed the specificity and affinity of HAP1 binding to the PKG Iβ N terminus. These data fully support that HAP1 is a GKAP, anchoring specifically to the cGMP-dependent protein kinase isoform Iβ, and provide further evidence that also PKG spatiotemporal signaling is largely controlled by anchoring proteins.

Tiam1 (T-cell lymphoma invasion and metastasis 1) is a guanine nucleotide exchange factor that specifically controls the activity of the small GTPase Rac, a key regulator of cell adhesion, proliferation, and survival. Here, we report that in response to mitogens, Tiam1 is degraded by the ubiquitin-proteasome system via the SCF(βTrCP) ubiquitin ligase. Mitogenic stimulation triggers the binding of Tiam1 to the F-box protein βTrCP via its degron sequence and subsequent Tiam1 ubiquitylation and proteasomal degradation. The proteolysis of Tiam1 is prevented by βTrCP silencing, inhibition of CK1 and MEK, or mutation of the Tiam1 degron site. Expression of a stable Tiam1 mutant that is unable to interact with βTrCP results in sustained activation of the mTOR/S6K signaling and increased apoptotic cell death. We propose that the SCF(βTrCP)-mediated degradation of Tiam1 controls the duration of the mTOR-S6K signaling pathway in response to mitogenic stimuli.

The PRIME-XS consortium is a pan-European infrastructure for proteomics. As a prologue to this special issue of Molecular & Cellular Proteomics on the research activities of the PRIME-XS consortium, we, as the guest editors of this issue, provide an overview of the structure and activities of this consortium, which is funded by the European Union's 7th Framework Programme for Research and Technological Development.

Protein phosphorylation is a widespread process forming the mechanistic basis of cellular signaling. Up to now, different aspects, for example, site-specificity, kinetics, role of co-factors, and structure-function relationships have been typically investigated by multiple techniques that are incompatible with one another. The approach introduced here maximizes the amount of information gained on protein (complex) phosphorylation while minimizing sample handling. Using high-resolution native mass spectrometry on intact protein (assemblies) up to 150 kDa we track the sequential incorporation of phosphate groups and map their localization by peptide LC-MS/MS. On two model systems, the protein kinase G and the interplay between Aurora kinase A and Bora, we demonstrate the simultaneous monitoring of various aspects of the phosphorylation process, namely the effect of different cofactors on PKG autophosphorylation and the interaction of AurA and Bora as both an enzyme-substrate pair and physical binding partners.

Unambiguous identification of tandem mass spectra is a cornerstone in mass-spectrometry-based proteomics. As the study of post-translational modifications (PTMs) by means of shotgun proteomics progresses in depth and coverage, the ability to correctly identify PTM-bearing peptides is essential, increasing the demand for advanced data interpretation. Several PTMs are known to generate unique fragment ions during tandem mass spectrometry, the so-called diagnostic ions, which unequivocally identify a given mass spectrum as related to a specific PTM. Although such ions offer tremendous analytical advantages, algorithms to decipher MS/MS spectra for the presence of diagnostic ions in an unbiased manner are currently lacking. Here, we present a systematic spectral-pattern-based approach for the discovery of diagnostic ions and new fragmentation mechanisms in shotgun proteomics datasets. The developed software tool is designed to analyze large sets of high-resolution peptide fragmentation spectra independent of the fragmentation method, instrument type, or protease employed. To benchmark the software tool, we analyzed large higher-energy collisional activation dissociation datasets of samples containing phosphorylation, ubiquitylation, SUMOylation, formylation, and lysine acetylation. Using the developed software tool, we were able to identify known diagnostic ions by comparing histograms of modified and unmodified peptide spectra. Because the investigated tandem mass spectra data were acquired with high mass accuracy, unambiguous interpretation and determination of the chemical composition for the majority of detected fragment ions was feasible. Collectively we present a freely available software tool that allows for comprehensive and automatic analysis of analogous product ions in tandem mass spectra and systematic mapping of fragmentation mechanisms related to common amino acids.

Mature human adenovirus particles contain four minor capsid proteins, in addition to the three major capsid proteins (penton base, hexon and fiber) and several proteins associated with the genomic core of the virion. Of the minor capsid proteins, VI plays several crucial roles in the infection cycle of the virus, including hexon nuclear targeting during assembly, activation of the adenovirus proteinase (AVP) during maturation and endosome escape following cell entry. VI is translated as a precursor (pVI) that is cleaved at both the N- and C-termini by AVP. Whereas the role of the C-terminal fragment of pVI, pVIc, is well established as an important co-factor of AVP, the role of the N-terminal fragment, pVIn, is currently elusive. In fact, the fate of pVIn following proteolytic cleavage is completely unknown. Here, we use a combination of proteomics-based peptide identification, native mass spectrometry and hydrogen-deuterium exchange mass spectrometry to show that pVIn is associated with mature human adenovirus, where it binds at the base of peripentonal hexons in a pH-dependent manner. Our findings suggest a possible role for pVIn in targeting pVI to hexons for proper assembly of the virion and timely release of the membrane lytic mature VI molecule.

Post-translational modifications (PTMs) play an important role in the regulation of protein function. Mass spectrometry based proteomics experiments nowadays identify tens of thousands of PTMs in a single experiment. A wealth of data has therefore become publically available. Evidently the biological function of each PTM is the key question to be addressed; however, such analyses focus primarily on single PTM events. This ignores the fact that PTMs may act in concert in the regulation of protein function, a process termed PTM crosstalk. Relatively little is known on the frequency and functional relevance of crosstalk between PTM sites. In a bioinformatics approach, we extracted PTMs occurring in proximity in the protein sequence from publically available databases. These PTMs and their flanking sequences were subjected to stringent motif searches, including a scoring for evolutionary conservation. Our unprejudiced approach was able to detect a respectable set of motifs, of which about half were described previously. Among these we could add many new proteins harboring these motifs. We extracted also several novel motifs, which through their widespread appearance and high conservation may pinpoint at previously nonannotated concerted PTM actions. By employing network analyses on these proteins, we propose putative functional roles for these novel motifs with two PTM sites in close proximity.

TFAP4, a basic helix-loop-helix transcription factor that regulates the expression of a multitude of genes involved in the regulation of cellular proliferation, stemness, and epithelial-mesenchymal transition, is up-regulated in colorectal cancer and a number of other human malignancies. We have found that, during the G2 phase of the cell division cycle, TFAP4 is targeted for proteasome-dependent degradation by the SCF(βTrCP) ubiquitin ligase. This event requires phosphorylation of TFAP4 on a conserved degron. Expression of a stable TFAP4 mutant unable to interact with βTrCP results in a number of mitotic defects, including chromosome missegregation and multipolar spindles, which eventually lead to the activation of the DNA damage response. Our findings reveal that βTrCP-dependent degradation of TFAP4 is required for the fidelity of mitotic division.

