Silicone implant surface microtopography modulates inflammation and tissue repair in capsular fibrosis.

Excessive fibrous capsule formation around silicone mammary implants (SMI) involves immune reactions to silicone. Capsular fibrosis, a common SMI complication linked to host responses, worsens with specific implant topographies. Our study with 10 patients investigated intra- and inter-individually, reduced surface roughness effects on disease progression, wound responses, chronic inflammation, and capsular composition. The results illuminate the significant impact of surface roughness on acute inflammatory responses, fibrinogen accumulation, and the subsequent fibrotic cascade. The reduction of surface roughness to an average roughness of 4 µm emerges as a promising approach for mitigating detrimental immune reactions, promoting healthy wound healing, and curbing excessive fibrosis. The identified proteins adhering to rougher surfaces shed light on potential mediators of pro-inflammatory and pro-fibrotic processes, further emphasizing the need for meticulous consideration of surface design. The composition of the implant capsule and the discovery of intracapsular HSP60 expression highlight the intricate web of stress responses and immune activation that can impact long-term tissue outcomes.

Surface Topography, Microbial Adhesion, and Immune Responses in Silicone Mammary Implant-Associated Capsular Fibrosis.

Breast cancer is the most common cancer in women globally, often necessitating mastectomy and subsequent breast reconstruction. Silicone mammary implants (SMIs) play a pivotal role in breast reconstruction, yet their interaction with the host immune system and microbiome remains poorly understood. This study investigates the impact of SMI surface topography on host antimicrobial responses, wound proteome dynamics, and microbial colonization. Biological samples were collected from ten human patients undergoing breast reconstruction with SMIs. Mass spectrometry profiles were analyzed for acute and chronic wound proteomes, revealing a nuanced interplay between topography and antimicrobial response proteins. 16S rRNA sequencing assessed microbiome dynamics, unveiling topography-specific variations in microbial composition. Surface topography alterations influenced wound proteome composition. Microbiome analysis revealed heightened diversity around rougher SMIs, emphasizing topography-dependent microbial invasion. In vitro experiments confirmed staphylococcal adhesion, growth, and biofilm formation on SMI surfaces, with increased texture correlating positively with bacterial colonization. This comprehensive investigation highlights the intricate interplay between SMI topography, wound proteome dynamics, and microbial transmission. The findings contribute to understanding host-microbe interactions on SMI surfaces, essential for optimizing clinical applications and minimizing complications in breast reconstruction.

Complement C7 and clusterin form a complex in circulation.

Introduction: The complement system is part of innate immunity and is comprised of an intricate network of proteins that are vital for host defense and host homeostasis. A distinct mechanism by which complement defends against invading pathogens is through the membrane attack complex (MAC), a lytic structure that forms on target surfaces. The MAC is made up of several complement components, and one indispensable component of the MAC is C7. The role of C7 in MAC assembly is well documented, however, inherent characteristics of C7 are yet to be investigated. Methods: To shed light on the molecular characteristics of C7, we examined the properties of serum-purified C7 acquired using polyclonal and novel monoclonal antibodies. The properties of serum­purified C7 were investigated through a series of proteolytic analyses, encompassing Western blot and mass spectrometry. The nature of C7 protein-protein interactions were further examined by a novel enzyme-linked immunosorbent assay (ELISA), as well as size­exclusion chromatography. Results: Protein analyses showcased an association between C7 and clusterin, an inhibitory complement regulator. The distinct association between C7 and clusterin was also demonstrated in serum-purified clusterin. Further assessment revealed that a complex between C7 and clusterin (C7-CLU) was detected. The C7-CLU complex was also identified in healthy serum and plasma donors, highlighting the presence of the complex in circulation. Discussion: Clusterin is known to dissociate the MAC structure by binding to polymerized C9, nevertheless, here we show clusterin binding to the native form of a terminal complement protein in vivo. The presented data reveal that C7 exhibits characteristics beyond that of MAC assembly, instigating further investigation of the effector role that the C7-CLU complex plays in the complement cascade.

Skeletal muscle proteome analysis underpins multifaceted mitochondrial dysfunction in Friedreich's ataxia.

