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Amino groups in proteins can react with aldehyde groups in aldoses or keto groups in ketoses, e.g., d-glucose and d-fructose, yielding Schiff bases that rearrange to more stable Amadori and Heyns products, respectively. Analytical strategies to identify and quantify each glycation product in the presence of the corresponding isomer are challenged by similar physicochemical properties, impeding chromatographic separations, and by identical masses including very similar fragmentation patterns in tandem mass spectrometry. Thus, we studied the separation of seven peptide families, each consisting of unmodified, glucated, and fructated 15mer to 22mer peptides using reversed-phase (RP) and hydrophilic interaction chromatography (HILIC). In RP-HPLC using acidic acetonitrile gradients, unglycated peptides eluted ~ 0.1 to 0.8 min after the corresponding glycated peptides with four of seven peptides being baseline separated. Isomeric glucated and fructated peptides typically coeluted, although two late-eluting peptides were partially separated. Neutral eluents (pH 7.2) improved the chromatographic resolution (Rs), especially in the presence of phosphate, providing good and often even baseline separations for six of the seven isomeric glycated peptide pairs with fructated peptides eluting earlier (Rs = 0.7 to 1.5). Some glucated and unmodified peptides coeluted, but they can be distinguished by mass spectrometry. HILIC separated glycated and unmodified peptides well, whereas glucated and fructated peptides typically coeluted. In conclusion, HILIC efficiently separated unmodified and the corresponding glycated peptides, while isomeric Amadori and Heyns peptides were best separated by RP-HPLC using phosphate buffered eluents.

Objectives: Klebsiella pneumoniae is an emerging invasive pathogen in humans and pigs. Resistance against multiple antibiotics in this species is a major health concern and the development of new antibiotics is urgently needed. The objective of this study was to investigate the effects of proline-rich antimicrobial peptides (PrAMPs) on the survival of K. pneumoniae strains in porcine blood. Methods: We established a bactericidal assay with K. pneumoniae in fresh blood drawn from 4-week-old piglets. PrAMPs, namely the apidaecins Api137 and Api802 as well as the oncocin Onc112, were added to ex vivo-infected whole blood samples in order to study their bactericidal effects and, in the case of Api137, also immune responses. Results: A porcine invasive and a human iucA+rmpA+ K. pneumoniae strain showed prominent proliferation in porcine blood. Application of Api137 resulted in a dose-dependent prominent bactericidal effect killing the invasive porcine K. pneumoniae strain. Addition of 8 μg/mL Api137 also resulted in complete killing of the human iucA+rmpA+ strain. Cytotoxicity, haemolysis and induction of the pro-inflammatory cytokine tumour necrosis factor-alpha (TNFα) in K. pneumoniae-infected porcine blood treated with Api137 was comparable with values obtained after application of 10 μg/mL cefquinome. Conclusion: We describe a new non-rodent model for invasive K. pneumoniae bacteraemia and present promising data for the PrAMP Api137 for the control of infection with hypervirulent K. pneumoniae strains.

d-glucose and d-fructose present in blood, tissues, and organs of all mammals can react with amino groups, leading to glucated (Amadori) and fructated (Heyns) products, i.e., proteins glycated at lysine residues. While typically present at low concentration in humans, metabolic diseases including diabetes elevate sugar levels, favoring glycation and consecutive reactions leading to advanced glycation end products (AGEs) linked to diabetic complications and cardiovascular diseases. Analytical methods able to differentiate and to individually quantify Amadori- and Heyns-modified proteins in complex sample mixtures, e.g., serum, are still very limited. Here, we show that the reported and supposedly specific neutral losses displayed in tandem mass spectra of Heyns peptides cannot be used for a reliable differentiation as they were also observed for Amadori peptides. However, the combination of several neutral loss signals in fragment ion ratios at both precursor and fragment ion signals allowed the differentiation and relative quantitation of coeluting isomeric Amadori and Heyns peptides at different concentrations and peptide ratios. This was also true for digested human plasma. Thus, the presented strategy allows the quantitation of Amadori and Heyns peptides in complex samples, especially by spiking isotope-labeled peptides. This will allow searching for glucated and fructated biomarkers in clinical samples.

The rapid development, approval, and production of vaccines against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in less than 1 year after the first reports of a new infectious disease was a real game changer, providing 80%–90% efficacy in preventing severe etiopathologies of the coronavirus disease 2019 (COVID-19). These vaccines induce an immune response against the SARS-CoV-2 spike (S) protein located on the surface of the virus particle. Antibodies (Abs) recognizing the S-protein can inhibit binding of the virus via the S-protein to the angiotensin-converting enzyme-2 (ACE-2) receptor expressed on different human cells, especially when these Abs bind to the interaction site, the so-called receptor-binding domain (RBD). We have expressed the RBDs of wild-type SARS-CoV-2 and five variants of concern (VOCs) to test the immune response in people before vaccination with mRNA vaccines BNT162b2 and mRNA-1273 and after up to three vaccinations using in-house ELISA and inhibition assays. The methods of both assays are provided. Both vaccines initiated similarly high IgG titers after two vaccinations against the wild-type and even two VOC-RBDs (alpha and delta) and strongly inhibited the corresponding RBD-ACE-2 binding. The IgG titers and inhibition of ACE-2 binding were lower for beta and gamma RBDs and much lower for omicron RBD. The third vaccination after 6 months strongly increased both the IgG titers and the neutralizing effect against all variants, especially for omicron, leading to 63% ± 13% neutralization potential. Importantly, neutralization linearly increased with the IgG titers.

Background The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered the worldwide coronavirus disease 2019 (COVID-19) pandemic. Serological assays for the detection of SARS-CoV-2 infections are important to understand the immune response in patients and to obtain epidemiological data about the number of infected people, especially to identify asymptomatic persons not aware of a past infection. Methods We recombinantly produced SARS-CoV-2 nucleocapsid (N)-protein in Escherichia coli. We used the purified protein to develop an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of SARS-CoV-2 specific antibodies. This ELISA method was optimized and validated with serum samples collected from 113 patients with RT-PCR-confirmed SARS-CoV-2 infections including hospitalized COVID-19 patients and 1500 control sera mostly collected before 2015 with different clinical background. Results The optimized N-protein-ELISA provided a sensitivity of 89.7% (n = 68) for samples collected from patients with confirmed SARS-CoV-2 infections and mild to severe symptoms more than 14 days after symptom onset or a positive PCR test. The antibody levels remained low for serum samples collected in the first six days (n = 23) and increased in the second week (n = 22) post symptom onset or PCR confirmation. At this early phase, the ELISA provided a sensitivity of 39.1% and 86.4%, respectively, reflecting the time of an IgG immune response against pathogens. The assay specificity was 99.3% (n = 1500; 95% CI 0.995–0.999). Serum samples from persons with confirmed antibody titers against human immunodeficiency viruses 1/2, parvovirus B19, hepatitis A/B virus, cytomegalovirus, Epstein Barr virus, and herpes simplex virus were tested negative. Conclusions We conclude that the N-protein-based ELISA developed here is well suited for the sensitive and specific serological detection of SARS-CoV-2 specific IgG antibodies in human serum for symptomatic infections. It may also prove useful to identify previous SARS-CoV-2 infections in vaccinated people, as all currently approved vaccines rely on the SARS-CoV-2 spike (S-) protein.

