The Promiscuity of Squalene Synthase-Like Enzyme: Dehydrosqualene Synthase, a Natural Squalene Hyperproducer?

Dehydrosqualene synthase (CrtM), as a squalene synthase-like enzyme from Staphylococcus aureus, can naturally utilize farnesyl diphosphate to produce dehydrosqualene (C30H48). However, no study has documented the natural production of squalene (C30H50) by CrtM. Here, based on an HPLC-Q-Orbitrap-MS/MS study, we report that the expression of crtM in vitro or in Bacillus subtilis 168 both results in the output of squalene, dehydrosqualene, and phytoene (C40H64). Notably, wild-type CrtM exhibits a significantly higher squalene yield compared to squalene synthase (SQS) from Bacillus megaterium with an approximately 2.4-fold increase. Moreover, the examination of presqualene diphosphate's stereostructures in both CrtM and SQS enzymes provides further understanding into the presence of multiple identified terpenoids. In summary, this study not only provides insights into the promiscuity demonstrated by squalene synthase-like enzymes but also highlights a new strategy of utilizing CrtM as a potential replacement for SQS in cell factories, thereby enhancing squalene production.

A propensity score-matched analysis on the impact of patient and surgical factors on early periprosthetic joint infection in minimally invasive anterolateral and transgluteal total hip arthroplasty.

INTRODUCTION: Increased risk of periprosthetic joint infection (PJI) in minimally invasive (MIS) total hip arthroplasty (THA) is still debated. This study aimed to identify differences in surgical and patient-related risk factors for PJI between an MIS anterolateral approach and transgluteal-modified Hardinge approach. METHODS: A retrospective cohort of 5315 THAs performed between 2006 and 2019 at a single institution was screened. Short stem THAs performed via an MIS anterolateral approach in the supine position and standard straight stem THAs performed via a transgluteal modified Hardinge approach were included. Propensity score matching was performed to control for selection bias. After matching, 1405 (34.3%) short stem THAs implanted via MIS anterolateral approach and 2687 (65.7%) straight stem THAs implanted via a transgluteal modified Hardinge approach were included. The risk of PJI due to patient-specific and surgical factors was retrospectively analyzed using chi-square test and multivariate regression analysis. RESULTS: PJI occurred in 1.1% in both MIS anterolateral and transgluteal approach (p = 0.823). Multivariate regression showed an increased infection risk for patients with a BMI between 35 and 39.99 kg/m2 (OR 6.696; CI 1.799-24.923; p = 0.005), which could not be demonstrated for transgluteal approach (OR 0.900; CI 0.900-4.144; p = 0.93). A BMI ≥ 40 kg/m2 (OR 14.150; CI 2.416-82.879; p = 0.003) was detected as a risk factor for PJI only in anterolateral approach. Increased operation time ≥ 121 min showed a significantly increased risk for PJI in the general cohort (OR 6.989; CI1.286-37.972; p = 0.024). CONCLUSION: Minimally invasive anterolateral and transgluteal THA show a comparable rate of early PJI within the first year of index surgery. A BMI of ≥ 35 kg/m2 was detected as a clear risk factor for infection in the anterolateral approach. Prolonged operation time ≥ 121 min increases the risk of PJI regardless of approach.

DNAJB6-Containing Extracellular Vesicles as Chaperone Delivery Systems: A Proteomic Analysis.

Cell-derived extracellular vesicles (EVs) are effectors of cell-to-cell communication that are in the spotlight as promising candidates for in vivo drug delivery because of their ability to enter cells and deliver cargo. For example, proteins of interest can be loaded into EVs to mediate protein transfer into target cells. To determine causality between EV content and function, which is also important to assess the clinical safety of EVs, it is crucial to comprehensively characterize their complete molecular composition. Here, we investigated EVs loaded with the chaperone protein DNAJB6. Chaperone proteins assist in protein folding and have been suggested to alleviate protein aggregation diseases, such as Alzheimer's disease and Huntington's disease. We analyzed and compared the proteome of EVs isolated from wildtype HEK293T cells with that of EVs from HEK 293T cells overexpressing DNAJB6-WT or loss-of-function mutant DNAJB6-M3. Comprehensive analysis of proteomics data showed enhanced levels of DNAJB6 as well as protein-folding-related proteins in EVs derived from DNAJB6-overexpression cells. Interestingly, upregulation of a chaperone and its protein-folding-related proteins resulted in downregulation of another chaperone plus its related proteins, and vice versa. This implies the presence of compensatory mechanisms in the cellular expression of chaperones. Collectively, we provide the proteomic EV signatures underlying EV mediated DNAJB6 transmission by HEK293T cells, with the aim of establishing a causal relationship between EV protein content and EV function.