Friedreich's ataxia (FRDA) is a severe multisystemic disorder caused by a deficiency of the mitochondrial protein frataxin. While some aspects of FRDA pathology are developmental, the causes underlying the steady progression are unclear. The inaccessibility of key affected tissues to sampling is a main hurdle. Skeletal muscle displays a disease phenotype and may be sampled in vivo to address open questions on FRDA pathophysiology. Thus, we performed a quantitative mass spectrometry-based proteomics analysis in gastrocnemius skeletal muscle biopsies from genetically confirmed FRDA patients (n = 5) and controls. Obtained data files were processed using Proteome Discoverer and searched by Sequest HT engine against a UniProt human reference proteome database. Comparing skeletal muscle proteomics profiles between FRDA and controls, we identified 228 significant differentially expressed (DE) proteins, of which 227 were downregulated in FRDA. Principal component analysis showed a clear separation between FRDA and control samples. Interactome analysis revealed clustering of DE proteins in oxidative phosphorylation, ribosomal elements, mitochondrial architecture control, and fission/fusion pathways. DE findings in the muscle-specific proteomics suggested a shift toward fast-twitching glycolytic fibers. Notably, most DE proteins (169/228, 74%) are target of the transcription factor nuclear factor-erythroid 2. Our data corroborate a mitochondrial biosignature of FRDA, which extends beyond a mere oxidative phosphorylation failure. Skeletal muscle proteomics highlighted a derangement of mitochondrial architecture and maintenance pathways and a likely adaptive metabolic shift of contractile proteins. The present findings are relevant for the design of future therapeutic strategies and highlight the value of skeletal muscle-omics as disease state readout in FRDA.

PKN1 Exerts Neurodegenerative Effects in an In Vitro Model of Cerebellar Hypoxic-Ischemic Encephalopathy via Inhibition of AKT/GSK3ß Signaling.

We recently identified protein kinase N1 (PKN1) as a negative gatekeeper of neuronal AKT protein kinase activity during postnatal cerebellar development. The developing cerebellum is specifically vulnerable to hypoxia-ischemia (HI), as it occurs during hypoxic-ischemic encephalopathy, a condition typically caused by oxygen deprivation during or shortly after birth. In that context, activation of the AKT cell survival pathway has emerged as a promising new target for neuroprotective interventions. Here, we investigated the role of PKN1 in an in vitro model of HI, using postnatal cerebellar granule cells (Cgc) derived from Pkn1 wildtype and Pkn1-/- mice. Pkn1-/- Cgc showed significantly higher AKT phosphorylation, resulting in reduced caspase-3 activation and improved survival after HI. Pkn1-/- Cgc also showed enhanced axonal outgrowth on growth-inhibitory glial scar substrates, further pointing towards a protective phenotype of Pkn1 knockout after HI. The specific PKN1 phosphorylation site S374 was functionally relevant for the enhanced axonal outgrowth and AKT interaction. Additionally, PKN1pS374 shows a steep decrease during cerebellar development. In summary, we demonstrate the pathological relevance of the PKN1-AKT interaction in an in vitro HI model and establish the relevant PKN1 phosphorylation sites, contributing important information towards the development of specific PKN1 inhibitors.

Enzymatic Cleavage of Stx2a in the Gut and Identification of Pancreatic Elastase and Trypsin as Possible Main Cleavers.

Shiga toxins (Stxs), especially the Stx2a subtype, are the major virulence factors involved in enterohemorrhagic Escherichia coli (EHEC)-associated hemolytic uremic syndrome (eHUS), a life-threatening disease causing acute kidney injury, especially in children. After oral transmission and colonization in the gut, EHEC release Stx. Intracellular cleavage of the Stx A subunit, when followed by reduction, boosts the enzymatic activity that causes damage to targeted cells. This cleavage was assumed to be mostly mediated by furin during Stx intracellular trafficking. To investigate whether this cleavage could occur in the intestine, even prior to entering target cells, Stx2a A subunit structure (intact or cleaved) was characterized after its exposure to specific host factors present in human stool. The molecular weight of Stx2a A subunit/fragments was determined by immunoblotting after electrophoretic separation under reducing conditions. In this study, it was demonstrated that Stx2a is cleaved by certain human stool components. Trypsin and chymotrypsin-like elastase 3B (CELA3B), two serine proteases, were identified as potential candidates that can trigger the extracellular cleavage of Stx2a A subunit directly after its secretion by EHEC in the gut. Whether the observed cleavage indeed translates to natural infections and plays a role in eHUS pathogenesis has yet to be determined. If so, it seems likely that a host's protease profile could affect disease development by changing the toxin's biological features.