Background Colorectal cancer, one of the most common malignancies worldwide, is associated with a high mortality rate, mainly caused by metastasis. Comparative metagenome-wide association analyses of healthy individuals and cancer patients suggest a role for the human intestinal microbiota in tumor progression. However, the microbial molecules involved in host-microbe communication are largely unknown, with current studies mainly focusing on short-chain fatty acids and amino acid metabolites as potential mediators. Quorum sensing peptides are not yet considered in this context since their presence in vivo and their ability to affect host cells have not been reported so far. Results Here, we show that EntF*, a metabolite of the quorum sensing peptide EntF produced by Enterococcus faecium, is naturally present in mice bloodstream. Moreover, by using an orthotopic mouse model, we show that EntF* promotes colorectal cancer metastasis in vivo, with metastatic lesions in liver and lung tissues. In vitro tests suggest that EntF* regulates E-cadherin expression and consequently the epithelial-mesenchymal transition, via the CXCR4 receptor. In addition, alanine-scanning analysis indicates that the first, second, sixth, and tenth amino acid of EntF* are critical for epithelial-mesenchymal transition and tumor metastasis. Conclusion Our work identifies a new class of molecules, quorum sensing peptides, as potential regulators of host-microbe interactions. We prove, for the first time, the presence of a selected quorum sensing peptide metabolite in a mouse model, and we demonstrate its effects on colorectal cancer metastasis. We believe that our work represents a starting point for future investigations on the role of microbiome in colorectal cancer metastasis and for the development of novel bio-therapeutics in other disease areas.

Sarcopenia-the accelerated loss of muscle mass, strength and function with ageing-represents an important health challenge with reduced quality of life and increased mortality. Gut microbiota has been suggested to contribute to this age-associated muscle wasting but the underlying mechanisms are still unclear. Here, we uncover the quorum sensing peptide iAM373 as a hitherto unknown contributor to sarcopenia.

ObjectivesTo investigate the impact on efficiency and quality of preprostatectomy multidisciplinary therapy conferences (MDT) at Karolinska University Hospital related to the use of a digital solution compared with standard of care. Further, to explore whether gains in MDT efficiency and quality impact oncological or functional patient outcomes.MethodsWe conducted a prospective, observational study of preoperative prostate cancer MDT at Karolinska between February 2017 and March 2021, including 1329 patients. We compared efficiency and quality of the standard MDT and the MDT using the digital solution IntelliSpace Precision Medicine Multidisciplinary Team Orchestrator (ISPM) based on the previously used MDT-MODe approach. Clinical and patient-reported functional outcomes were derived from the medical records and the Swedish National Prostate Cancer Register.ResultsWhile ISPM was used during the MDT meeting, the time spent per patient was reduced by 24% (pDiscussion and conclusionTo increase the value of the MDT, all data relevant for treatment decision need to be purposefully presented and compiled, which also enables secondary use of the data.The use of a digital solution during preoperative MDTs for prostate cancer decision making at Karolinska University Hospital improved the efficiency and quality of this multidisciplinary team meeting without impacting patient outcomes.

Cryptococcus neoformans, an opportunistic fungal pathogen ubiquitously present in the environment, causes cryptococcal meningitis (CM) mainly in immunocompromised patients, such as AIDS patients. We aimed to identify disease-associated cryptococcal protein antigens targeted by the human humoral immune response. Therefore, we used sera from Colombian CM patients, with or without HIV infection, and from healthy individuals living in the same region. Serological analysis revealed increased titers of anti-cryptococcal IgG in HIV-negative CM patients, but not HIV-positive CM patients, compared to healthy controls. In contrast, titers of anti-cryptococcal IgM were not affected by CM. Furthermore, we detected pre-existing IgG and IgM antibodies even in sera from healthy individuals. The observed induction of anti-cryptococcal IgG but not IgM during CM was supported by analysis of sera from C. neoformans-infected mice. Stronger increase in IgG was found in wild type mice with high lung fungal burden compared to IL-4Rα-deficient mice showing low lung fungal burden. To identify the proteins targeted by human anti-cryptococcal IgG antibodies, we applied a quantitative 2D immunoproteome approach identifying cryptococcal protein spots preferentially recognized by sera from CM patients or healthy individuals followed by mass spectrometry analysis. Twenty-three cryptococcal proteins were recombinantly expressed and confirmed to be immunoreactive with human sera. Fourteen of them were newly described as immunoreactive proteins. Twelve proteins were classified as disease-associated antigens, based on significantly stronger immunoreactivity with sera from CM patients compared to healthy individuals. The proteins identified in our screen significantly expand the pool of cryptococcal proteins with potential for (i) development of novel anti-cryptococcal agents based on implications in cryptococcal virulence or survival, or (ii) development of an anti-cryptococcal vaccine, as several candidates lack homology to human proteins and are localized extracellularly. Furthermore, this study defines pre-existing anti-cryptococcal immunoreactivity in healthy individuals at a molecular level, identifying target antigens recognized by sera from healthy control persons.

Increased PSA-based screening for prostate cancer has resulted in a growing number of diagnosed cases. However, around half of these are ‘indolent’, neither metastasizing nor leading to disease specific death. Treating non-progressing tumours with invasive therapies is currently regarded as unnecessary over-treatment with patients being considered for conservative regimens, such as active surveillance (AS). However, this raises both compliance and protocol issues. Great clinical benefit could accrue from a biomarker able to predict long-term patient outcome accurately at the time of biopsy and initial diagnosis. Here we delineate the translation of a laboratory discovery through to the precision development of a clinically validated, novel prognostic biomarker assay (InformMDx™). This centres on determining transcript levels for phosphodiesterase-4D7 (PDE4D7), an enzyme that breaks down cyclic AMP, a signalling molecule intimately connected with proliferation and androgen receptor function. Quantifiable detection of PDE4D7 mRNA transcripts informs on the longitudinal outcome of post-surgical disease progression. The risk of post-surgical progression increases steeply for patients with very low ‘PDE4D7 scores’, while risk decreases markedly for those patients with very high ‘PDE4D7 scores’. Combining clinical risk variables, such as the Gleason or CAPRA (Cancer of the Prostate Risk Assessment) score, with the ‘PDE4D7 score’ further enhances the prognostic power of this personalized, precision assessment. Thus the ‘PDE4D7 score’ has the potential to define, more effectively, appropriate medical intervention/AS strategies for individual prostate cancer patients.

Aims High levels of glucose and reactive carbonyl intermediates of its degradation pathway such as methylglyoxal (MG) may contribute to diabetic complications partly via increased generation of reactive oxygen species (ROS). This study focused on glutathione peroxidase-1 (GPx1) expression and the impact of carbonylation as an oxidative protein modification on GPx1 abundance and activity in human umbilical vein endothelial cells (HUVEC) under conditions of mild to moderate oxidative stress. Results High extracellular glucose and MG enhanced intracellular ROS formation in HUVECs. Protein carbonylation was only transiently augmented pointing to an effective antioxidant defense in these cells. Nitric oxide synthase expression was decreased under hyperglycemic conditions but increased upon exposure to MG, whereas superoxide dismutase expression was not significantly affected. Increased glutathione peroxidase (GPx) activity seemed to compensate for a decrease in GPx1 protein due to enhanced degradation via the proteasome. Mass spectrometry analysis identified Lys-114 as a possible carbonylation target which provides a vestibule for the substrate H2O2 and thus enhances the enzymatic reaction. Innovation Oxidative protein carbonylation has so far been associated with functional inactivation of modified target proteins mainly contributing to aging and age-related diseases. Here, we demonstrate that mild oxidative stress and subsequent carbonylation seem to activate protective cellular redox signaling pathways whereas severe oxidative stress overwhelms the cellular antioxidant defense leading to cell damage. Conclusions This study may contribute to a better understanding of redox homeostasis and its role in the development of diabetes and related vascular complications., Graphical abstract fx1