Peptide and protein self-assembly resulting in the formation of amyloidogenic aggregates is generally thought of as a pathological event associated with severe diseases. However, amyloid formation may also provide a basis for advanced bionanomaterials, since amyloid fibrils combine unique material-like properties that make them very useful for design of new types of conducting nanowires, bioactive ligands, and biodegradable coatings as drug-encapsulating materials. The morphology of the supramolecular aggregates determines the properties and application range of these bionanomaterials. An important parameter to control the supramolecular morphology, is the overall charge of the peptide, which is related to the pH of the environment. Herein, we describe the design, synthesis and morphological analysis of a series of N-terminally functionalized Aβ(16-22) peptides (∼1/4Lys-Leu-Val-Phe-Phe-Ala-Glu-OH), that underwent a pH-induced polymorphism, ranging from lamellar sheets, helical tapes, peptide nanotubes, and amyloid fibrils as was observed by transmission electron microscopy. Infrared spectroscopy and wide angle X-ray scattering studies showed that peptide self-assembly was driven by β-sheet formation, and that the supramolecular morphology was directed by subtle variations in electrostatic interactions. Finally, a structural model and hierarchy of self-assembly of a peptide nanotube, assembled at pH 1, is proposed.

The synthesis of a new triazacyclophane scaffold (TACO scaffold) containing three selectively deprotectable amines is described. The TACO scaffold is conformationally more constrained than our frequently used TAC scaffold, due to introduction of a substituent on the para position of the benzoic acid hinge, which prevents ring flipping and makes it more attractive than the TAC scaffold for preparation of artificial receptor molecules or for mimicking discontinuous epitopes toward protein mimics when more preorganization is required.

There is an urgent need to develop reliable strategies for the rapid assembly of complex oligosaccharides. This paper presents a set of strategically selected orthogonal protecting groups, glycosyl donors modified by a (S)-phenylthiomethylbenzyl ether at C-2, and a glycosyl acceptor containing a fluorous tag, which makes it possible to rapidly prepare complex branched oligosaccharides of biological importance. The C-2 auxiliary controlled the 1,2-cis anomeric selectivity of the various galactosylations. The orthogonal protecting groups, 2-naphthylmethyl ether (Nap) and levulinic ester (Lev), made it possible to generate glycosyl acceptors and allowed the installation of a crowded branching point. After the glycosylations, the chiral auxiliary could be removed using acidic conditions, which was compatible with the presence of the orthogonal protecting groups Lev and Nap, thereby allowing the efficient installation of 1,2-linked glycosides. The light fluorous tag made it possible to purify the compounds by a simple filtration method using silica gel modified by fluorocarbons. The set of building blocks was successfully employed for the preparation of the carbohydrate moiety of the GPI anchor of Trypanosoma brucei, which is a parasite that causes sleeping sickness in humans and similar diseases in domestic animals.

Protein kinase A (PKA) in vertebrates is localized to specific locations in the cell via A-kinase anchoring proteins (AKAPs). The regulatory subunits of the four PKA isoforms (RIα, RIβ, RIIα, and RIIβ) each form a homodimer, and their dimerization domain interacts with a small helical region present in each of the more than 40 AKAPs reported so far. This allows for tight anchoring of PKA and efficient communication with other signaling proteins that interact with the AKAP scaffold in a spatial and temporal manner. The hydrophobic interaction surfaces of the PKA-R dimer and several AKAP helices have been investigated in great detail. Despite this knowledge, not every suggested AKAP has had its binding motif specified. Here we created an efficient bioinformatic tool, termed THAHIT, to accurately map the PKA binding motif and/or additional motifs of all previously reported AKAPs. Moreover, THAHIT predicts its specificity toward PKA-RIα and/or PKA-RIIα binding. To verify the validity of these newly predicted anchoring sites and their putative specificities, we used computational modeling approaches (HADDOCK), biochemical affinity studies (fluorescence anisotropy), and cellular colocalization studies. We further demonstrate the potential of THAHIT to identify novel AKAPs in cAMP-based chemical proteomics discovery data sets, and the human proteome. We retrieved numerous novel AKAP candidates, including a never reported 330 kDa AKAP observed in heart tissue, which we further characterized biochemically as a PKA-RIIα binder. Altogether, THAHIT provides a comprehensive overview of known and novel PKA-AKAP interaction domains and their PKA-R specificities.

Somatic cell reprogramming to a pluripotent state continues to challenge many of our assumptions about cellular specification, and despite major efforts, we lack a complete molecular characterization of the reprograming process. To address this gap in knowledge, we generated extensive transcriptomic, epigenomic and proteomic data sets describing the reprogramming routes leading from mouse embryonic fibroblasts to induced pluripotency. Through integrative analysis, we reveal that cells transition through distinct gene expression and epigenetic signatures and bifurcate towards reprogramming transgene-dependent and -independent stable pluripotent states. Early transcriptional events, driven by high levels of reprogramming transcription factor expression, are associated with widespread loss of histone H3 lysine 27 (H3K27me3) trimethylation, representing a general opening of the chromatin state. Maintenance of high transgene levels leads to re-acquisition of H3K27me3 and a stable pluripotent state that is alternative to the embryonic stem cell (ESC)-like fate. Lowering transgene levels at an intermediate phase, however, guides the process to the acquisition of ESC-like chromatin and DNA methylation signature. Our data provide a comprehensive molecular description of the reprogramming routes and is accessible through the Project Grandiose portal at http://www.stemformatics.org.

Ultra-high pressure liquid chromatography (UHPLC) systems combined with state-of-the-art mass spectrometers have pushed the limit of deep proteome sequencing to new heights making it possible to identify thousands of proteins in a single LC-MS experiment within a few hours. The recently released EASY-spray technology allows one to implement nano-UHPLC with straightforwardness. In this work we initially characterized the EASY-spray containing a 50 cm column containing 7500 proteins. We report, here, that with this fast online SCX-RP UHPLC-MS/MS workflow, the proteome coverage can be substantially extended without significantly compromising the analysis time and sample usage.

In response to genotoxic stress, DNA damage checkpoints maintain the integrity of the genome by delaying cell cycle progression to allow for DNA repair. Here we show that the degradation of the basic helix-loop-helix (bHLH) transcription factor DEC1, a critical regulator of cell fate and circadian rhythms, controls the DNA damage response. During unperturbed cell cycles, DEC1 is a highly unstable protein that is targeted for proteasome-dependent degradation by the SCF(βTrCP) ubiquitin ligase in cooperation with CK1. Upon DNA damage, DEC1 is rapidly induced in an ATM/ATR-dependent manner. DEC1 induction results from protein stabilization via a mechanism that requires the USP17 ubiquitin protease. USP17 binds and deubiquitylates DEC1, markedly extending its half-life. Subsequently, during checkpoint recovery, DEC1 proteolysis is reestablished through βTrCP-dependent ubiquitylation. Expression of a degradation-resistant DEC1 mutant prevents checkpoint recovery by inhibiting the downregulation of p53. These results indicate that the regulated degradation of DEC1 is a key factor controlling the DNA damage response.