Probing the Conformational States of Thimet Oligopeptidase in Solution.

Thimet oligopeptidase (TOP) is a metallopeptidase involved in the metabolism of oligopeptides inside and outside cells of various tissues. It has been proposed that substrate or inhibitor binding in the TOP active site induces a large hinge-bending movement leading to a closed structure, in which the bound ligand is enclosed. The main goal of the present work was to study this conformational change, and fluorescence techniques were used. Four active TOP mutants were created, each equipped with a single-Trp residue (fluorescence donor) and a p-nitro-phenylalanine (pNF) residue as fluorescence acceptor at opposite sides of the active site. pNF was biosynthetically incorporated with high efficiency using the amber codon suppression technology. Inhibitor binding induced shorter Donor-Acceptor (D-A) distances in all mutants, supporting the view that a hinge-like movement is operative in TOP. The activity of TOP is known to be dependent on the ionic strength of the assay buffer and D-A distances were measured at different ionic strengths. Interestingly, a correlation between the D-A distance and the catalytic activity of TOP was observed: the highest activities corresponded to the shortest D-A distances. In this study for the first time the hinge-bending motion of a metallopeptidase in solution could be studied, yielding insight about the position of the equilibrium between the open and closed conformation. This information will contribute to a more detailed understanding of the mode of action of these enzymes, including therapeutic targets like neurolysin and angiotensin-converting enzyme 2 (ACE2).

Evaluating the use of an Unmanned Aerial Vehicle (UAV)-based active AirCore system to quantify methane emissions from dairy cows.

Enteric fermentation and manure methane emissions from livestock are major anthropogenic greenhouse gas emissions. In general, direct measurements of farm-scale methane emissions are scarce due to the source complexity and the limitations of existing atmospheric sampling methods. Using an innovative UAV-based active AirCore system, we have performed accurate atmospheric measurements of CH4 mole fractions downwind of a dairy cow farm in the Netherlands on four individual days during the period from March 2017 to March 2019. The total CH4 emission rates from the farm were determined using the UAV-based mass balance approach to be 1.1-2.4 g/s. After subtracting estimated emission factors of manure onsite, we derived the enteric emission factors to be 0.20-0.51 kgCH4/AU/d (1 AU = 500 kg animal weight) of dairy cows. We show that the uncertainties of the estimates were dominated by the variabilities in the wind speed and the angle between the wind and the flight transect. Furthermore, nonsimultaneous sampling in the vertical direction of the plume is one of the main limiting factors to achieving accurate estimate of the CH4 emissions from the farm. In addition, a N2O tracer release experiment at the farm was performed when both a UAV and a mobile van were present to simultaneously sample the N2O tracer and the CH4 plumes from the farm, improving the source quantification with a correction factor of 1.04 and 1.22 for the inverse Gaussian approach and for the mass balance approach, respectively. The UAV-based active AirCore system is capable of providing useful estimates of CH4 emissions from dairy cow farms. The uncertainties of the estimates can be improved when combined with accurate measurements of local wind speed and direction or when combined with a tracer approach.

Identification of damage associated molecular patterns and extracellular matrix proteins as major constituents of the surface proteome of lung implantable silicone/nitinol devices.