Glycation and glycoxidation of proteins and peptides have been intensively studied and are considered as reliable diagnostic biomarkers of hyperglycemia and early stages of type II diabetes. However, glucose can also react with primary amino groups present in other cellular components, such as aminophospholipids (aminoPLs). Although it is proposed that glycated aminoPLs can induce many cellular responses and contribute to the development and progression of diabetes, the routes of their formation and their biological roles are only partially revealed. The same is true for the influence of glucose-derived modifications on the biophysical properties of PLs. Here we studied structural, signaling, and biophysical properties of glycated and glycoxidized phosphatidylethanolamines (PEs). By combining high resolution mass spectrometry and nuclear magnetic resonance spectroscopy it was possible to deduce the structures of several intermediates indicating an oxidative cleavage of the Amadori product yielding glycoxidized PEs including advanced glycation end products, such as carboxyethyl- and carboxymethyl-ethanolamines. The pro-oxidative role of glycated PEs was demonstrated and further associated with several cellular responses including activation of NFκB signaling pathways. Label free proteomics indicated significant alterations in proteins regulating cellular metabolisms. Finally, the biophysical properties of PL membranes changed significantly upon PE glycation, such as melting temperature (Tm), membrane surface charge, and ion transport across the phospholipid bilayer.

Advanced glycation end products (AGEs) are posttranslational modifications formed non-enzymatically from the reaction of carbohydrates and their degradation products with proteins. Accumulation of AGEs is associated with the progression of severe diabetic complications, for example, and elevated tissue levels of AGEs might even predict these pathologies. As AGE formation is often site-specific, mapping of these modification sites may reveal more sensitive and specific markers than the global tissue level. Here, 42 AGE modifications were identified in a bottom-up proteomic approach by tandem mass spectrometry, which corresponded to 36 sites in 22 high to medium abundant proteins in individual plasma samples obtained from type 2 diabetes mellitus (T2DM) patients with long disease duration (>10 years). Major modifications were glarg (11 modification sites) and carboxymethylation (5) of arginine and formylation (8), acetylation (7), and carboxymethylation (7) of lysine residues. Relative quantification of these sites in plasma samples obtained from normoglycemic individuals (n = 47) and patients with T2DM being newly diagnosed (n = 47) or of medium (2–5 years, n = 20) and long disease duration (>10 years, n = 20) did not reveal any significant differences.

Protein glycation refers to the reversible reaction between aldoses (or ketoses) and amino groups yielding relatively stable Amadori (or Heyns) products. Consecutive oxidative cleavage reactions of these products or the reaction of amino groups with other reactive substances (e.g. α-dicarbonyls) yield advanced glycation end products (AGEs) that can alter the structures and functions of proteins. AGEs have been identified in all organisms, and their contents appear to rise with some diseases, such as diabetes and obesity. Here, we report a pilot study using highly sensitive and specific proteomics approach to identify and quantify AGE modification sites in plasma proteins by reversed phase HPLC mass spectrometry in tryptic plasma digests. In total, 19 AGE modification sites corresponding to 11 proteins were identified in patients with type 2 diabetes mellitus under poor glycemic control. The modification degrees of 15 modification sites did not differ among cohorts of normoglycemic lean or obese and type 2 diabetes mellitus patients under good and poor glycemic control. The contents of two amide-AGEs in human serum albumin and apolipoprotein A-II were significantly higher in patients with poor glycemic control, although the plasma levels of both proteins were similar among all plasma samples. These two modification sites might be useful to predict long term, AGE-related complications in diabetic patients, such as impaired vision, increased arterial stiffness, or decreased kidney function.

Reactive oxygen species (ROS) can oxidize virtually all cellular components. In proteins cysteine, methionine, tryptophan, and tyrosine residues are most prone to oxidation and their oxidized forms are thus considered as biomarkers of oxidative protein damages. Ultraviolet radiation and some endogenous ROS can produce tyrosine radicals reacting with other tyrosine residues yielding intra- or intermolecular cross-links in proteins. These 3,3'-dityrosines can be quantified by their characteristic fluorescence, but analytical methods to identify the modification sites in proteins are still missing. Although mass spectrometry (MS) is routinely used to map other post-translational modifications, the analysis of dityrosines is challenged by simultaneous fragmentations of both cross-linked peptide chains producing complex tandem mass spectra. Additionally, the fragmentation patterns differ from linear peptides. Here, we studied the fragmentation behavior of dityrosine cross-linked peptides obtained by incubating three peptides (AAVYHHFISDGVR, TEVSSNHVLIYLDK, and LVAYYTLIGASGQR) with horseradish peroxidase in the presence of hydrogen peroxide. Homo- and hetero-dimerization via dityrosine was monitored by fluorescence spectroscopy and MS. The fragmentation characteristics of dityrosine-linked peptides were studied on an ESI-LTQ-Orbitrap-MS using collision induced dissociation, which allowed localizing the cross-linked positions and provided generic rules to identify this oxidative modification. When human serum albumin oxidized with 50-fold molar excess of HOCl in phosphate buffer saline was analyzed by nanoRPC-ESI-MS/MS, an automatic database search considering all possible (in-silico generated) tyrosine-containing peptides as dynamic modifications revealed four different types of oxidatively modified tyrosine residues including dityrosines linking ten different Tyr residues. The automatic database search was confirmed by manual interpretation of each tandem mass spectrum.

Background: The clinical metrics used to date to assess the progression risk of newly diagnosed prostate cancer patients only partly represent the true biological aggressiveness of the underlying disease. Objective: Validation of the prognostic biomarker phosphodiesterase-4D7 (PDE4D7) in predicting longitudinal biological outcomes in a historical surgery cohort to improve postsurgical risk stratification. Design, patients, and methods: RNA was extracted from biopsy punches of resected tumors from 550 patients. PDE4D7 was quantified using one-step quantitative reverse transcription-polymerase chain reaction. PDE4D7 scores were calculated by normalization of PDE4D7 to reference genes. Multivariate analyses were adjusted for clinical prognostic variables. Outcomes tested were: prostate-specific antigen relapse, start of salvage treatment, progression to metastases, overall mortality, and prostate cancer-specific mortality. The PDE4D7 score was combined with the clinical risk model Cancer of the Prostate Risk Assessment Postsurgical Score (CAPRA-S) using multivariate regression modeling; the combined score was tested in post-treatment progression free survival prediction. Outcome measurements and statistical analysis Correlations with outcomes were analyzed using multivariate Cox regression and logistic regression statistics. Results and limitations: The PDE4D7 score was significantly associated with time-to-prostate specific antigen failure after prostatectomy (hazard ratio [HR]: 0.53, 95% confidence interval [CI]: 0.41–0.67 for each unit increase, p < 0.0001). After adjustment for postsurgical prognostic variables the HR was 0.56 (95% CI: 0.43–0.73, p < 0.0001). The PDE4D7 score remained significant after adjusting the multi-variate analysis for the CAPRA-S model categories (HR = 0.54, 95% CI = 0.42–0.69, p < 0.0001). Combination of the PDE4D7 score with the CAPRA-S demonstrated a significant incremental value of 4–6% in 2-yr (p = 0.004) or 5-yr (p = 0.003) prediction of progression free survival after surgery. The combined model of PDE4D7 and CAPRA-S improves patient selection with very high risk of fast disease relapse after primary intervention. Conclusions: The PDE4D7 score has the potential to provide independent risk information and to restratify patients with clinical intermediate- to high-risk characteristics to a very low-risk profile. Patient summary In this report, we studied the potential of a novel biomarker to predict outcomes of a cohort of prostate cancer patients who underwent surgery more than 10 yr ago. We found that a gene called phosphodiesterase-4D7 added extra information to the available clinical data. We conclude that the measurement of this gene in tumor tissue may contribute to more effective treatment decisions.