Protein Networks Associated with Native Metabotropic Glutamate 1 Receptors (mGlu1) in the Mouse Cerebellum.

The metabotropic glutamate receptor 1 (mGlu1) plays a pivotal role in synaptic transmission and neuronal plasticity. Despite the fact that several interacting proteins involved in the mGlu1 subcellular trafficking and intracellular transduction mechanisms have been identified, the protein network associated with this receptor in specific brain areas remains largely unknown. To identify novel mGlu1-associated protein complexes in the mouse cerebellum, we used an unbiased tissue-specific proteomic approach, namely co-immunoprecipitation followed by liquid chromatography/tandem mass spectrometry analysis. Many well-known protein complexes as well as novel interactors were identified, including G-proteins, Homer, δ2 glutamate receptor, 14-3-3 proteins, and Na/K-ATPases. A novel putative interactor, KCTD12, was further investigated. Reverse co-immunoprecipitation with anti-KCTD12 antibodies revealed mGlu1 in wild-type but not in KCTD12-knock-out homogenates. Freeze-fracture replica immunogold labeling co-localization experiments showed that KCTD12 and mGlu1 are present in the same nanodomain in Purkinje cell spines, although at a distance that suggests that this interaction is mediated through interposed proteins. Consistently, mGlu1 could not be co-immunoprecipitated with KCTD12 from a recombinant mammalian cell line co-expressing the two proteins. The possibility that this interaction was mediated via GABAB receptors was excluded by showing that mGlu1 and KCTD12 still co-immunoprecipitated from GABAB receptor knock-out tissue. In conclusion, this study identifies tissue-specific mGlu1-associated protein clusters including KCTD12 at Purkinje cell synapses.

Quantitative Proteomic Characterization of Foreign Body Response towards Silicone Breast Implants Identifies Chronological Disease-Relevant Biomarker Dynamics.

The etiology of exaggerated fibrous capsule formation around silicone mammary implants (SMI) is multifactorial but primarily induced by immune mechanisms towards the foreign material silicone. The aim of this work was to understand the disease progression from implant insertion and immediate tissue damage response reflected in (a) the acute wound proteome and (b) the adsorption of chronic inflammatory wound proteins at implant surfaces. An intraindividual relative quantitation TMT-liquid chromatography-tandem mass spectrometry approach was applied to the profile wound proteome formed around SMI in the first five days post-implantation. Compared to plasma, the acute wound profile resembled a more complex composition comprising plasma-derived and locally differentially expressed proteins (DEPs). DEPs were subjected to a functional enrichment analysis, which revealed the dysregulation of signaling pathways mainly involved in immediate inflammation response and ECM turnover. Moreover, we found time-course variations in protein enrichment immediately post-implantation, which were adsorbed to SMI surfaces after 6-8 months. Characterization of the expander-adhesive proteome by a label-free approach uncovered a long-term adsorbed acute wound and the fibrosis-associated proteome. Our findings propose a wound biomarker panel for the early detection and diagnosis of excessive fibrosis that could potentially broaden insights into the characteristics of fibrotic implant encapsulation.

Platelet TAU is Associated with Changes in Depression and Alzheimer's Disease.

BACKGROUND: Platelets (thrombocytes) are small anuclear cells that play an important role in blood clotting. They are activated and dysfunctional in brain disorders, such as Alzheimer's disease (AD) and depression. Platelets express the amyloid-precursor protein (APP) and release beta-amyloid40 into the blood. Recent evidence reports that platelets also express the microtubule-associated protein tau. In this study, we further characterized the molecular appearance of tau and examined its alterations in patients with neurocognitive impairment. METHODS: Platelets were isolated from patients with AD, mild cognitive impairment (MCI) or depression and compared to healthy controls. Subsequently, FACS analysis was employed to characterize platelets for platelet surface P-selectin (CD62P). In order to enhance the detection levels, samples were pooled (15 samples per group) and analyzed by Lumipulse Assay, Western blots, and mass spectrometry. RESULTS: Tau is expressed in human platelets and tau levels were decreased in platelets isolated from patients with AD and depression. Additionally, phospho-tau-181 was slightly increased in patients with depression. We show that tau is highly fragmented (20-40 kDa) in the platelet extracts using Western blot analysis. The mass spectrometry data did not show a clear identification of tau in the pooled platelet samples. CONCLUSIONS: Our data reveal that tau is found in platelets, possibly in a highly fragmented form. Tau levels may be used as a potential diagnostic approach to differentiate AD and depression from healthy controls.