Summary The scarcity of primordial germ cells (PGCs) in the developing mammalian embryo hampers robust biochemical analysis of the processes that underlie early germ cell formation. Here, we demonstrate that DAZL, a germ cell-specific RNA binding protein, is a robust PGC marker during in vitro germ cell development. Using Dazl-GFP reporter ESCs, we demonstrate that DAZL plays a central role in a large mRNA/protein interactive network that blocks the translation of core pluripotency factors, including Sox2 and Sall4, as well as of Suz12, a polycomb family member required for differentiation of pluripotent cells. Thus, DAZL limits both pluripotency and somatic differentiation in nascent PGCs. In addition, we observed that DAZL associates with mRNAs of key Caspases and similarly inhibits their translation. This elegant fail-safe mechanism ensures that, whereas loss of DAZL results in prolonged expression of pluripotency factors, teratoma formation is avoided due to the concomitant activation of the apoptotic cascade., Graphical Abstract, Highlights • DAZL colocalizes with a network of translational inhibitors in granular structures • DAZL interacts with mRNA transcripts of key pluripotency genes and Caspases • Loss of DAZL function leads to prolonged expression of pluripotency genes • Loss of DAZL leads to expression of Caspases, resulting in apoptosis in PGCs, In this article, Geijsen and colleagues demonstrate that DAZL acts as a translational suppressor of pluripotency, differentiation, and apoptosis in developing primordial germ cells. As such, DAZL limits pluripotency while simultaneously preventing somatic differentiation and provides an elegant fail-safe mechanism into the PGC system, in which the loss of pluripotency regulation simultaneously triggers germ cell death and prevents germ cell tumor formation.

The cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) is an elementary molecule involved in both acute and chronic modulation of cardiac function. Substantial research in recent years has highlighted the importance of A-kinase anchoring proteins (AKAP) therein as they act as the backbones of major macromolecular signalling complexes of the β-adrenergic/cAMP/PKA pathway. This review discusses the role of AKAP-associated protein complexes in acute and chronic cardiac modulation by dissecting their role in altering the activity of different ion channels, which underlie cardiac action potential (AP) generation. In addition, we review the involvement of different AKAP complexes in mechanisms of cardiac remodelling and arrhythmias.

A single step immobilization-polymerization strategy of a highly active antimicrobial peptide into a soft hydrogel network on a poly(ethylene terephthalate) surface using thiol-ene chemistry is described. The bactericidal hydrogel was molecularly characterized via Coomassie and Lowry assay protein staining agents as well as by X-ray photoelectron spectroscopy. The bactericidal activity was established against Staphylococcus aureus and Staphylococcus epidermidis, two bacterial strains commonly associated with biomaterial infections. To gain further insight into the biological stability, the hydrogels were incubated with human serum prior to activity testing without loss of activity. These studies revealed a promising bactericidal hydrogel with good stability under physiological conditions.

The ectopic expression of Oct4, Klf4, c-Myc and Sox2 (OKMS) transcription factors allows reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). The reprogramming process, which involves a complex network of molecular events, is not yet fully characterized. Here we perform a quantitative mass spectrometry-based analysis to probe in-depth dynamic proteome changes during somatic cell reprogramming. Our data reveal defined waves of proteome resetting, with the first wave occurring 48 h after the activation of the reprogramming transgenes and involving specific biological processes linked to the c-Myc transcriptional network. A second wave of proteome reorganization occurs in a later stage of reprogramming, where we characterize the proteome of two distinct pluripotent cellular populations. In addition, the overlay of our proteome resource with parallel generated -omics data is explored to identify post-transcriptionally regulated proteins involved in key steps during reprogramming.

MicroRNAs (miRNAs) are critical to somatic cell reprogramming into induced pluripotent stem cells (iPSCs), however, exactly how miRNA expression changes support the transition to pluripotency requires further investigation. Here we use a murine secondary reprogramming system to sample cellular trajectories towards iPSCs or a novel pluripotent 'F-class' state and perform small RNA sequencing. We detect sweeping changes in an early and a late wave, revealing that distinct miRNA milieus characterize alternate states of pluripotency. miRNA isoform expression is common but surprisingly varies little between cell states. Referencing other omic data sets generated in parallel, we find that miRNA expression is changed through transcriptional and post-transcriptional mechanisms. miRNA transcription is commonly regulated by dynamic histone modification, while DNA methylation/demethylation consolidates these changes at multiple loci. Importantly, our results suggest that a novel subset of distinctly expressed miRNAs supports pluripotency in the F-class state, substituting for miRNAs that serve such roles in iPSCs.

Outcomes of comparative evaluations of enrichment methods for phosphopeptides depend highly on the experimental protocols used, the operator, the source of the affinity matrix, and the samples analyzed. Here, we attempt such a comparative study exploring a very large synthetic library containing thousands of serine, threonine, and tyrosine phosphorylated peptides, being present in roughly equal abundance, along with their nonphosphorylated counterparts, and use an optimized protocol for enrichment by TiO2 and Ti(4+)-immobilized metal affinity chromatography (IMAC) by a single operator. Surprisingly, our data reveal that there are minimal differences between enrichment of phosphopeptides by TiO2 and Ti(4+)-IMAC when considering biochemical and biophysical parameters such as peptide length, sequence surrounding the site, hydrophobicity, and nature of the amino acid phosphorylated. Similar results were obtained when evaluating a tryptic digest of a cellular lysate, representing a more natural source of phosphopeptides. All the data presented are available via ProteomeXchange with the identifier PXD000759.

Phosphorylated tyrosine hydroxylase (TH) can form complexes with 14-3-3 proteins, resulting in enzyme activation and stabilization. Although TH was among the first binding partners identified for these ubiquitous regulatory proteins, the binding stoichiometry and the activation mechanism remain unknown. To address this, we performed native mass spectrometry analyses of human TH (nonphosphorylated or phosphorylated on Ser19 (TH-pS19), Ser40 (TH-pS40), or Ser19 and Ser40 (TH-pS19pS40)) alone and together with 14-3-3γ. Tetrameric TH-pS19 (224 kDa) bound 14-3-3γ (58.3 kDa) with high affinity (Kd = 3.2 nM), generating complexes containing either one (282.4 kDa) or two (340.8 kDa) dimers of 14-3-3. Electron microscopy also revealed one major population of an asymmetric complex, consistent with one TH tetramer and one 14-3-3 dimer, and a minor population of a symmetric complex of one TH tetramer with two 14-3-3 dimers. Lower phosphorylation stoichiometries (0.15-0.54 phosphate/monomer) produced moderate changes in binding kinetics, but native MS detected much less of the symmetric TH:14-3-3γ complex. Interestingly, dephosphorylation of [(32)P]-TH-pS19 was mono-exponential for low phosphorylation stoichiometries (0.18-0.52), and addition of phosphatase accelerated the dissociation of the TH-pS19:14-3-3γ complex 3- to 4-fold. All together this is consistent with a model in which the pS19 residues in the TH tetramer contribute differently in the association to 14-3-3γ. Complex formation between TH-pS40 and 14-3-3γ was not detected via native MS, and surface plasmon resonance showed that the interaction was very weak. Furthermore, TH-pS19pS40 behaved similarly to TH-pS19 in terms of binding stoichiometry and affinity (Kd = 2.1 nM). However, we found that 14-3-3γ inhibited the phosphorylation rate of TH-pS19 by PKA (3.5-fold) on Ser40. We therefore conclude that Ser40 does not significantly contribute to the binding of 14-3-3γ, and rather has reduced accessibility in the TH:14-3-3γ complex. This adds to our understanding of the fine-tuned physiological regulation of TH, including hierarchical phosphorylation at multiple sites.