Polo-like kinase 1 (Plk1), a validated cancer target, harbors a protein-protein interaction domain referred to as the polo-box domain (PBD), in addition to its enzymatic domain. Although functional inhibition either of the enzymatic domain or of the PBD has been shown to inhibit Plk1, so far there have been no reports of bifunctional agents with the potential to target both protein domains. Here we report the development of Plk1 inhibitors that incorporate both an ATP-competitive ligand of the enzymatic domain, derived from BI 2536, and a functional inhibitor of the PBD, based either on the small molecule poloxin-2 or on a PBD-binding peptide. Although these bifunctional agents do not seem to bind both protein domains simultaneously, the most potent compound displays low-nanomolar activity against the Plk1 PBD, with excellent selectivity over the PBDs of Plk2 and Plk3. Our data provide insights into challenges and opportunities relating to the optimization of Plk1 PBD ligands as potent Plk1 inhibitors.

Adiponectin, an adipose tissue-excreted adipokine plays protective roles in metabolic and cardiovascular diseases and exerts anti-cancer activities, partially by interfering with leptin-induced signaling. Previously we identified the active site in the adiponectin protein, and generated both a nanomolar monomeric agonist of the adiponectin receptor (10-mer ADP355) and an antagonist (8-mer ADP400) to modulate various adiponectin receptor-mediated cellular functions. As physiologically circulating adiponectin forms multimeric complexes, we also generated an agonist dimer with improved biodistribution and in vitro efficacy. In the current report, we attempted to optimize the monomeric agonist structure. Neither extension of the peptide up to 14-mer analogs nor reinstallation of native residues in permissible positions enhanced significantly the activity profile. The only substitutions that resulted in 5-10-fold improved agonistic activity were the replacement of turn-forming Gly4 and Tyr7 residues with Pro and Hyp, respectively, yielding the more active native β-sheet structure. All peptides retained good stability in human serum exhibiting half-lives >2 h. The cellular efficacy and stability rankings among the peptides followed expected structure-activity relationship trends. To investigate whether simultaneous activation of adiponectin pathways and inhibition of leptin-induced signals can result in cytostatic and anti-oncogenic signal transduction processes, we developed a chimera of the leptin receptor antagonist peptide Allo-aca (placed to the N-terminus) and ADP355 (at the C-terminus). The in vitro anti-tumor activity and intracellular signaling of the chimera were dominated by the more active Allo-aca component. The ADP355 part, however, reversed unfavorable in vivo metabolic effects of the leptin receptor antagonist.

Aims Free fatty acids (FFA) mediate adverse metabolic effects such as downregulated carbohydrate metabolisms, providing causal links between obesity and the development of type 2 diabetes mellitus (T2DM). Here, we investigated the plasma concentrations of FFA alone and in combination with protein glycation as potential diagnostic and prognostic biomarkers of T2DM. Materials and methods EDTA-plasma obtained from 48 newly diagnosed male T2DM patients, 48 long-term controlled (24 male and 24 female) T2DM patients, 20 prediabetic male T2DM patients and two age-matched control cohorts (48 non-diabetic (ND) men; 24 male and 24 female ND partipants) were analysed for a set of clinical parameters including FFA. Glycation sites were quantified after tryptic digestion using tandem mass spectrometry. Results Median plasma concentrations of FFA were almost three-fold higher in samples obtained from newly diagnosed (long-term controlled) T2DM patients than in those obtained from the control group, providing diagnostic sensitivity (SN) of 92% (85%) and specificity (SP) of 90% (88%). When combined with the glycation level of lysine-141 of haptoglobin, diagnostic accuracy improved further for newly diagnosed (SN, 94%; SP 96%) and long-term controlled (SN, 85%; SP, 94%) T2DM patients (HbA1c: SN, 88%; SP, 96%). A prospective pilot study evaluating the prognostic value revealed initially low FFA levels for pre-diabetic patients that increased in the following four years in patients whose prediabetic state worsened or who developed T2DM. Conclusions FFA levels are elevated in newly diagnosed and long-term controlled T2DM patients, providing high diagnostic accuracy of 87% and 91%, respectively, which improved further when combined with the glycation degree of lysine-141 in haptoglobin. Additionally, FFA showed higher mean fold-changes than HbA1c or FPG in subjects developing T2DM, indicating higher sensitivity towards the progression of the disease.

Background Type 2 diabetes mellitus (T2DM) is the most common lifestyle disease affecting all countries. Due to its asymptomatic onset, it is often diagnosed after irreversible vascular complications have been initiated. Therefore, specific markers characteristic for very early disease stages and suitable for early diagnostics are required. Glycation of plasma proteins, such as human serum albumin (HSA), has been often suggested as marker. However, the total glycation degree of HSA does not provide sufficient information about short-term fluctuations of blood glucose concentrations due to the large number of glycation sites. Analysis of individual modification sites might be more informative, but methods for reliable quantifications are still missing. Objective The main objective of this study was to establish and qualify a method of analysis applicable to sensitive and precise quantification of glycations sites in plasma proteins. Methods Plasma samples obtained from diabetic patients and non-diseased individuals were separated from low-molecular weight compounds, digested with trypsin, enriched for glycated peptides by boronic acid affinity chromatography (BAC), desalted by solid phase extraction (SPE), and separated by RP-HPLC coupled online to ESI-QqQ-MS. Quantification relied on multiple reaction monitoring (MRM) of multiple glycation sites identified in plasma proteins using a stable isotope dilution approach or internal standardization. Results The data presented here suggests high selectivity and precision (relative standard deviations below 10%) of the overall approach appearing to be well suited for the identification of prospective biomarkers. Six glycated peptides corresponding to different glycation sites of HSA were present in plasma samples obtained from T2DM patients at significantly higher levels than in non-diabetic men matched for age. Additionally, each of the studied glycation site of HSA appeared to be affected at different degrees. Conclusion The presented approach enables the sensitive and robust quantification of prospective T2D biomarkers promising for clinical diagnostics.

Kefir has a long tradition in human nutrition due to its presupposed health promoting effects. To investigate the potential contribution of bioactive peptides to the physiological effects of kefir, comprehensive analysis of the peptide profile was performed by nano-ESI-LTQ-Orbitrap MS coupled to nano-ultrahigh-performance liquid chromatography. Thus, 257 peptides were identified, mainly released from ß-casein, followed by ?S1-, ?-, and ?S2-casein. Most (236) peptides were uniquely detected in kefir, but not in raw milk indicating that the fermentation step does not only increase the proteolytic activity 1.7- to 2.4-fold compared to unfermented milk, but also alters the composition of the peptide fraction. The influence of the microflora was determined by analyzing kefir produced from traditional kefir grains or commercial starter culture. Kefir from starter culture featured 230 peptide sequences and showed a significantly, 1.4-fold higher proteolytic activity than kefir from kefir grains with 127 peptides. A match of 97 peptides in both varieties indicates the presence of a typical kefir peptide profile that is not influenced by the individual composition of the microflora. Sixteen of the newly identified peptides were previously described as bioactive, including angiotensin-converting enzyme (ACE)-inhibitory, antimicrobial, immunomodulating, opioid, mineral binding, antioxidant, and antithrombotic effects. Biological significance: The present study describes a comprehensive peptide profile of kefir comprising 257 sequences. The peptide list was used to identify 16 bioactive peptides with ACE-inhibitory, antioxidant, antithrombotic, mineral binding, antimicrobial, immunomodulating and opioid activity in kefir. Furthermore, it was shown that a majority of the kefir peptides were not endogenously present in the raw material milk, but were released from milk caseins by proteases of the microbiota and are therefore specific for the product. Consequently, the proteolytic activity and the composition of the peptide profile can be controlled by the applied microflora (grains or starter culture). On the other hand, a considerable portion of the peptide profile was identified to be typical for kefir in general and independent from production parameters.In summary, the generated kefir peptide profile helped to reveal its origin and to identify bioactive peptides in kefir, which may advance the understanding of health benefits of this food product. The results further indicate that subsets of the kefir peptide list can be used as markers to control food authenticity, for example, to distinguish different types of kefir. © 2015 Elsevier B.V., Türkiye Bilimsel ve Teknolojik Araştirma Kurumu: 1100797 01DL12033, The present study was funded by the Intensified Cooperation Program (IntenC) for the Promotion of German-Turkish Higher Education Research of the International Bureau of the German Federal Ministry of Education and Research (grant No. 01DL12033 ) and Turkey's Scientific and Technological Research Council (TÜBITAK, grant No. 1100797 ). The funders were not involved in study design, the collection, analysis and interpretation of data, writing the report or in the decision to submit the article for publication.