N-chlorotaurine is highly active against respiratory viruses including SARS-CoV-2 (COVID-19) in vitro.

N-chlorotaurine (NCT) a long-lived oxidant generated by leukocytes, can be synthesized chemically and applied topically as an anti-infective to different body sites, including the lung via inhalation. Here, we demonstrate the activity of NCT against viruses causing acute respiratory tract infections, namely severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza viruses, and respiratory syncytial virus (RSV). Virucidal activity of NCT was tested in plaque assays, confirmed by RT-qPCR assays. Attack on virus proteins was investigated by mass spectrometry. NCT revealed broad virucidal activity against all viruses tested at 37°C and pH 7. A significant reduction in infectious particles of SARS-CoV-2 isolates from early 2020 by 1 log10 was detected after 15 min of incubation in 1% NCT. Proteinaceous material simulating body fluids enhanced this activity by transchlorination mechanisms (1 -2 log10 reduction within 1-10 min). Tested SARS-CoV-2 variants B.1.1.7 (Alpha) und B.1.351 (Beta) showed a similar susceptibility. Influenza virus infectious particles were reduced by 3 log10 (H3N2) to 5 log10 (H1N1pdm), RSV by 4 log10 within a few min. Mass spectrometry of NCT-treated SARS-CoV-2 spike protein and 3C-like protease, influenza virus haemagglutinin and neuraminidase, and RSV fusion glycoprotein disclosed multiple sites of chlorination and oxidation as the molecular mechanism of action. Application of 1.0% NCT as a prophylactic and therapeutic strategy against acute viral respiratory tract infections deserves comprehensive clinical investigation.

Expression of transport proteins in the rete mirabile of european silver and yellow eel.

BACKGROUND: In physoclist fishes filling of the swimbladder requires acid secretion of gas gland cells to switch on the Root effect and subsequent countercurrent concentration of the initial gas partial pressure increase by back-diffusion of gas molecules in the rete mirabile. It is generally assumed that the rete mirabile functions as a passive exchanger, but a detailed analysis of lactate and water movements in the rete mirabile of the eel revealed that lactate is diffusing back in the rete. In the present study we therefore test the hypothesis that expression of transport proteins in rete capillaries allows for back-diffusion of ions and metabolites, which would support the countercurrent concentrating capacity of the rete mirabile. It is also assumed that in silver eels, the migratory stage of the eel, the expression of transport proteins would be enhanced. RESULTS: Analysis of the transcriptome and of the proteome of rete mirabile tissue of the European eel revealed the expression of a large number of membrane ion and metabolite transport proteins, including monocarboxylate and glucose transport proteins. In addition, ion channel proteins, Ca2+-ATPase, Na+/K+-ATPase and also F1F0-ATP synthase were detected. In contrast to our expectation in silver eels the expression of these transport proteins was not elevated as compared to yellow eels. A remarkable number of enzymes degrading reactive oxygen species (ROS) was detected in rete capillaries. CONCLUSIONS: Our results reveal the expression of a large number of transport proteins in rete capillaries, so that the back diffusion of ions and metabolites, in particular lactate, may significantly enhance the countercurrent concentrating ability of the rete. Metabolic pathways allowing for aerobic generation of ATP supporting secondary active transport mechanisms are established. Rete tissue appears to be equipped with a high ROS defense capacity, preventing damage of the tissue due to the high oxygen partial pressures generated in the countercurrent system.

Exposing the High Heterogeneity of Circulating Pro B-Type Natriuretic Peptide Fragments in Healthy Individuals and Heart Failure Patients.