Over the last two decades, native mass spectrometry (MS) has emerged as a valuable tool to study intact proteins and noncovalent protein complexes. Studied experimental systems range from small-molecule (drug)-protein interactions, to nanomachineries such as the proteasome and ribosome, to even virus assembly. In native MS, ions attain high m/z values, requiring special mass analyzers for their detection. Depending on the particular mass analyzer used, instrumental mass resolution does often decrease at higher m/z but can still be above a couple of thousand at m/z 5000. However, the mass resolving power obtained on charge states of protein complexes in this m/z region is experimentally found to remain well below the inherent instrument resolution of the mass analyzers employed. Here, we inquire into reasons for this discrepancy and ask how native MS would benefit from higher instrumental mass resolution. To answer this question, we discuss advantages and shortcomings of mass analyzers used to study intact biomolecules and biomolecular complexes in their native state, and we review which other factors determine mass resolving power in native MS analyses. Recent examples from the literature are given to illustrate the current status and limitations.

AIMS: Platelets are a natural source of growth factors, cytokines and chemokines, that regulate angiogenesis and inflammation. It has been suggested that differential release of pro- and anti-angiogenic growth factors from platelet α-granules by protease-activated receptors (PAR) 1 and 4 may be important for the regulation of angiogenesis. We aimed to compare the releasates of unstimulated platelets with PAR-1- and PAR-4-stimulated platelets. METHODS AND RESULTS: The release of β-thromboglobulin, platelet factor (PF)-4, thrombospondin, platelet-derived growth factor (PDGF)-A/B, regulated and normal T-cell expressed and secreted (RANTES/CCL5), endostatin, CXCL12, and vascular endothelial growth factor (VEGF) was measured with enzyme-linked immunosorbent assay (ELISA). Mass spectrometry (MS)-based quantitative proteomics identified 93 proteins from platelets stimulated with PAR-1 and PAR-4. A strong correlation between the factors released after either stimulus was observed (Spearman's r 0.94, P < 0.001). Analysis with ELISA showed that stimulation with PAR-1 or PAR-4 lead to non-differential release of β-thromboglobulin, PF-4, thrombospondin, PDGF-A/B, RANTES/CCL5, endostatin, CXCL12, and VEGF. Release of thrombospondin was slightly lower after PAR-1 stimulation (7.2 μg/mL), compared with PAR-4 induced release (9.8 μg/mL; P < 0.05). CONCLUSIONS: Both ELISA on established α-granule proteins and MS-based quantitative proteomics showed that the most abundant α-granule proteins are released in similar quantities from platelets after stimulation with either PAR-1 or PAR-4. Our findings provide evidence against the hypothesis that PAR-1 and PAR-4 stimulation of platelets trigger differential release of alpha-granule, but further studies are needed to draw conclusions for physiological conditions.

Using high-resolution MS-based proteomics in combination with multiple protease digestion, we profiled, with on average 90% sequence coverage, all 13 viral proteins present in an human adenovirus (HAdV) vector. This in-depth profile provided multiple peptide-based evidence on intrinsic protease activity affecting several HAdV proteins. Next, the generated peptide library was used to develop a targeted proteomics method using selected reaction monitoring (SRM) aimed at quantitative profiling of the stoichiometry of all 13 proteins present in the HAdV. We also used this method to probe the release of specific virus proteins initiated by thermal stimulation, mimicking the early stage of HAdV disassembly during entry into host cells. We confirmed the copy numbers of the most well characterized viral capsid components and established the copy numbers for proteins whose stoichiometry has so far not been accurately defined. We also found that heating HAdV induces the complete release of the penton base and fiber proteins as well as a substantial release of protein VIII and VI. For these latter proteins, maturational proteolysis by the adenoviral protease leads to the differential release of fragments with certain peptides being fully released and others largely retained in the AdV particles. This information is likely to be beneficial for the ongoing interpretation of high resolution cryoEM and x-ray electron density maps.

The protease-chaperone DegP undergoes secondary through quaternary structural changes, regulating function and preventing indiscriminate proteolysis. Several structures of DegP oligomers have been observed, including the resting state 6-mer and the 12-mer and 24-mer active states. However, the precise events of the transition between the resting and active states still need to be elucidated. We used native mass spectrometry to demonstrate that binding of multiple substrate-mimicking peptide ligands to the DegP resting state occurs prior to the transition to an active conformation. This transition occurred at a 6-mer occupancy of 40\% for each peptide ligand. We observed ligand-specific 9-mer formation with a maximum load of 9 peptides, whereas other substrates led to 12-mers accommodating 24 peptides. Based on these data, we present a model for the initial steps of substrate-induced transitions from the resting to active states of DegP. \copyright 2014 Elsevier Ltd.

SummaryMost double-stranded DNA (dsDNA) viruses, including bacteriophages and herpesviruses, rely on a staged assembly process of capsid formation. A viral protease is required for many of them to disconnect scaffolding domains/proteins from the capsid shell, therefore priming the maturation process. We used the bacteriophage HK97 as a model system to decipher the molecular mechanisms underlying the recruitment of the maturation protease by the assembling procapsid and the influence exerted onto the latter. Comparisons of the procapsid with and without protease using single-particle cryoelectron microscopy reconstructions, hydrogen/deuterium exchange coupled to mass spectrometry, and native mass spectrometry demonstrated that the protease interacts with the scaffolding domains within the procapsid interior and stabilizes them as well as the whole particle. The results suggest that the thermodynamic consequences of protease packaging are to shift the equilibrium between isolated coat subunit capsomers and procapsid in favor of the latter by stabilizing the assembled particle before making the process irreversible through proteolysis of the scaffolding domains.

An ultimate goal in biology is to fully understand how a cell, or even a whole organism, works. Ideally, such knowledge might be used to develop models that predict the responses of cells to specific cues or diseases. A first step towards this goal is to identify and characterize all the molecular players present in cells. The Human Genome Project was probably one of the most ambitious scientific endeavors so far and provided the first essential pieces in this puzzle. The availability of the 3 billion base pairs that make up our DNA generated worldwide excitement as this information might lead to understanding the molecular mechanisms of human pathologies. However, soon thereafter the complexity embedded in our genetic code was already realized. A surprising finding was the low percentage of DNA (less than 2% of the genome) coding for proteins—roughly 20.000 human genes. However, recent analysis indicates that 80% of the human genome is functional and either transcribed, binding to regulatory proteins, or associated with other biochemical functions. Although genomic information is vital, it does not touch upon proteins, the main molecular effectors of cells. Every researcher will agree that the analysis of the proteome is of more relevance, but still this has been less exploited due to technical hurdles and by the fact that the proteome is inherently several magnitudes more complex. Whereas the genome is nearly identical in every cell of the human body and also relatively constant over the lifetime of an organism, the proteome of every cell is very different and changes dramatically over time (Figure 1). Notwithstanding these challenges, the field of proteomics has witnessed tremendous developments over the last decade, primarily through advances in mass spectrometry and bioinformatics, and is now somewhat coming up to par with genomics and transcriptomics technologies. This is evidenced by two recent reports in Nature from a German team led by Bernard K ster and a USA/India-based collaboration headed by Akhilesh Pandey, who independently initiated an unprecedented effort with the aim of identifying all the human proteins encoded in the genome. To this end, both laboratories performed extensive proteomic analyses onmore than 70 human tissues and body fluids and more than 150 cell lines. Although the two teams used a very similar MS-centric workflow, some differences exist between these two studies, especially in the depth of the analyses. While Pandey et al. performed around 2000 mass spectrometric (LC-MS) runs, K ster et al. carried out more than 6000 analyses and made use of another 10000 measurements publicly available in proteomic repositories. Assuming an average of two hours per run, the instrument time used to acquire these data would reach an astonishing number of 34000 h (4.3 years if only one mass spectrometer had been used). The analysis of all the data resulted in the identification of 946000 and 293000 nonredundant unique peptide sequences in K ster s and Pandey s studies, respectively. Strikingly, and despite the significant difference in depth, the two studies found evidence for a nearly identical number of protein-coding genes: 18097 (K ster) and 17294 (Pandey). Although a careful comparison of the two studies is still needed, a first conclusion can be drawn: the unequivocal existence of protein translation for 90–95% of the human genes. This is a highly relevant finding, as previously almost one-third of the human genes had been barely annotated, and there was no experimental evidence that they could lead to proteins. Another relevant discovery derived from these studies concerns the extent of alternative splicing in the generation of protein isoforms. It is clear that the number of genes does not correlate with the complexity of an organism (C. elegans for instance has 20500 genes) and it has been suggested that alternative splicing might increase the repertoire of functional proteins. However, these proteomic studies could only identify as many as 9000 of the 67000 isoforms annotated in Uniprot. Although some of these isoforms may produce only one unique peptide, decreasing the likelihood of observation by proteomics, these data could also support the idea that there is a dominant isoform per gene. Both studies confirmed the existence of a core proteome present in all tissues, made up of “housekeeping [*] Prof. Dr. A. J. R. Heck Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University Padualaan 8. 3584 CH Utrecht (The Netherlands) E-mail: a.j.r.heck@uu.nl