Glycation refers to a nonenzymatic post-translational modification formed by the reaction of amino groups and reducing sugars. Consecutive oxidation and degradation can produce advanced glycation end products (AGEs), such as N(ε)-(carboxyethyl)lysine (CEL) and N(ε)-(carboxymethyl)lysine (CML). Although CEL and CML are considered to be markers of arteriosclerosis, diabetes mellitus, and aging, the modified proteins and the exact modification sites are mostly unknown due to their low frequency and a lack of enrichment strategies. Here, we report characteristic fragmentation patterns of CML- and CEL-containing peptides and two modification-specific reporter ions for each modification (CML, m/z 142.1 and 187.1; CEL, m/z 156.1 and 201.1). The protocol allowed sensitive and selective precursor ion scans to detect the modified peptides in complex sample mixtures. The corresponding m/z values identified eight CEL/CML-modification sites in glycated human serum albumin (HSA) by targeted nano-RPC-MS/MS. The same strategy revealed 21 CML sites in 17 different proteins, including modified lysine residues 88 and 396 of human serum albumin, in a pooled plasma sample that was obtained from patients with type 2 diabetes mellitus.

Lipids are dynamic constituents of biological systems, rapidly responding to any changes in physiological conditions. Thus, there is a large interest in lipid-derived markers for diagnostic and prognostic applications, especially in translational and systems medicine research. As lipid identification remains a bottleneck of modern untargeted lipidomics, we developed LipidHunter, a new open source software for the high-throughput identification of phospholipids in data acquired by LC-MS and shotgun experiments. LipidHunter resembles a workflow of manual spectra annotation. Lipid identification is based on MS/MS data analysis in accordance with defined fragmentation rules for each phospholipid (PL) class. The software tool matches product and neutral loss signals obtained by collision-induced dissociation to a user-defined white list of fatty acid residues and PL class-specific fragments. The identified signals are tested against elemental composition and bulk identification provided via LIPID MAPS search. Furthermore, LipidHunter provides information-rich tabular and graphical reports allowing to trace back key identification steps and perform data quality control. Thereby, 202 discrete lipid species were identified in lipid extracts from rat primary cardiomyocytes treated with a peroxynitrite donor. Their relative quantification allowed the monitoring of dynamic reconfiguration of the cellular lipidome in response to mild nitroxidative stress. LipidHunter is available free for download at https://bitbucket.org/SysMedOs/lipidhunter .

Glucose can modify proteins in human blood, forming early glycation products (e.g., Amadori compounds), which can slowly degrade to advanced glycation endproducts (AGEs). AGEs contribute significantly to complications of diabetes mellitus and, thus, represent markers of advanced disease stages. They are, however, currently unsuitable for early diagnosis and therapeutic monitoring. Here, we report sensitive strategies to identify and relatively quantify protein glycation sites in human plasma samples obtained from type 2 diabetes mellitus (T2DM) patients and age-matched nondiabetic individuals using a bottom-up approach. Specifically, Amadori peptides were enriched from tryptic digests by boronic acid affinity chromatography, separated by reversed-phase chromatography, and analyzed on-line by high-resolution mass spectrometry. Among the 52 Amadori peptides studied here were 20 peptides resembling 19 glycation sites in six human proteins detected at statistically significantly higher levels in T2DM than in the normoglycemic controls. Four positions appeared to be unique for T2DM within the detection limit. All 19 glycation sites represent promising new biomarker candidates for early diagnosis of T2DM and adequate therapeutic control, as they may indicate early metabolic changes preceding T2DM.

Background: Isoforms of the PDE4 family of cAMP-specific phosphodiesterases (PDEs) are expressed in a cell type-dependent manner and contribute to underpinning the paradigm of intracellular cAMP signal compartmentalisation. Here we identify the differential regulation of the PDE4D7 isoform during prostate cancer progression and uncover a role in controlling prostate cancer cell proliferation. Methods: PDE4 transcripts from 19 prostate cancer cell lines and xenografts were quantified by qPCR. PDE4D7 expression was further investigated because of its significant downregulation between androgen-sensitive (AS) and androgen-insensitive (AI) samples. Western blot analysis, PDE activity assay, immunofluorescent staining and cAMP responsive FRET assays were used to investigate the sub-plasma membrane localisation of a population of PDE4D7 in VCaP (AS) and PC3 (AI) cell lines. Disruption of this localisation pattern using dominant-negative protein expression and siRNA knockdown showed that PDE4D7 acts in opposition to proliferative signalling as assessed by electrical impedance-based proliferation assays. Results: Here we identify the differential regulation of the PDE4D7 isoform during prostate cancer progression. PDE4D7 is highly expressed in AS cells and starkly downregulated in AI samples. The significance of this downregulation is underscored by our finding that PDE4D7 contributes a major fraction of cAMP degrading PDE activity tethered at the plasma membrane and that displacement of PDE4D7 from this compartment leads to an increase in the proliferation of prostate cancer cells. PDE4D7 mRNA expression is not, however, directly regulated by the androgen receptor signalling axis despite an overlapping genomic structure with the androgen responsive gene PART1. PDE4D7, which locates to the plasma membrane, acts to supress aberrant non-steroidal growth signals within the prostate or AS metastasis. Conclusions: PDE4D7 expression is significantly downregulated between AS and AI cell phenotypes. This change in expression potentially provides a novel androgen-independent biomarker and manipulation of its activity or its expression may provide therapeutic possibilities and insights into contributory aspects of the complex molecular pathology of prostate cancer.

Proline-rich antimicrobial peptides (PrAMPs) freely penetrate through the outer membrane into the periplasm of Gram-negative bacteria, before they are actively translocated by a permease/transporter-mediated uptake into the cytoplasm where they are reported to inhibit chaperone DnaK. Here we have studied the PrAMP apidaecin 1b, which is produced in honey bees in response to bacterial infections, and optimized apidaecin analogs for their bacterial uptake. The peptides were labeled with 5(6)-carboxyfluorescein and their internalization in Escherichia coli and Klebsiella pneumoniae was visualized by fluorescence microscopy and quantified by flow cytometry for four different time points over an incubation period of 4 h. Apidaecin 1b entered only 40% to 50% of the cells at detectable quantities, whereas designer peptides Api88, Api134 and Api155 entered more than 95% of the bacteria within 30 min at around fourfold higher quantities than the native peptide. Interestingly, a shortened version designated as (1-17)Api88, bound DnaK as efficiently as the 18-residue long Api88 and entered the bacteria at similar kinetics as Api88, but was unable to inhibit the bacterial growth. Similar conflicts with currently proposed mechanisms of PrAMPs were also obtained for some Ala-substituted analogs and reverse apidaecin sequences. Although peptides with C-terminal amides enter the cells much more efficiently than homologous C-terminal acids, this improved cell penetration does not improve the antibacterial activities. These studies suggest that PrAMPs utilize additional modes of action to kill sensitive organisms.