BACKGROUND: The high molecular complexity of variably O-glycosylated and degraded pro B-type natriuretic peptide (proBNP) derived molecular forms challenges current immunoassays. Antibodies used show pronounced differences in cross-reactivities with these circulating fragments, which still need to be better characterized on a molecular level. To pave the way for advanced quantitative assays in the future, it is critical to fully understand these circulating forms. METHODS: Plasma samples were collected from 8 heart failure (HF) patients and 2 healthy controls. NT-proBNP and proBNP were purified by immunoprecipitation and analyzed by nano-flow liquid chromatography coupled to high-resolution mass spectrometry. Fragments formed during proteolysis in solution digestion were distinguished from naturally occurring peptides by using an 18O stable isotope labeling strategy. RESULTS: We detected 16 previously unknown circulating fragments of proBNP peptides (9 of which are located in the N-terminal and 7 in the C-terminal region), revealing a more advanced state of degradation than previously known. Two of these fragments are indicative of either unidentified processing modes or a far-reaching C-terminal degradation (or a combination thereof) of the precursor proBNP. CONCLUSIONS: Our results further restrict ideal target epitopes for immunoassay antibodies and expand the current thinking of diversity, degradation, and processing of proBNP, as well as the distribution of circulating forms.

Multiplex Genetic Engineering Exploiting Pyrimidine Salvage Pathway-Based Endogenous Counterselectable Markers.

Selectable markers are indispensable for genetic engineering, yet their number and variety are limited. Most selection procedures for prototrophic cells rely on the introduction of antibiotic resistance genes. New minimally invasive tools are needed to facilitate sophisticated genetic manipulations. Here, we characterized three endogenous genes in the human fungal pathogen Aspergillus fumigatus for their potential as markers for targeted genomic insertions of DNAs of interest (DOIs). Since these genes are involved in uptake and metabolization of pyrimidines, resistance to the toxic effects of prodrugs 5-fluorocytosine and 5-fluorouracil can be used to select successfully integrated DOIs. We show that DOI integration, resulting in the inactivation of these genes, caused no adverse effects with respect to nutrient requirements, stress resistance, or virulence. Beside the individual use of markers for site-directed integration of reporter cassettes, including the 17-kb penicillin biosynthetic cluster, we demonstrate their sequential use by inserting three genes encoding fluorescent proteins into a single strain for simultaneous multicolor localization microscopy. In addition to A. fumigatus, we validated the applicability of this novel toolbox in Penicillium chrysogenum and Fusarium oxysporum Enabling multiple targeted insertions of DOIs without the necessity for exogenous markers, this technology has the potential to significantly advance genetic engineering.IMPORTANCE This work reports the discovery of a novel genetic toolbox comprising multiple, endogenous selectable markers for targeted genomic insertions of DNAs of interest (DOIs). Marker genes encode proteins involved in 5-fluorocytosine uptake and pyrimidine salvage activities mediating 5-fluorocytosine deamination as well as 5-fluorouracil phosphoribosylation. The requirement for their genomic replacement by DOIs to confer 5-fluorocytosine or 5-fluorouracil resistance for transformation selection enforces site-specific integrations. Due to the fact that the described markers are endogenously encoded, there is no necessity for the exogenous introduction of commonly employed markers such as auxotrophy-complementing genes or antibiotic resistance cassettes. Importantly, inactivation of the described marker genes had no adverse effects on nutrient requirements, growth, or virulence of the human pathogen Aspergillus fumigatus Given the limited number and distinct types of selectable markers available for the genetic manipulation of prototrophic strains such as wild-type strains, we anticipate that the proposed methodology will significantly advance genetic as well as metabolic engineering of fungal species.

Application of CE-MS for the analysis of histones and histone modifications.

The analysis, identification and quantification of histones and their post-translational modifications plays a central role in chromatin research and in studying epigenetic regulations during physiological processes. In the last decade analytical strategies based on mass spectrometry have been greatly improved for providing a global view of single modification abundances or to determine combinatorial patterns of modifications. Presented here is a newly developed strategy for histone protein analysis and a number of applications are illustrated with an emphasis on PTM characterization. Capillary electrophoresis is coupled to mass spectrometry (CE-MS) and has proven to be a very promising concept as it enables to study intact histones (top-down proteomics) as well as the analysis of enzymatically digested proteins (bottom-up proteomics). This technology combines highly efficient low-flow CE separations with ionization in a single device and offers an orthogonal separation principle to conventional LC-MS analysis, thus expanding the existing analytical repertoire in a perfect manner.