Disulfide bond identification is important for a detailed understanding of protein structures, which directly affect their biological functions. Here we describe an integrated workflow for the fast and accurate identification of authentic protein disulfide bridges. This novel workflow incorporates acidic proteolytic digestion using pepsin to eliminate undesirable disulfide reshuffling during sample preparation and a novel search engine, SlinkS, to directly identify disulfide-bridged peptides isolated via electron transfer higher energy dissociation (EThcD). In EThcD fragmentation of disulfide-bridged peptides, electron transfer dissociation preferentially leads to the cleavage of the S-S bonds, generating two intense disulfide-cleaved peptides as primary fragment ions. Subsequently, higher energy collision dissociation primarily targets unreacted and charge-reduced precursor ions, inducing peptide backbone fragmentation. SlinkS is able to provide the accurate monoisotopic precursor masses of the two disulfide-cleaved peptides and the sequence of each linked peptide by matching the remaining EThcD product ions against a linear peptide database. The workflow was validated using a protein mixture containing six proteins rich in natural disulfide bridges. Using this pepsin-based workflow, we were able to efficiently and confidently identify a total of 31 unique Cys-Cys bonds (out of 43 disulfide bridges present), with no disulfide reshuffling products detected. Pepsin digestion not only outperformed trypsin digestion in terms of the number of detected authentic Cys-Cys bonds, but, more important, prevented the formation of artificially reshuffled disulfide bridges due to protein digestion under neutral pH. Our new workflow therefore provides a precise and generic approach for disulfide bridge mapping, which can be used to study protein folding, structure, and stability.

The identification of peptides presented by human leukocyte antigen (HLA) class I is tremendously important for the understanding of antigen presentation mechanisms under healthy or diseased conditions. Currently, mass spectrometry-based methods represent the best methodology for the identification of HLA class I-associated peptides. However, the HLA class I peptide repertoire remains largely unexplored because the variable nature of endogenous peptides represents difficulties in conventional peptide fragmentation technology. Here, we substantially enhanced (about threefold) the identification success rate of peptides presented by HLA class I using combined electron-transfer/higher-energy collision dissociation (EThcD), reporting over 12,000 high-confident (false discovery rate

Drug purity and affinity are essential attributes during development and production of therapeutic proteins. In this work, capillary electrophoresis (CE) was used to determine both the affinity and composition of the biotechnologically produced "nanobody" EGa1, the binding fragment of a heavy-chain-only antibody. EGa1 is an antagonist of the epidermal growth factor receptor (EGFR), which is overexpressed on the surface of tumor cells. Using a background electrolyte (BGE) of 50mM sodium phosphate (pH 8.0) in combination with a polybrene-poly(vinylsulfonic acid) capillary coating, CE analysis of EGa1 showed the presence of at least three components. Affinity of the EGa1 components towards the extracellular domain of EGFR was assessed by adding different concentrations (0-12 nM) of the receptor to the BGE while measuring the effective electrophoretic mobility of the respective EGa1 components. Binding curves obtained by plotting electrophoretic mobility shifts as a function of receptor concentration, yielded dissociation constants (Kd) of 1.65, 1.67, and 1.75 nM for the three components, respectively; these values were comparable to the Kd of 2.1 nM obtained for the bulk EGa1 product using a cellular assay. CE with mass spectrometry (MS) detection using a BGE of 25 mM ammonium acetate (pH 8.0) revealed that the EGa1 sample comprised of significant amounts of deamidated, bisdeamidated and N-terminal pyroglutamic acid products. CE-MS using a BGE of 100mM acetic acid (pH 2.8) in combination with a polybrene-dextran sulfate-polybrene capillary coating demonstrated the additional presence of minor products related to incomplete removal of the signal peptide from the produced nanobody. Combining the results obtained from affinity CE and CE-MS, it is concluded that the EGa1 nanobody product is heterogeneous, comprising highly-related proteins that exhibit very similar affinity towards EGFR.

Nature uses bottom-up approaches for the controlled assembly of highly ordered hierarchical structures with defined functionality, such as organelles, molecular motors, and transmembrane pumps. The field of bionanotechnology draws inspiration from nature by utilizing biomolecular building blocks such as DNA, proteins, and lipids, for the (self-) assembly of new structures for applications in biomedicine, optics, or electronics. Among the toolbox of available building blocks, proteins that form cage-like structures, such as viruses and virus-like particles, have been of particular interest since they form highly symmetrical assemblies and can be readily modified genetically or chemically both on the outer or inner surface. Bacterial encapsulins are a class of nonviral protein cages that self-assemble in vivo into stable icosahedral structures. Using teal fluorescent proteins (TFP) engineered with a specific native C-terminal docking sequence, we report the molecular self-sorting and selective packaging of TFP cargo into bacterial encapsulins during in vivo assembly. Using native mass spectrometry techniques, we show that loading of either monomeric or dimeric TFP cargo occurs with unprecedented high fidelity and exceptional loading accuracy. Such self-assembling systems equipped with self-sorting capabilities would open up exciting opportunities in nanotechnology, for example, as artificial (molecular storage or detoxification) organelles or as artificial cell factories for in situ biocatalysis.

Accurate mass analysis can provide useful information on the stoichiometry and composition of protein-based particles, such as virus-like assemblies. For applications in nanotechnology and medicine, such nanoparticles are loaded with foreign cargos, making accurate mass information essential to define the cargo load. Here, we describe modifications to an Orbitrap mass spectrometer that enable high mass analysis of several virus-like nanoparticles up to 4.5 MDa in mass. This allows the accurate determination of the composition of virus-like particles. The modified instrument is utilized to determine the cargo load of bacterial encapsulin nanoparticles that were engineered to encapsulate foreign cargo proteins. We find that encapsulin packages from 8 up to 12 cargo proteins, thereby quantifying cargo load but also showing the ensemble spread. In addition, we determined the previously unknown stoichiometry of the three different splice variants of the capsid protein in adeno-associated virus (AAV) capsids, showing that symmetry is broken and assembly is heterogeneous and stochastic. These results demonstrate the potential of high-resolution mass analysis of protein-based nanoparticles, with widespread applications in chemical biology and nanotechnology.