Lung implantable devices have been widely adopted as mechanical interventions for a wide variety of pulmonary pathologies. Despite successful initial treatment, long-term efficacy can often be impacted by fibrotic or granulation tissue formation at the implant sites. This study aimed to explore the lung-device interface by identifying the adhered proteome on lung devices explanted from patients with severe emphysema. In this study, scanning electron microscopy is used to visualize the adhesion of cells and proteins to silicone and nitinol surfaces of explanted endobronchial valves. By applying high-resolution mass-spectrometry, the surface proteome of eight explanted valves is characterized, identifying 263 unique protein species to be mutually adsorbed on the valves. This subset is subjected to gene enrichment analysis, matched with known databases and further validated using immunohistochemistry. Enrichment analyses reveal dominant clusters of functionally-related ontology terms associated with coagulation, pattern recognition receptor signaling, immune responses, cytoskeleton organization, cell adhesion and migration. Matching results show that extracellular matrix proteins and damage-associated molecular patterns are cardinal in the formation of the surface proteome. This is the first study investigating the composition of the adhered proteome on explanted lung devices, setting the groundwork for hypothesis generation and further exploration. STATEMENT OF SIGNIFICANCE: This is the first study investigating the composition of the adhered proteome on explanted lung devices. Lung implantable devices have been widely adopted as mechanical interventions for pulmonary pathologies. Despite successful initial treatment, long-term efficacy can often be impacted by fibrotic or granulation tissue formation around the implant sites. We identified the adhered proteome on explanted lung devices using several techniques. We identified 263 unique protein species to be mutually adsorbed on explanted lung devices. Pathway analyses revealed that these proteins are associated with coagulation, pattern recognition receptor signaling, immune responses, cytoskeleton organization, cell adhesion and migration. Furthermore, we identified that especially extracellular matrix proteins and damage-associated molecular patterns were cardinal in the formation of the surface proteome.

The USP7-TRIM27 axis mediates non-canonical PRC1.1 function and is a druggable target in leukemia.

In an attempt to unravel functionality of the non-canonical PRC1.1 Polycomb complex in human leukemogenesis, we show that USP7 and TRIM27 are integral components of PRC1.1. USP7 interactome analyses show that PRC1.1 is the predominant Polycomb complex co-precipitating with USP7. USP7 inhibition results in PRC1.1 disassembly and loss of chromatin binding, coinciding with reduced H2AK119ub and H3K27ac levels and diminished gene transcription of active PRC1.1-controlled loci, whereas H2AK119ub marks are also lost at PRC1 loci. TRIM27 and USP7 are reciprocally required for incorporation into PRC1.1, and TRIM27 knockdown partially rescues USP7 inhibitor sensitivity. USP7 inhibitors effectively impair proliferation in AML cells in vitro, also independent of the USP7-MDM2-TP53 axis, and MLL-AF9-induced leukemia is delayed in vivo in human leukemia xenografts. We propose a model where USP7 counteracts TRIM27 E3 ligase activity, thereby maintaining PRC1.1 integrity and function. Moreover, USP7 inhibition may be a promising new strategy to treat AML patients.

The Isotopic Ac-IP Tag Enables Multiplexed Proteome Quantification in Data-Independent Acquisition Mode.

Data-independent acquisition (DIA) is an increasingly used approach for quantitative proteomics. However, most current isotope labeling strategies are not suitable for DIA as they lead to more complex MS2 spectra or severe ratio distortion. As a result, DIA suffers from a lower throughput than data-dependent acquisition (DDA) due to a lower level of multiplexing. Herein, we synthesized an isotopically labeled acetyl-isoleucine-proline (Ac-IP) tag for multiplexed quantification in DIA. Differentially labeled peptides have distinct precursor ions carrying the quantitative information but identical MS2 spectra since the isotopically labeled Ac-Ile part leaves as a neutral loss upon collision-induced dissociation, while fragmentation of the peptide backbone generates regular fragment ions for identification. The Ac-IP-labeled samples can be analyzed using general DIA liquid chromatography-mass spectrometry settings, and the data obtained can be processed with established approaches. Relative quantification requires deconvolution of the isotope envelope of the respective precursor ions. Suitability of the Ac-IP tag is demonstrated with a triplex-labeled yeast proteome spiked with bovine serum albumin that was mixed at 10:5:1 ratios, resulting in measured ratios of 9.7:5.3:1.1.

A Versatile Isobaric Tag Enables Proteome Quantification in Data-Dependent and Data-Independent Acquisition Modes.