Hyperosmotic stress: in situ chromatin phase separation.

Dehydration of cells by acute hyperosmotic stress has profound effects upon cell structure and function. Interphase chromatin and mitotic chromosomes collapse ("congelation"). HL-60/S4 cells remain ~100% viable for, at least, 1 hour, exhibiting shrinkage to ~2/3 their original volume, when placed in 300mM sucrose in tissue culture medium. Fixed cells were imaged by immunostaining confocal and STED microscopy. At a "global" structural level (µm), mitotic chromosomes congeal into a residual gel with apparent (phase) separations of Ki67, CTCF, SMC2, RAD21, H1 histones and HMG proteins. At an "intermediate" level (sub-µm), radial distribution analysis of STED images revealed a most probable peak DNA density separation of ~0.16 µm, essentially unchanged by hyperosmotic stress. At a "local" structural level (~1-2 nm), in vivo crosslinking revealed essentially unchanged crosslinked products between H1, HMG and inner histones. Hyperosmotic cellular stress is discussed in terms of concepts of mitotic chromosome structure and liquid-liquid phase separation.

Investigating capillary electrophoresis-mass spectrometry for the analysis of common post-translational modifications.

Capillary electrophoresis coupled to mass spectrometry is a very efficient analytical method for the analysis of post-translational modifications because of its high separation efficiency and high detection sensitivity. Here we applied CE-MS using three differently coated separation capillaries for in-depth analysis of a set of 70 synthetic post-translationally modified peptides (including phosphorylation, acetylation, methylation, and nitration). We evaluated the results in terms of peptide detection and separation characteristics and found that the use of a neutrally coated capillary resulted in highest overall signal intensity of singly modified peptides. In contrast, the use of a bare-fused silica capillary was superior in the identification of multi-phosphorylated peptides (12 out of 15 were identified). Fast separations of approximately 12 min could be achieved using a positively coated capillary, however, at the cost of separation efficiency. A comparison to nanoLC-MS revealed that multi-phosphorylated peptides interact with the RP material very poorly so that these peptides were either washed out or elute as very broad peaks from the nano column which results in a reduced peptide identification rate (7 out of 15). Moreover, the methods applied were found to be very well suited for the analysis of the acetylated, nitrated and methylated peptides. All 36 synthetic peptides, which exhibit one of those modifications, could be identified regardless of the method applied. As a final step in this study and as a proof of principle, the phosphoproteome enriched from PC-12 pheochromocytoma cells was analyzed by CE-MS resulting in 5686 identified and 4088 quantified phosphopeptides. We compared the characterized analytes to those identified by a nanoLC-MS proteomics study and found that less than one third of the phosphopeptides were identical, which demonstrates the benefit by combining different approaches quite impressively.

Enhancing Proteomic Throughput in Capillary Electrophoresis-Mass Spectrometry by Sequential Sample Injection.

In this study we demonstrate the potential of sequential injection of samples in capillary electrophoresis-mass spectrometry for rapid and sensitive proteome characterization of human lymphoblastic T-cells (line CCRF-CEM). Proteins were extracted, enzymatically digested, and the resulting peptides fractionated by RP-HPLC. Twenty fractions were thereafter analyzed by CE-MS within a single MS analysis. The CE-MS method was designed so that every 10 min a new fraction was injected into the CE system. Without any rinsing or equilibration steps we were able to generate a continuous stream of peptides feeding the mass analyzer. In 250 min, the total analysis time of a single sequential injection experiment, we were able to identify roughly 28 000 peptide sequences counting for 4800 proteins. These numbers could be increased to 62 000 peptides and more than 6100 proteins identified, when performing three experiments analyzing a total of 60 fractions, all within 12.5 h. We found that the electrophoretic mobility of peptides can be used to trace back peptides and assign them to the fraction they originate from.

Inactivation of microbicidal active halogen compounds by sodium thiosulphate and histidine/methionine for time-kill assays.