Analysis of the size and mass of nanoparticles, whether they are natural biomacromolecular or synthetic supramolecular assemblies, is an important step in the characterization of such molecular species. In recent years, electrospray ionization (ESI) has emerged as a technology through which particles with masses up to 100 MDa can be ionized and transferred into the gas phase, preparing them for accurate mass analysis. Here we review currently used methodologies, with a clear focus on native mass spectrometry (MS). Additional complementary methodologies are also covered, including ion-mobility analysis, nanomechanical mass sensors, and charge-detection MS. The literature discussed clearly demonstrates the great potential of ESI-based methodologies for the size and mass analysis of nanoparticles, including very large naturally occurring protein assemblies. The analytical approaches discussed are powerful tools in not only structural biology, but also nanotechnology.

Synthesis of biogenic membranes requires transbilayer movement of lipid-linked sugar molecules. This biological process, which is fundamental in prokaryotic cells, remains as yet not clearly understood. In order to obtain insights into the molecular basis of its mode of action, we analyzed the structure-function relationship between Lipid II, the important building block of the bacterial cell wall, and its inner membrane-localized transporter FtsW. Here, we show that the predicted transmembrane helix 4 of Escherichia coli FtsW (this protein consists of 10 predicted transmembrane segments) is required for the transport activity of the protein. We have identified two charged residues (Arg(145) and Lys(153)) within this segment that are specifically involved in the flipping of Lipid II. Mutating these two amino acids to uncharged ones affected the transport activity of FtsW. This was consistent with loss of in vivo activity of the mutants, as manifested by their inability to complement a temperature-sensitive strain of FtsW. The transport activity of FtsW could be inhibited with a Lipid II variant having an additional size of 420 Da. Reducing the size of this analog by about 274 Da resulted in the resumption of the transport activity of FtsW. This suggests that the integral membrane protein FtsW forms a size-restricted porelike structure, which accommodates Lipid II during transport across the bacterial cytoplasmic membrane.

Fritless SPE on-line coupled to CE with UV and MS detection (SPE-CE-UV and SPE-CE-MS) was evaluated for the analysis of opioid peptides. A microcartridge of 150 μm id was packed with a C18 sorbent (particle size > 50 μm), which was retained between a short inlet capillary and a separation capillary (50 μm id). Several experimental parameters were optimized by SPE-CE-UV using solutions of dynorphin A (DynA), endomorphin 1 (End1), and methionine-enkephaline (Met). A microcartridge length of 4 mm was selected, sample was loaded for 10 min at 930 mbar and the retained peptides were eluted with 67 nL of an acidic hydro-organic solution. Using SPE-CE-MS, peak area and migration time repeatabilities for the three opioid peptides were 12-27% and 4-5%, respectively. SPE recovery was lower for the less hydrophobic DynA (22%) than for End1 (66%) and Met (78%) and linearity was satisfactory in all cases between 5 and 60 ng/mL. The LODs varied between 0.5 and 1.0 ng/mL which represent an enhancement of two orders of magnitude when compared with CE-MS. Cerebrospinal fluid (CSF) samples spiked with the opioid peptides were analyzed to demonstrate the applicability to biological samples. Peak area and migration time repeatabilities were similar to the standard solutions and the opioid peptides could be detected down to 1.0 ng/mL.

Streptococcus pneumoniae, the pneumococcus, is an important human pathogen causing considerable morbidity and mortality worldwide. This thesis addresses several interactions between pneumococcus and man. The first part of the thesis deals with the host immune response against pneumococci. We studied the anticapsular antibody response during pneumonia and found that only 45% of adult pneumococcal pneumonia patients mount a significant serotype-specific antibody response. We showed that corticosteroid use during pneumonia does not affect anticapsular pneumococcal antibody formation. In the second part of the thesis diagnostic options for pneumococcal pneumonia are discussed. We investigated the measurement of antibodies as an alternative method to detect pneumococcal pneumonia as well as two additional newly developed techniques: a quantitative PCR followed by capsular sequence typing, and a serotype-specific urinary antigen assay. Using all three techniques we were able to detect the pneumococcus in samples of 56% more patients compared to conventional methods. Furthermore, we investigated whether a urinary antigen test (UAT) could serve as a method to diagnose invasive pneumococcal pneumonia, classically defined as pneumonia in which S. pneumoniae is cultured from a normally sterile body site, by comparing the outcome of bacteraemic and non-bacteraemic/UAT-positive patients. A non-significant association with mortality was found for bacteraemia. In the third part of the thesis various techniques for distinguishing and typing cultured pneumococci are described. Two molecular serotyping techniques, a real-time multiplex PCR (rmPCR) assay detecting 21 different serotypes/-groups and a sequetyping assay, based on the sequence of the cpsB gene within the pneumococcal capsulation locus, were compared. The rmPCR assay was shown to perform well for the 21 serotypes/-groups included in the assay and for serotype mixtures. The sequetyping assay performs well, with specific exceptions, and may be more useful for regions where vaccine serotypes are less common. To gain more insight in the genetic background of pneumococci, we developed a mixed genome microarray containing 470 DNA fragments and evaluated genetic differences between 445 invasive and non-invasive pneumococcal isolates. The microarray was able to genetically distinguish pneumococci at a deeper level than the typing techniques generally used. The fourth and last part of the thesis focuses on pneumococcal vaccination. We have studied the impact of the seven-valent pneumococcal conjugate vaccine (PCV7) introduction in the Netherlands on the incidence and presentation of invasive pneumococcal disease (IPD). Four years after introduction of the vaccine, significant declines in overall IPD incidence were observed in children

Peptide-based materials constitute a class of molecules that play an important role in many biological processes and are utilized by many organisms to interact with their environment. One of the most well-known examples is spider silk, a material produced by web-spinning spiders composed of repeating stretches of amino acids that has a very high tensile strength. Many peptide-based materials are composed of repeating stretches of certain amino acids that confer function and (macroscopic) structure to these materials. In the first three research chapters a peptide material based on naturally occurring antifreeze molecules from arctic fish is described. In nature this material is composed of repeating stretches of a glycosylated tripeptide. Our mimic is synthesized by a copper-catalyzed polymerization reaction from a modified glycotripeptide with polymerization handles. This polymerization reaction introduces a non-natural element in the peptide backbone that was known to mimic the natural amide bond in previously published examples to a certain extent. A one-pot reduction and polymerization yielded a mixture of oligomers that could be separated by size. Upon analysis of the antifreeze activity of the resulting antifreeze mimics by an ice recrystallization experiment, the polymeric material was shown to have a strongly reduced antifreeze activity compared to the natural compound and a synthetic derivative synthesized previously. Structural analysis by circular dichroism showed a similar structure to the previously described and potent synthetic antifreeze glycopeptide, indicating that a small structural change can abolish the potent antifreeze properties of such molecules. The chemistry for introducing a urea moiety that can be incorporated into the antifreeze glycopeptides to enhance the interaction with the ice-lattice is described, using a 4-chlorophenyl glycoside synthon that can react with amines under mild conditions to form a ureido glycoside linkage. As a case study, this chemistry was used to synthesize a urea-containing glycopolymer based on the previously synthesized mimic. In chapter five a modification of the naturally occurring antibiotic nisin is described, wherein commercially available nisin was conjugated on the C-terminus with propargylamine, a handle for copper catalyzed click chemistry. In a second step this modified nisin could be coupled to fluorescent reporter molecules, and as a proof-of-concept, a small molecule with two azide handles. The successful synthesis of the dimeric nisin structure and retention of antimicrobial activity of all conjugates indicated the possibility to easily incorporate nisin in a functionalized (peptide) material or surface to obtain antimicrobial peptide based materials. In chapter six a dicysteine motif is described, which easily oxidizes to a cyclocystine. Initially found in a peptide derived from Tamm-Horsfall protein (F991) that was indicated to bind free-light chains, it became apparent that in serum this peptide rapidly formed the cyclocystine moiety. Attempts to use this structural motif in the beta-sheet forming human islet amyloid polypeptide as an oxidative switch are described. Preliminary results show a distorted structure by circular dichroism, but the resulting peptide still formed similar structures in aqueous buffer. Further attempts at increasing the change upon oxidation are described.