Quantifying proteins based on peptide-coupled reporter ions is a multiplexed quantitative strategy in proteomics that alleviates the problem of ratio distortion caused by peptide cofragmentation, as commonly observed in other reporter-ion-based approaches, such as TMT and iTRAQ. Data-independent acquisition (DIA) is an attractive alternative to data-dependent acquisition (DDA) due to its better reproducibility. While multiplexed labeling is widely used in DDA, it is rarely used in DIA, presumably because current approaches lead to more complex MS2 spectra, severe ratio distortion, or to a reduction in quantification accuracy and precision. Herein, we present a versatile acetyl-alanine-glycine (Ac-AG) tag that conceals quantitative information in isobarically labeled peptides and reveals it upon tandem MS in the form of peptide-coupled reporter ions. Since the peptide-coupled reporter ion is precursor-specific while fragment ions of the peptide backbone originating from different labeling channels are identical, the Ac-AG tag is compatible with both DDA and DIA. By isolating the monoisotopic peak of the precursor ion in DDA, intensities of the peptide-coupled reporter ions represent the relative ratios between constituent samples, whereas in DIA, the ratio can be inferred after deconvoluting the peptide-coupled reporter ion isotopes. The proteome quantification capability of the Ac-AG tag was demonstrated by triplex labeling of a yeast proteome spiked with bovine serum albumin (BSA) over a 10-fold dynamic range. Within this complex proteomics background, BSA spiked at 1:5:10 ratios was detected at ratios of 1.00:4.87:10.13 in DDA and 1.16:5.20:9.64 in DIA.

Impact of spatial proximity on territoriality among human skin bacteria.

Bacteria display social behavior and establish cooperative or competitive interactions in the niches they occupy. The human skin is a densely populated environment where many bacterial species live. Thus, bacterial inhabitants are expected to find a balance in these interactions, which eventually defines their spatial distribution and the composition of our skin microbiota. Unraveling the physiological basis of the interactions between bacterial species in organized environments requires reductionist analyses using functionally relevant species. Here, we study the interaction between two members of our skin microbiota, Bacillus subtilis and Staphylococcus epidermidis. We show that B. subtilis actively responds to the presence of S. epidermidis in its proximity by two strategies: antimicrobial production and development of a subpopulation with migratory response. The initial response of B. subtilis is production of chlorotetain, which degrades the S. epidermidis at the colony level. Next, a subpopulation of B. subtilis motile cells emerges. Remarkably this subpopulation slides towards the remaining S. epidermidis colony and engulfs it. A slow response back from S. epidermidis cells give origin to resistant cells that prevent both attacks from B. subtilis. We hypothesized that this niche conquering and back-down response from B. subtilis and S. epidermidis, respectively, which resembles other conflicts in nature as the ones observed in animals, may play a role in defining the presence of certain bacterial species in the specific microenvironments that these bacteria occupy on our skin.

A Collision-Induced Dissociation Cleavable Isobaric Tag for Peptide Fragment Ion-Based Quantification in Proteomics.

Quantifying peptides based on unique peptide fragment ions avoids the issue of ratio distortion that is commonly observed for reporter ion-based quantification approaches. Herein, we present a collision-induced dissociation-cleavable, isobaric acetyl-isoleucine-proline-glycine (Ac-IPG) tag, which conserves the merits of quantifying peptides based on unique fragments while reducing the complexity of the b-ion series compared to conventional fragment ion-based quantification methods thus facilitating data processing. Multiplex labeling is based on selective N-terminal dimethylation followed by derivatization of the ε-amino group of the C-terminal Lys residue of LysC peptides with isobaric Ac-IPG tags having complementary isotope distributions on Pro-Gly and Ac-Ile. Upon fragmentation between Ile and Pro, the resulting y ions, with the neutral loss of Ac-Ile, can be distinguished between the different labeling channels based on different numbers of isotope labels on the Pro-Gly part and thus contain the information for relative quantification, while b ions of different labeling channels have the same m/z values. The proteome quantification capability of this method was demonstrated by triplex labeling of a yeast proteome spiked with bovine serum albumin (BSA) over a 10-fold dynamic range. With the yeast proteins as the background, BSA was detected at ratios of 1.14:5.06:9.78 when spiked at 1:5:10 ratios. The raw mass data is available on the ProteomeXchange with the identifier PXD 018790.

Selective Maleylation-Directed Isobaric Peptide Termini Labeling for Accurate Proteome Quantification.