Rapid inactivation of antimicrobial test agents after exact incubation times with microorganisms is required in time-kill assays. Sodium thiosulphate and a combination of methionine and histidine were compared for neutralisation of active halogen compounds. Test oxidants were mixed with surplus sodium thiosulphate (3%-6%) or histidine/methionine (1% each) in phosphate-buffered saline and incubated for different times, followed by addition of Staphylococcus aureus, Escherichia coli, or Pseudomonas aeruginosa at 1000CFU/ml. After further incubation, quantitative cultures were performed. Thiosulphate did not sufficiently inactivate chlorine and bromine compounds, indicated by a 10-fold (S. aureus) up to >100-fold (E. coli, P. aeruginosa) reduction of CFU. This was particularly true for high concentrations of the oxidants of about 50mM, for highly reactive agents (HOCl and bromamine T) more than for chloramine T and N-chlorotaurine, and for short pre-incubation times before addition of the bacteria. By contrast, histidine/methionine proved to be suitable for chloramines and bromamine T and for low concentrations of HOCl (0.07%). HOCl at 0.7% could neither be inactivated completely by thiosulphate nor by histidine/methionine. In contrast to chlorine and bromine compounds, iodine was neutralized by thiosulphate, but not by histidine/methionine. Histidine/methionine is superior to inactivate chlorine and bromine and should replace sodium thiosulphate at least in killing tests with high concentrations of these disinfectants. Inclusion of a short reaction time (maximum one minute) of test oxidant and neutralising substance before addition of bacteria is decisive in inactivation tests to obtain reliable results.

Identification of Novel Site-Specific Alterations in the Modification Level of Myelin Basic Protein Isolated from Mouse Brain at Different Ages Using Capillary Electrophoresis-Mass Spectrometry.

Myelin basic protein (MBP) is a multifunctional protein involved in maintaining the stability and integrity of the myelin sheath by a variety of interactions with membranes and other proteins. MBP is subjected to extensive posttranslational modifications (PTMs) that are known to be crucial for the regulation of these interactions. Here, we report capillary electrophoresis-mass spectrometric (CE-MS) analysis for the separation and identification of MBP peptides that incorporate the same PTM at different sites, creating multiple localization variants, and the ability to analyze challenging modifications such as asparagine and glutamine deamidation, isomerization, and arginine citrullination. Moreover, we observed site-specific alterations in the modification level of MBP purified from brain of mice of different age. In total, we identified 40 modifications at 33 different sites, which include both previously reported and seven novel modifications. The identified modifications include Nα-terminal acetylation, mono- and dimethylation, phosphorylation, oxidation, deamidation, and citrullination. Notably, some new sites of arginine methylation overlap with the sites of citrullination. Our results highlight the need for sensitive and efficient techniques for a comprehensive analysis of PTMs.

Exploiting charge differences for the analysis of challenging post-translational modifications by capillary electrophoresis-mass spectrometry.

Reversed-phase high-performance liquid chromatography (RP-HPLC) in combination with mass spectrometry (MS) is typically employed for mapping modifications in proteins and peptides. Here we applied a low-flow capillary electrophoresis (CE) -electrospray ionization interface coupled to Orbitrap mass spectrometers to analyze challenging modifications such as asparagine deamidation, aspartate isomerization, arginine citrullination, and phosphopeptide isomers. We achieved excellent resolution of asparagine (Asn), aspartic acid (Asp) and isoaspartic acid (iso-Asp) containing peptides using a synthetic peptide mixture. The migration order in CE enabled a clear assignment of in vitro deamidation/isomerization sites in a protein standard mixture of intermediate complexity (48 proteins) as well as the determination of the in vivo deamidation rate of histone H1.0 directly in a crude nuclear protein fraction. Besides these well-known modifications citrullination, a post-translational modification which changes the positively charged guanidinium group of arginine to the uncharged ureido group of citrulline, was investigated. Applying CE-MS for fast and sensitive analyses of various post-translational modifications of intact and enzymatically digested histone H4, we were able to detect a variety of citrullinated proteoforms. MS/MS analysis with electron transfer dissociation (ETD) fragmentation identified the presence of deiminated Arg at position 3 and 17 of histone H4. Moreover, based on CE-MS, isobaric mono-phosphorylated peptides obtained in the course of a kinase activity study were separated and individual positional isomers quantified.