The interaction between proteins is important in all biological functions. In practically every cellular process protein complexes have been identified as essential components. Defects or disturbance in the regulation of protein-protein interactions are responsible for many diseases. Therefore, the development of modulators of protein-protein interactions has become an important topic in drug design. Peptide-based mimics of protein binding sites are believed to be promising candidates for modulating protein-protein interactions. The binding sites of proteins consist of discontinuous epitopes. Mimicry of discontinuous epitopes is a challenging task because of the complex nature of these epitopes. In order to achieve good mimics, the multiple binding segments of the native protein should be combined into one single molecule by employing a molecular scaffold approach. Moreover, discontinuous epitopes are often present as loop-like structures. Therefore, it is believed that it is necessary to use cyclic peptides in discontinuous epitope mimicry. The aim of this thesis was the development of a general applicable method for the mimicry of discontinuous epitopes employing scaffolded cyclic peptides. Although the mimicry of discontinuous protein binding sites is gaining more and more interest, the design and synthesis of molecules, which are functionally capable of mimicking the epitopes of natural proteins, is still a great challenge. In addition, most examples of discontinuous epitope mimicry by scaffolded cyclic peptides concern bicyclic systems, only few approaches allow a regioselective attachment of more than two pre-organized ligands to a molecular scaffold. With respect to design and synthesis of scaffolded cyclic peptides mainly three issues are involved: 1) molecular scaffold, 2) cyclic peptides containing a handle for conjugation, 3) an efficient conjugation method for attaching the cyclic peptides to the scaffold. These topics were studied in this thesis.

Metabolomics encompasses the identification and quantification of low-molecular weight endogenous metabolites in biological samples, such as cells, tissues and body fluids. The identities, concentrations and fluxes of metabolites result from gene expression, protein expression and environmental factors (e.g. bacteria, diet, drugs), which together determine the phenotype of an organism. Therefore, profiling of metabolites present in biological samples provides information on biochemical processes and phenotypes of organisms. Various analytical techniques have been developed to analyze different classes of metabolites, including capillary electrophoresis (CE) and hydrophilic interaction chromatography (HILIC) coupled to mass spectrometry (MS), which are eminently suited for profiling highly polar compounds. This thesis describes the optimization, evaluation and comparison of CE–MS and HILIC–MS using negative electrospray ionization for the profiling of anionic metabolites. Particular attention was paid to sensitivity, reproducibility, separation selectivity and metabolite coverage. It is demonstrated that the use of triethylamine in the background electrolyte and sheath liquid in CE–MS is an effective way to improve the limits of detection of anionic metabolites significantly as compared to earlier developed CE–MS methods applying ammonium acetate. The increase in signal intensities allows the detection of low-abundant metabolites, thereby increasing the metabolite coverage. This is of utmost importance in metabolic profiling studies. The optimization of the mobile phase composition, the gradient of the mobile phase and sample solvent in HILIC–MS resulted in a large separation window with good peak shapes and low limits of detection of anionic metabolites. Detection limits obtained with HILIC–MS were up to 80 times lower compared to CE–MS, mainly due to the larger injection volume. Moreover, the migration and retention times of anionic metabolites were considerably different, demonstrating the different separation selectivities of CE–MS and HILIC–MS. The applicability of the optimal CE–MS and HILIC–MS methods has been tested by analyzing urine samples in studies on antibiotic-treated rats and patients with inborn errors of metabolism. Reproducible metabolite profiles were obtained as demonstrated by the repetitive analysis of a quality control sample. In general, more molecular features were obtained with HILIC–MS. However, CE–MS also revealed a large number of metabolite features, which were not detected with HILIC–MS. Using multivariate data analysis, different groups of urine samples could be discerned based on obtained CE–MS and HILIC–MS data, and potential biomarkers were revealed. These discriminatory compounds were putatively identified based on accurate mass and MS/MS experiments. Some of the biomarker candidates were observed with both CE–MS and HILIC–MS. Several anionic metabolites were also uniquely revealed by one of the applied techniques. This demonstrates that these analytical techniques can provide important and complementary information in metabolic profiling studies.

Hepatitis B virus core-antigen (capsid protein) and e-antigen (an immune regulator) have almost complete sequence identity, yet the dimeric proteins (termed Cp149d and Cp(-10)149d , respectively) adopt quite distinct quaternary structures. Here we use hydrogen deuterium exchange-mass spectrometry (HDX-MS) to study their structural properties. We detect many regions that differ substantially in their HDX dynamics. Significantly, whilst all regions in Cp(-10)149d exchange by EX2-type kinetics, a number of regions in Cp149d were shown to exhibit a mixture of EX2- and EX1-type kinetics, hinting at conformational heterogeneity in these regions. Comparison of the HDX of the free Cp149d with that in assembled capsids (Cp149c ) indicated increased resistance to exchange at the C-terminus where the inter-dimer contacts occur. Furthermore, evidence of mixed exchange kinetics were not observed in Cp149c , implying a reduction in flexibility upon capsid formation. Cp(-10)149d undergoes a drastic structural change when the intermolecular disulphide bridge is reduced, adopting a Cp149d -like structure, as evidenced by the detected HDX dynamics being more consistent with Cp149d in many, albeit not all, regions. These results demonstrate the highly dynamic nature of these similar proteins. To probe the effect of these structural differences on the resulting antigenicity, we investigated binding of the antibody fragment (Fab E1) that is known to bind a conformational epitope on the four-helix bundle. Whilst Fab E1 binds to Cp149c and Cp149d , it does not bind non-reduced and reduced Cp(-10)149d , despite unhindered access to the epitope. These results imply a remarkable sensitivity of this epitope to its structural context.

Synthetic mimics of protein surfaces have the potential to become inhibitors of protein-protein interactions or even synthetic vaccines. However, the synthesis of these complicated molecular constructs is still difficult. Here we describe an efficient and versatile synthesis of protein mimics containing up to three different cyclic peptides. Using a sequential native chemical ligation strategy, peptide loops containing a thioester handle were introduced onto a triazacyclophane scaffold bearing orthogonal protected cysteine residues.