Isobaric peptide termini labeling (IPTL) is an attractive protein quantification method because it provides more accurate and reliable quantification information than traditional isobaric labeling methods (e.g., TMT and iTRAQ) by making use of the entire fragment-ion series instead of only a single reporter ion. The multiplexing capacity of published IPTL implementations is, however, limited to three. Here, we present a selective maleylation-directed isobaric peptide termini labeling (SMD-IPTL) approach for quantitative proteomics of LysC protein digestion. SMD-IPTL extends the multiplexing capacity to 4-plex with the potential for higher levels of multiplexing using commercially available 13C/15N labeled amino acids. SMD-IPTL is achieved in a one-pot reaction in three consecutive steps: (1) selective maleylation at the N-terminus; (2) labeling at the ε-NH2 group of the C-terminal Lys with isotopically labeled acetyl-alanine; (3) thiol Michael addition of an isotopically labeled acetyl-cysteine at the maleylated N-terminus. The isobarically labeled peptides are fragmented into sets of b- and y-ion clusters upon LC-MS/MS, which convey not only sequence information but also quantitative information for every labeling channel and avoid the issue of ratio distortion observed with reporter-ion-based approaches. We demonstrate the SMD-IPTL approach with a 4-plex labeled sample of bovine serum albumin (BSA) and yeast lysates mixed at different ratios. With the use of SMD-IPTL for labeling and a narrow precursor isolation window of 0.8 Th with an offset of -0.2 Th, accurate ratios were measured across a 10-fold mixing range of BSA in a background of yeast proteome. With the yeast proteins mixed at ratios of 1:5:1:5, BSA was detected at ratios of 0.94:2.46:4.70:9.92 when spiked at 1:2:5:10 ratios with an average standard deviation of peptide ratios of 0.34.

The use of metabolic profiling to identify insulin resistance in veal calves.

Heavy veal calves (4-6 months old) are at risk of developing insulin resistance and disturbed glucose homeostasis. Prolonged insulin resistance could lead to metabolic disorders and impaired growth performance. Recently, we discovered that heavy Holstein-Friesian calves raised on a high-lactose or high-fat diet did not differ in insulin sensitivity, that insulin sensitivity was low and 50% of the calves could be considered insulin resistant. Understanding the patho-physiological mechanisms underlying insulin resistance and discovering biomarkers for early diagnosis would be useful for developing prevention strategies. Therefore, we explored plasma metabolic profiling techniques to build models and discover potential biomarkers and pathways that can distinguish between insulin resistant and moderately insulin sensitive veal calves. The calves (n = 14) were classified as insulin resistant (IR) or moderately insulin sensitive (MIS) based on results from a euglycemic-hyperinsulinemic clamp, using a cut-off value (M/I-value <4.4) to identify insulin resistance. Metabolic profiles of fasting plasma samples were analyzed using reversed phase (RP) and hydrophilic interaction (HILIC) liquid chromatography-mass spectrometry (LC-MS). Orthogonal partial least square discriminant analysis was performed to compare metabolic profiles. Insulin sensitivity was on average 2.3x higher (P <0.001) in MIS than IR group. For both RP-LC-MS and HILIC-LC-MS satisfactory models were build (R2Y >90% and Q2Y >66%), which allowed discrimination between MIS and IR calves. A total of 7 and 20 metabolic features (for RP-LC-MS and HILIC-LC-MS respectively) were most responsible for group separation. Of these, 7 metabolites could putatively be identified that differed (P <0.05) between groups (potential biomarkers). Pathway analysis indicated disturbances in glycerophospholipid and sphingolipid metabolism, the glycine, serine and threonine metabolism, and primary bile acid biosynthesis. These results demonstrate that plasma metabolic profiling can be used to identify insulin resistance in veal calves and can lead to underlying mechanisms.

Specific Affinity Enrichment of Electrochemically Cleaved Peptides Based on Cu(II)-Mediated Spirolactone Tagging.