Optical tweezers-assisted dynamic force spectroscopy is employed to investigate specific receptor-ligand interactions on the level of single binding events. In particular, we analyze binding of the phosphorylation-specific monoclonal antibody (mAb) HPT-101 to synthetic tau-peptides with two potential phosphorylation sites (Thr231 and Ser235), being the most probable markers for Alzheimer's disease. Whereas the typical interpretation of enzyme-linked immunosorbent assay (ELISA) suggests that this monoclonal antibody binds exclusively to the double-phosphorylated tau-peptide, we show here by DFS that the specificity of only mAb HPT-101 is apparent. In fact, binding occurs also to each sort of monophosphorylated peptide. Therefore, we characterize the unbinding process by analyzing the measured rupture force distributions, from which the lifetime of the bond without force τ0, its characteristic length xts, and the free energy of activation ΔG are extracted for the three mAb/peptide combinations. This information is used to build a simple theoretical model to predict features of the unbinding process for the double-phosphorylated peptide purely based on data on the monophosphorylated ones. Finally, we introduce a method to combine binding and unbinding measurements to estimate the relative affinity of the bonds. The values obtained for this quantity are in accordance with ELISA, showing how DFS can offer important insights about the dynamic binding process that are not accessible with this common and widespread assay.

Proline-rich antimicrobial peptides (PrAMPs) from insects and mammals have recently been evaluated for their pharmaceutical potential in treating systemic bacterial infections. Besides the native peptides, several shortened, modified, or even artificial sequences were highly effective in different murine infection models. Most recently, we showed that the 18-residue-long peptide Api88, an optimized version of apidaecin 1b, was efficient in two different animal infection models using the pathogenic Escherichia coli strains ATCC 25922 and Neumann, with a promising safety margin. Here, we show that Api88 is degraded relatively fast upon incubation with mouse serum, by cleavage of the C-terminal leucine residue. To improve its in vitro characteristics, we aimed to improve its serum stability. Replacing the C-terminal amide by the free acid or substituting Arg-17 with l -ornithine or l -homoarginine increased the serum stabilities by more than 20-fold (half-life, ∼4 to 6 h). These analogs were nontoxic to human embryonic kidney (HEK 293), human hepatoma (HepG2), SH-SY5Y, and HeLa cells and nonhemolytic to human erythrocytes. The binding constants of all three analogs with the chaperone DnaK, which is proposed as the bacterial target of PrAMPs, were very similar to that of Api88. Of all the analogs tested, Api137 (Gu-ONNRPVYIPRPRPPHPRL; Gu is N , N , N ′, N ′-tetramethylguanidino) appeared most promising due to its high antibacterial activity, which was very similar to Api88. Positional alanine and d -amino acid scans of Api137 indicated that substitutions of residues 1 to 13 had only minor effects on the activity against an E. coli strain, whereas substitutions of residues 14 to 18 decreased the activity dramatically. Based on the significantly improved resistance to proteolysis, Api137 appears to be a very promising lead compound that should be even more efficient in vivo than Api88.

Thermal treatment preserves the microbiological safety of milk, but also induces Maillard reactions modifying for example proteins. The purpose of this study was evaluating the influence of consumer behaviors (storage and heating) on protein glycation degrees in bovine milk products. Lactosylation and hexosylation sites were identified in ultra-high temperature (UHT), lactose-free pasteurized, and lactose-free UHT milk (ULF) and infant formula (IF) using tandem mass spectrometry (electron transfer dissociation). Overall, 303 lactosylated and 199 hexosylated peptides were identified corresponding to 170 lactosylation (31 proteins) and 117 hexosylation sites (25 proteins). In quantitative terms, storage increased lactosylation up to fourfold in UHT and IF and hexosylation up to elevenfold in ULF and threefold in IF. These levels increased additionally twofold when the stored samples were heated (40°C). In conclusion, storage and heating appear to influence protein glycation levels in milk at similar or even higher degrees than industrial processing.

We report on quality control performed in the context of a large-scale, multi-institutional study of the immune response in blood samples from prostate cancer patients. The measurements were performed by two commercially available techniques/services: protein arrays and an automated bead-based ELISA-like technique on 871 patient samples. The project started with a wide screen using standard arrays with 8302 protein sequences for 113 patients, followed by three studies using custom arrays with 215 selected protein sequences. These studies were followed up by three studies using the bead-based approach on 57 protein sequences chosen from the 215 selected before. We find similar responses in plasma and serum samples. Samples from the two European projects from which the samples originated also appeared comparable. In the data from the high-density standard arrays, we see for ~12% of the protein sequences high cross-correlation (R(2) > 0.8) with signals from unrelated protein sequences that are physically nearby on the array, suggesting production issues. The custom array and bead-based techniques both have good reproducibility, but the techniques do not agree with each other for ~50% of the protein sequences measured. We discuss the consequences of the observed data quality for the design and interpretation of the study.

The emergence of multiple-drug-resistant (MDR) bacterial pathogens in hospitals (nosocomial infections) presents a global threat of growing importance, especially for Gram-negative bacteria with extended spectrum β-lactamase (ESBL) or the novel New Delhi metallo-β-lactamase 1 (NDM-1) resistance. Starting from the antibacterial peptide apidaecin 1b, we have optimized the sequence to treat systemic infections with the most threatening human pathogens, such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. The lead compound Api88 enters bacteria without lytic effects at the membrane and inhibits chaperone DnaK at the substrate binding domain with a K(D) of 5 μmol/L. The Api88-DnaK crystal structure revealed that Api88 binds with a seven residue long sequence (PVYIPRP), in two different modes. Mice did not show any sign of toxicity when Api88 was injected four times intraperitoneally at a dose of 40 mg/kg body weight (BW) within 24 h, whereas three injections of 1.25 mg/kg BW and 5 mg/kg BW were sufficient to rescue all animals in lethal sepsis models using pathogenic E. coli strains ATCC 25922 and Neumann, respectively. Radioactive labeling showed that Api88 enters all organs investigated including the brain and is cleared through both the liver and kidneys at similar rates. In conclusion, Api88 is a novel, highly promising, 18-residue peptide lead compound with favorable in vitro and in vivo properties including a promising safety margin.

Reactive oxygen species (ROS) can oxidize proteins at almost any amino acid residue. Whereas some modifications are reversible within the cells, the higher oxidation states are especially irreversible. These irreversible post translational modifications are widely used as biomarkers of oxidative stress, such as protein carbonylation, which refers to aldehydes, ketones and lactams as ‘reactive carbonyl groups’. This study relied on a set of synthetic peptides containing a C-terminal aldehyde (arginal) or modification with pyruvic acid (ketone) or 4-hydroxynonenal (aldehyde) at lysine or histidine residues, as well as peptides containing pyroglutamic acid (oxidation product of proline) and 2-amino-3-butyric acid (oxidation product of threonine). The carbonylation sites were specifically derivatized with 2,4-dinitrophenylhydrazine (DNPH) and the fragmentation behavior of the products investigated in electrospray ionization (ESI-) MS. Importantly, the DNPH-labeled carbonylated peptides showed favorable ionization behaviors in negative ion mode ESI, providing a sensitive detection method. Regular peptides were mostly discriminated under these conditions. Among the fragmentation techniques tested for the negatively charged ions, pulsed Q dissociation provided three diagnostic ions at m/z values 152.0, 163.1 and 179.0, specific for DNPH-modified peptides. These marker ions were successfully applied to detect the carbonylated model peptides in a spiked tryptic digest of bovine serum albumin and a complex protein mixture obtained from HeLa cells.