Synthetic mimics of discontinuous epitopes may have a wide range of potential applications, including synthetic vaccines and inhibition of protein-protein interactions. However, synthetic access to these relatively complex peptide molecular constructs is limited. This paper describes a versatile convergent strategy for the construction of protein mimics presenting three different cyclic peptides. Using an orthogonal alkyne protection strategy, peptide loops were introduced successively onto a triazacyclophane scaffold via Cu(I)-catalyzed azide alkyne cycloaddition. This method provides rapid access to protein mimics requiring different peptide segments for their interaction and activity.

The work described in this thesis initially focusses on the discovery of a novel PKA-RI specific AKAP: small membrane AKAP (smAKAP). Afterwards we centre on the structural interaction between smAKAP and PKA-RI to reveal the first PKA-RI specific AKAP bound to PKA-RI crystal structure. Interestingly, a novel self-inhibition mechanism was discovered which allows PKA to block its binding to AKAPs under certain restrictions. In order to fully understand the specific limitations associated with binding a study centering on the PKA-RI and PKA-RII interactions with AKAPs was performed. In Chapter 2, recent literature on the structural interface between PKA-RI/RII and AKAPs is reviewed. Most of these structural studies involve either X-ray crystallography, three-dimensional NMR, binding affinity assays and various other biochemical methods. In Chapter 3, the discovery and initial characterization of a novel AKAP termed smAKAP is described. Via binding affinity assays and imaging techniques it is shown that smAKAP is PKA-RI specific. The intracellular location of smAKAP at the plasma membrane is shown by means of fluorescence imaging and advanced electron microscopy. In Chapter 4, structural techniques such as hydrogen/deuterium exchange and X-ray crystallography are applied to probe the interaction interface between smAKAP and PKA-RI. Additionally, via a phosphoproteomics study it was shown that in the middle of the A-kinase binding domain of smAKAP there is a putative PKA phosphorylation site. Upon phosphorylation of this site, PKA cannot bind to smAKAP anymore. A mechanistic model on how this disruption occurs is presented. In Chapter 5, a novel bioinformatic tool, THAHIT (THe AKAP/amphipathic Helix Identification Tool), is able to predict PKA-RIα and/or PKA-RIIα binding domains. This software package is based on currently known and well-established PKA-RIα and PKA-RIIα binding motifs. After applying it on all known AKAPs, numerous new PKA-RIα and PKA-RIIα binding domains in these AKAPs were found and/or narrowed down. Several of these were confirmed via conservation (BlastP), in silico docking studies using HADDOCK and in vitro binding studies using fluorescence anisotropy. In addition, several cAMP pull-downs were investigated for potential novel AKAPs using THAHIT. Here we propose a novel very large AKAP: vlAKAP.

The present work focuses on the assignment of uronic acid stereochemistry in heparan sulfate (HS) oligomers. The structural elucidation of HS glycosaminoglycans is the subject of considerable importance due to the biological and biomedical significance of this class of carbohydrates. They are highly heterogeneous due to their non-template biosynthesis. Advances in tandem mass spectrometry activation methods, particularly electron detachment dissociation (EDD), have led to the development of methods to assign sites of sulfo modification in glycosaminoglycan oligomers, but there are few reports of the assignment of uronic acid stereochemistry, necessary to distinguish glucuronic acid (GlcA) from iduronic acid (IdoA). While preceding studies focused on uronic acid epimers with no sulfo modification, the current work extends the assignment of the hexuronic acid stereochemistry to 2-O-sulfo uronic acid epimeric tetrasaccharides. The presence of a 2-O-sulfo group on the central uronic acid was found to greatly influence the formation of B3, C2, Z2, and Y1 ions in glucuronic acid and Y2 and 1,5X2 for iduronic acid. The intensity of these peaks can be combined to yield diagnostic ratios (DR), which can be used to confidently assign the uronic acid stereochemistry.

Circadian timing in cyanobacteria is determined by the Kai system consisting of KaiA, KaiB, and KaiC. Interactions between Kai proteins change the phosphorylation status of KaiC, defining the phase of circadian timing. The KaiC-KaiB interaction is crucial for the circadian rhythm to enter the dephosphorylation phase but it is not well understood. Using mass spectrometry to characterize Kai complexes, we found that KaiB forms monomers, dimers, and tetramers. The monomer is the unit that interacts with KaiC, with six KaiB monomers binding to one KaiC hexamer. Hydrogen-deuterium exchange MS reveals structural changes in KaiC upon binding of KaiB in both the CI and CII domains, showing allosteric coupling upon KaiB binding. Based on this information we propose a model of the KaiB-KaiC complex and hypothesize that the allosteric changes observed upon complex formation relate to coupling KaiC ATPase activity with KaiB binding and to sequestration of KaiA dimers into KaiCBA complexes.

A novel strategy to prepare bisubstrate based inhibitors for histone acetyltransferases is presented. To obtain these, azido peptides derived from histone H3 incorporating either a serine or a phosphoserine residue were connected to a propargyl coenzyme A derivative through copper catalyzed click chemistry. The resulting inhibitors were tested with therapeutically relevant acetyltransferase PCAF. Increased potency of the phosphoserine containing inhibitor was observed. The synthetic strategy presented may be used for developing bisubstrate based inhibitors against any acetyltransferase.

This article presents an overview of the design and application of coupled SPE-CE systems that have been reported in the literature between January 2011 and June 2013. The present paper is an update of three previous review papers covering the years 2000-2011 (Electrophoresis 2008, 29, 108-128; Electrophoresis 2010, 31, 44-54; Electrophoresis 2012, 33, 243-250). The use of in-line and on-line SPE-CE approaches is described in this review. Emerging technological developments, such as the use of in-line frit-free SPE and chip-based SPE for extraction of sample components prior to CE analysis, are outlined. Selected examples illustrate the applicability of SPE-CE in biomedical, pharmaceutical, and environmental analysis. A complete overview of recent SPE-CE studies is given in table format, providing information on sample type, SPE sorbent, coupling mode, detection mode, and LOD. Finally, some general conclusions and future perspectives are provided.

Red blood cells are rather unique body cells, since they have lost all organelles when mature, which results in lack of potential to replace proteins that have lost their function. They maintain only a few pathways for obtaining energy and reducing power for the key functions they need to fulfill. This makes RBCs highly sensitive to any aberration. If so, these RBCs are quickly removed from circulation, but if the RBC levels reduce extremely fast, this results in hemolytic anemia. Several causes of HA exist, and proteome analysis is the most straightforward way to obtain deeper insight into RBC functioning under the stress of disease. This should result in discovery of biomarkers, typical for each source of anemia. In this review, several challenges to generate in-depth RBC proteomes are described, like to obtain pure RBCs, to overcome the wide dynamic range in protein expression, and to establish which of the identified/quantified proteins are active in RBCs. The final challenge is to acquire and validate suited biomarkers unique for the changes that occur for each of the clinical questions; in red blood cell aging (also important for transfusion medicine), for thalassemias or sickle cell disease. Biomarkers for other hemolytic anemias that are caused by dysfunction of RBC membrane proteins (the RBC membrane defects) or RBC cytosolic proteins (the enzymopathies) are sometimes even harder to discover, in particular for the patients with RBC rare diseases with unknown cause. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.