Specific digestion of proteins is an essential step for mass spectrometry-based proteomics, and the chemical labeling of the resulting peptides is often used for peptide enrichment or the introduction of desirable tags. Electrochemical oxidation yielding specific cleavage C-terminal to tyrosine (Tyr) and tryptophan (Trp) residues provides a potential alternative to enzymatic digestion and a possibility for further chemical labeling by introducing reactive spirolactone moieties. However, spirolactone-containing peptides suffer from low stability due to hydrolysis and intramolecular side reactions. We found that Cu(II) ions stabilize the spirolactone and prevent intramolecular side reactions during chemical labeling, allowing efficient chemical tagging with a reduced excess of labeling reagent without intramolecular side reactions. On the basis of this reaction, we developed an analytical procedure combining electrochemical digestion, Cu(II)-mediated spirolactone biotinylation, and enrichment by avidin affinity chromatography with mass spectrometry. The method was optimized with the tripeptide LWL and subsequently applied to chicken egg white lysozyme, in which one biotinylated electrochemistry (EC)-cleaved peptide was identified after affinity enrichment. This proof-of-principle shows that specific enrichment of electrochemically cleaved spirolactone-containing peptides can be used for protein identification and notably that inclusion of Cu(II) ions is essential for stabilizing spirolactones for subsequent biotinylation.

The interaction of fluorescent nanodiamond probes with cellular media.

Fluorescent nanodiamonds (FNDs) are promising tools to image cells, bioanalytes and physical quantities such as temperature, pressure, and electric or magnetic fields with nanometer resolution. To exploit their potential for intracellular applications, the FNDs have to be brought into contact with cell culture media. The interactions between the medium and the diamonds crucially influence sensitivity as well as the ability to enter cells. The authors demonstrate that certain proteins and salts spontaneously adhere to the FNDs and may cause aggregation. This is a first investigation on the fundamental questions on how (a) FNDs interact with the medium, and (b) which proteins and salts are being attracted. A differentiation between strongly binding and weakly binding proteins is made. Not all proteins participate in the formation of FND aggregates. Surprisingly, some main components in the medium seem to play no role in aggregation. Simple strategies to prevent aggregation are discussed. These include adding the proteins, which are naturally present in the cell culture to the diamonds first and then inserting them in the full medium. Graphical abstractSchematic of the interaction of nanodiamonds with cell culture medium. Certain proteins and salts adhere to the diamond surface and lead to aggregation or to formation of a protein corona.

Semi-microbiological synthesis of an active lysinoalanine-bridged analog of glucagon-like-peptide-1.

Some modified glucagon-like-peptide-1 (GLP-1) analogs are highly important for treating type 2 diabetes. Here we investigated whether GLP-1 analogs expressed in Lactococcus lactis could be substrates for modification and export by the nisin dehydratase and transporter enzyme. Subsequently we introduced a lysinoalanine by coupling a formed dehydroalanine with a lysine and investigated the structure and activity of the formed lysinoalanine-bridged GLP-1 analog. Our data show: (i) GLP-1 fused to the nisin leader peptide is very well exported via the nisin transporter NisT, (ii) production of leader-GLP-1 via NisT is higher than via the SEC system, (iii) leader-GLP-1 exported via NisT was more efficiently dehydrated by the nisin dehydratase NisB than when exported via the SEC system, (iv) individual serines and threonines in GLP-1 are dehydrated by NisB to a significantly different extent, (v) an introduced Ser30 is well dehydrated and can be coupled to Lys34 to form a lysinoalanine-bridged GLP-1 analog, (vi) a lysinoalanine(30-34) variant's conformation shifts in the presence of 25% trifluoroethanol towards a higher alpha helix content than observed for wild type GLP-1 under identical condition, (vii) a lysinoalanine(30-34) GLP-1 variant has retained significant activity. Taken together the data extend knowledge on the substrate specificities of NisT and NisB and their combined activity relative to export via the Sec system, and demonstrate that introducing a lysinoalanine bridge is an option for modifying therapeutic peptides.

Efficient and Selective Chemical Labeling of Electrochemically Generated Peptides Based on Spirolactone Chemistry.

Specific digestion of proteins is an essential step for mass spectrometry-based proteomics, and the chemical labeling of the resulting peptides is often used for peptide enrichment or the introduction of desirable tags. Cleavage of the peptide bond following electrochemical oxidation of Tyr or Trp results in a spirolactone moiety at the newly formed C-terminus offering a handle for chemical labeling. In this work, we developed a highly efficient and selective chemical labeling approach based on spirolactone chemistry. Electrochemically generated peptide-spirolactones readily undergo an intramolecular rearrangement yielding isomeric diketopiperazines precluding further chemical labeling. A strategy was established to prevent intramolecular arrangement by acetylating the N-terminal amino group prior to electrochemical oxidation and cleavage allowing the complete and selective chemical labeling of the tripeptide LWL and the decapeptide ACTH 1-10 with amine-containing reagents. As examples, we show the successful introduction of a fluorescent label and biotin for detection or affinity enrichment. Electrochemical digestion of peptides and proteins followed by efficient chemical labeling constitutes a new, powerful tool in protein chemistry and protein analysis.