The proline-rich antimicrobial peptide oncocin is remarkably active in vitro against a number of important Gram-negative bacteria of concern to humans owing to their increasing resistance to antibiotics, i.e. Enterobacteriaceae ( Escherichia coli , Klebsiella pneumoniae and Enterobacter cloacae ) and non-fermenting species ( Acinetobacter baumannii and Pseudomonas aeruginosa ). Degradation of oncocin in mouse serum was investigated in this study. Several approaches to stabilise the main cleavage sites (C-terminal to Arg-15 and N-terminal to Arg-19) by substituting either or both arginine (Arg) residues with non-proteinogenic amino acids, i.e. α-amino-3-guanidino-propionic acid, homoarginine, nitro-arginine, N -methyl-arginine, β-homoarginine, d-arginine (d-Arg) or ornithine (Orn), were tested. These modifications were found to increase the half-life of oncocin in full mouse serum. For oncocin with two Orn residues in positions 15 and 19, the half-life in full serum increased from 25min to 3h. An increase of >8h was observed for oncocin with two d-Arg residues at these same positions. The antibacterial activities of these modified sequences were slightly better than the original oncocin sequence. Moreover, the three most stable analogues were found to be bactericidal against E. coli and were not toxic to HeLa cells or haemolytic to human erythrocytes.

Leptin, a multifunctional hormone, controls various processes in both the central nervous system and in peripheral tissues. Because of the presence of multiple leptin/receptor (ObR) interaction sites and diverse leptin activities, the literature lacks truly monofunctional leptin protein derivatives or fragments. To date, selective ObR antagonists have not been reported. We developed short, pharmacologically advantageous peptide analogs of ObR-binding site III of leptin that acted as selective ObR inhibitors without any partial agonistic activity. These reduced leptin-dependent growth and signaling in cancer cell lines at picomolar and low nanomolar concentrations. In immunocompromised mice the peptides suppressed the growth of rapidly proliferating orthotopic human breast cancer xenografts by 50% when administered either intraperitoneally (i.p.) or subcutaneously (s.c.) for 38 days at a 0.1 mg/kg/day dose. The peptides were distributed to the brain, and when added to growing C57BL/6 normal mice i.p., s.c., or orally, the lead antagonist accelerated normal weight increase without producing any toxic effects. Weight gain increases could not be observed after 10–12 days of treatment indicating that the mice became resistant to the central nervous system activity of leptin antagonists. However, in normal growing rats the intranasal administration at 0.1 mg/kg/day for 20 days resulted in a 2% net total body weight gain without signs of resistance induction. In addition to the potential of these peptides in drug development against primary and metastatic tumors and cachexia, our data confirm that resistance to leptin resides at the blood-brain barrier. © 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 96: 117–125, 2011.

The increasing prevalence of antibiotic-resistant bacteria is becoming a public health crisis. Antimicrobial peptides (AMPs) are a promising solution, because bacterial resistance is less likely. Quartz crystal microbalance with dissipation monitoring (QCM-D) is a versatile and valuable technique for investigation of these peptides. This article looks at the different approaches to the interpretation of QCM-D data, showing how to extract the maximum information from the data. Five AMPs of diverse charge, length and activity are used as case studies: caerin 1.1 wild-type, two caerin 1.1 mutants (Gly15Gly19-caerin 1.1 and Ala15Ala19-caerin 1.1), aurein 1.2 and oncocin. The interaction between the AMP and a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membrane is analysed inter alia using frequency-dissipation plots (∆f-∆D plots) to ascertain the mechanism of action of the AMP. The ∆f-∆D plot can then be used to provide a fingerprint for the AMP-membrane interaction. Building up a database of these fingerprints for all known AMPs will enable the relationship between AMP structure and membrane activity to be better understood, hopefully leading to the future development of antibiotics without bacterial resistance.

The separation of isomeric phosphorylated peptides is challenging and often impossible for multiphosphorylated isomers using chromatographic and capillary electrophoretic methods. In this study we investigated the separation of a set of single-, double-, and triple-phosphorylated peptides (corresponding to the human tau protein) by ion-pair reversed-phase chromatography (IP-RPC) and hydrophilic interaction chromatography (HILIC). In HILIC both hydroxyl and aminopropyl stationary phases were tested with aqueous acetonitrile in order to assess their separation efficiency. The hydroxyl phase separated the phosphopeptides very well from the unphosphorylated analogue, while on the aminopropyl phase even isomeric phosphopeptides attained baseline separation. Thus, up to seven phosphorylated versions of a given tau domain were separated. Furthermore, the low concentration of an acidic ammonium formate buffer allowed an online analysis with electrospray ionization tandem mass spectrometry (ESI-MS/MS) to be conducted, enabling peptide sequencing and identification of phosphorylation sites.

Glycation (or non-enzymatic glycosylation) is a common non-enzymatic covalent modification of human proteins. Glucose, the highest concentrated monosaccharide in blood, can reversibly react with amino groups of proteins to form Schiff bases that can rearrange to form relatively stable Amadori products. These can be further oxidized to advanced glycation end products (AGEs). Here, we analyzed the glycation patterns of human serum albumin (HSA) in plasma samples obtained from five patients with type 2 diabetes mellitus. Therefore, glycated peptides from a tryptic digest of plasma were enriched with m-aminophenylboronic acid (mAPBA) affinity chromatography. The glycated peptides were then further separated in the second dimension by RP-HPLC coupled on-line to an electrospray ionization (ESI) tandem mass spectrometer (MS/MS). Altogether, 18 Amadori peptides, encompassing 40% of the HSA sequence, were identified. The majority of the peptides were detected and relatively quantified in all five samples with a high reproducibility among the replicas. Eleven Lys-residues were glycated at similar quantities in all samples, with glycation site Lys(549) (K(Am(Glc))QTALVELVK) being the most abundant. In conclusion, the established mAPBA/nanoRP-HPLC-ESI-MS/MS approach could reproducibly identify and quantify glycation sites in plasma samples, potentially useful in diagnosis and therapeutic control.

Alzheimer's disease (AD) is the most common form of dementia. Neuropathologically, it is characterized by two major hallmarks: neurofibrillary tangles (NFT) formed from hyperphosphorylated versions of the tau-protein, and neuritic plaques (NP) containing mostly beta-amyloid peptides (A beta) that are formed from the amyloid precursor protein (APP) by enzymatic cleavage. Despite much progress in recent years, the causes of sporadic (i.e., nonfamiliar) AD are still unclear and its valid diagnosis still relies on autopsy. Clinically used biomarkers present in cerebrospinal fluid (CSF), that is, unphosphorylated or phosphorylated tau and A beta-peptides of different lengths, lack the necessary specificity and sensitivity. Here, we describe a novel strategy to characterize tau versions present in CSF with respect to their molecular mass and isoelectric point. Aliquots of 1 mL CSF (i.e., 700 to 1300 pg tau) from nondemented persons and histopathologically confirmed AD cases were depleted for six dominant proteins, separated by two-dimensional gel electrophoresis, and then electro-transferred onto PVDF-membranes. Tau was detected with monoclonal antibody (mAb) HT7 conjugated with horseradish peroxidase (HRP). In this way, a complex tau pattern was identified in CSF that was very similar to the tau preparations from autopsy brain samples. The presented strategy enables the analysis of the phosphorylation and processing status of tau in CSF samples from healthy people and patients diagnosed with different neurological disorders. This more-detailed information on circulating tau versions and their clearance rates may facilitate the development of new diagnostic tools.