Incorporation of tryptophan analogues into the lantibiotic nisin.

Lantibiotics are posttranslationally modified peptides with efficient inhibitory activity against various Gram-positive bacteria. In addition to the original modifications, incorporation of non-canonical amino acids can render new properties and functions to lantibiotics. Nisin is the most studied lantibiotic and contains no tryptophan residues. In this study, a system was constructed to incorporate tryptophan analogues into nisin, which included the modification machinery (NisBTC) and the overexpression of tryptophanyl-tRNA synthetase (TrpRS). Tryptophan and three different tryptophan analogues (5-fluoroTrp (5FW), 5-hydroxyTrp (5HW) and 5-methylTrp (5MeW)) were successfully incorporated at four different positions of nisin (I1W, I4W, M17W and V32W). The incorporation efficiency of tryptophan analogues into mutants I1W, M17W and V32W was over 97 %, while the mutant I4W showed relatively low incorporation efficiency (69-93 %). The variants with 5FW showed relatively higher production yield, while 5MeW-containing variants showed the lowest yield. The dehydration efficiency of serines or threonines was affected by the tryptophan mutants of I4W and V32W. The affinity of the peptides for the cation-ion exchange and reverse phase chromatography columns was significantly reduced when 5HW was incorporated. The antimicrobial activity of IIW and its 5FW analogue both decreased two times compared to that of nisin, while that of its 5HW analogue decreased four times. The 5FW analogue of I4W also showed two times decreased activity than nisin. However, the mutant M17W and its 5HW analogue both showed 32 times reduced activity relative to that of nisin.

Quantitative proteomics analyses of activation states of human THP-1 macrophages.

Macrophages display large functional and phenotypical plasticity. They can adopt a broad range of activation states depending on their microenvironment. Various surface markers are used to characterize these differentially polarized macrophages. However, this is not informative for the functions of the macrophage. In order to have a better understanding of the functional changes of macrophages upon differential polarization, we studied differences in LPS- and IL4-stimulated macrophages. The THP-1 human monocytic cell line, was used as a model system. Cells were labeled, differentiated and stimulated with either LPS or IL-4 in a quantitative SILAC proteomics set-up. The resulting sets of proteins were functionally clustered. LPS-stimulated macrophages show increased secretion of proinflammatory peptides, leading to increased pressure on protein biosynthesis and processing. IL4-stimulated macrophages show upregulation of cell adhesion and extracellular matrix remodeling. Our approach provides an integrated view of polarization-induced functional changes and proves useful for studying functional differences between subsets of macrophages. Moreover, the identified polarization specific proteins may contribute to a better characterization of different activation states in situ and their role in various inflammatory processes.

Significance of surface charge and shell material of superparamagnetic iron oxide nanoparticle (SPION) based core/shell nanoparticles on the composition of the protein corona.

As nanoparticles (NPs) are increasingly used in many applications their safety and efficient applications in nanomedicine have become concerns. Protein coronas on nanomaterials' surfaces can influence how the cell "recognizes" nanoparticles, as well as the in vitro and in vivo NPs' behaviors. The SuperParamagnetic Iron Oxide Nanoparticle (SPION) is one of the most prominent agents because of its superparamagnetic properties, which is useful for separation applications. To mimic surface properties of different types of NPs, a core-shell SPION library was prepared by coating with different surfaces: polyvinyl alcohol polymer (PVA) (positive, neutral and negative), SiO2 (positive and negative), titanium dioxide and metal gold. The SPIONs with different surfaces were incubated at a fixed serum : nanoparticle surface ratio, magnetically trapped and washed. The tightly bound proteins were quantified and identified. The surface charge has a great impact on protein adsorption, especially on PVA and silica where proteins preferred binding to the neutral and positively charged surfaces. The importance of surface material on protein adsorption was also revealed by preferential binding on TiO2 and gold coated SPION, even negatively charged. There is no correlation between the protein net charge and the nanoparticle surface charge on protein binding, nor direct correlation between the serum proteins' concentration and the proteins detected in the coronas.