A separation of mono, doubly and triply phosphorylated isomers was developed with CZE with an aqueous electrolyte containing 3.9 mol/L formic acid and 30% v/v trifluoroethanol. Thus a mixture of ten phosphopeptides corresponding to the human tau sequence 226-240 was separated within 70 min. Although peptides with different phosphorylation degrees, i.e. 0-3 phosphate groups, were well separated, some of the phosphopeptide isomers containing one or two phosphate groups were only partially separated. The electrolyte system is compatible with both MALDI- and ESI-MS, allowing a direct coupling, and thus could have some interesting applications in proteomics.

The receptor for advanced glycated end products (RAGE) is a multiligand receptor that is implicated in the pathogenesis of various diseases, including diabetic complications, neurodegenerative disorders, and inflammatory responses. The ability of RAGE to recognize advanced glycated end products (AGEs) formed by nonenzymatic glycoxidation of cellular proteins places RAGE in the category of pattern recognition receptors. The structural mechanism of AGE recognition was an enigma due to the diversity of chemical structures found in AGE-modified proteins. Here, using NMR spectroscopy we showed that the immunoglobulin V-type domain of RAGE is responsible for recognizing various classes of AGEs. Three distinct surfaces of the V domain were identified to mediate AGE-V domain interactions. They are located in the positively charged areas of the V domain. The first interaction surface consists of strand C and loop CC ', the second interaction surface consists of strand C ', strand F, and loop FG, and the third interaction surface consists of strand A ' and loop EF. The secondary structure elements of the interaction surfaces exhibit significant flexibility on the ms-micros time scale. Despite highly specific AGE-V domain interactions, the binding affinity of AGEs for an isolated V domain is low, approximately 10 microm. Using in-cell fluorescence resonance energy transfer we show that RAGE is a constitutive oligomer on the plasma membrane. We propose that constitutive oligomerization of RAGE is responsible for recognizing patterns of AGE-modified proteins with affinities less than 100 nm.

Pseudomonas aeruginosa is an opportunistic human pathogen that can cause a wide range of clinical symptoms and infections that are frequent in immunocompromised patients. In this study, we show that P. aeruginosa evades human complement attack by binding the human plasma regulators Factor H and Factor H-related protein-1 (FHR-1) to its surface. Factor H binds to intact bacteria via two sites that are located within short consensus repeat (SCR) domains 6–7 and 19–20, and FHR-1 binds within SCR domain 3–5. A P. aeruginosa Factor H binding protein was isolated using a Factor H affinity matrix, and was identified by mass spectrometry as the elongation factor Tuf. Factor H uses the same domains for binding to recombinant Tuf and to intact bacteria. Factor H bound to recombinant Tuf displayed cofactor activity for degradation of C3b. Similarly Factor H bound to intact P. aeruginosa showed complement regulatory activity and mediated C3b degradation. This acquired complement control was rather effective and acted in concert with endogenous proteases. Immunolocalization identified Tuf as a surface protein of P. aeruginosa. Tuf also bound plasminogen, and Tuf-bound plasminogen was converted by urokinase plasminogen activator to active plasmin. Thus, at the bacterial surface Tuf acts as a virulence factor and binds the human complement regulator Factor H and plasminogen. Acquisition of host effector proteins to the surface of the pathogen allows complement control and may facilitate tissue invasion.

The detection and identification of O-phosphorylation sites in proteins with mass spectrometry remains a challenge. A common approach to analyse these modifications is to enrich phosphopeptides by immobilized metal affinity chromatography (IMAC) prior to mass spectrometric analysis. In this study two commercially available IMAC kits based on Fe(III)-ions immobilized on magnetic beads and Ga(III)-ions immobilized on a chelate-resin, have been investigated and the binding efficiency of peptide mixtures containing non-phosphorylated, singly, doubly and triply phosphorylated peptides have been tested.

The opportunistic Gram-negative bacterium Pseudomonas aeruginosa has a low susceptibility to common antibiotics. Additionally, around 15% of all clinical isolates bear acquired resistance genes. Thus, the development of new antibiotics to combat this pathogen in pneumonia, urinary tract infections, and bacteremia, represents an urgent task. The activity spectrum of the proline-rich antimicrobial peptide apidaecin 1b, originally isolated from honeybees (Apis mellifera), was extended in previous studies to further human pathogens including P. aeruginosa. However, the in vitro activity of the optimized peptide Api137 is limited to diluted medium conditions. Thus, we synthesized 323 analogs of Api137 on cellulose membranes using the SPOT strategy by substituting each residue individually by 19 other amino acids or deleting the residue. The peptides were deprotected with trifluoroacetic acid and cleaved with aqueous trimethylamine as C-terminal acids providing around 30 μg crude peptide per spot. This amount allowed determining the minimal inhibitory concentrations in a microdilution broth assay. The most promising substitutions were selected to synthesize 44 doubly and triply substituted Api137 analogs on the membrane. The 19 best peptides were synthesized at a larger scale and purified. Eight triply substituted Api137 analogs were up to 16-fold more active against P. aeruginosa at high medium concentrations without losing activities against Klebsiella pneumoniae and Acinetobacter baumannii and only slightly against Escherichia coli. The eight most active Api137 analogs were non-hemolytic to human erythrocytes and non-toxic to HeLa cells.

The identification of lead molecules against multidrug-resistant bacteria ensuing the development of novel antimicrobial drugs is an urgent task. Proline-rich antimicrobial peptides are highly active in vitro and in vivo, but only against a few Gram-negative human pathogens, with rather weak activities against Pseudomonas aeruginosa and Staphylococcus aureus. This reduced level of efficacy could be related to inadequate uptake mechanisms or structural differences of the intracellular target proteins, i.e., the 70S ribosome or chaperone DnaK. Here we synthesized peptide arrays on cellulose membranes using cleavable linkers to release the free individual peptides for further antimicrobial tests. Thus, a library of singly substituted oncocin analogs was produced by replacing each residue by all other 19 canonical amino acids yielding a set of 361 individual peptides to be evaluated against a luminescent P. aeruginosa strain. Thirteen substitutions appeared promising and their improved antibacterial activities were confirmed for different bacteria after larger scale synthesis of these analogs. By combining two favorable substitutions into one peptide, we finally obtained an oncocin analog that was ten times more active against P. aeruginosa and even 100-fold more active against S. aureus than the original oncocin, providing minimal inhibitory concentrations of 4-8 and 0.5 µg/mL, respectively.

Neurofibrillary tangles, which represent a major pathological hallmark in Alzheimer's disease (AD), are deposits of the hyperphosphorylated microtubule-associated tau protein (PHF-tau). However, a link between the phosphorylation pattern and the cause or the progress of AD is still missing. The work reported here focused on PHF-tau specific local phosphorylation patterns at Thr212/Ser214 and Thr231/Ser235 using monoclonal antibodies (mAb) generated against correspondingly modified peptides. The binding motifs of the obtained six mAbs were characterized with non-, mono-, and double-phosphorylated peptides as well as terminally shortened sequences. Five mAbs stained neurofibrillary tangles, neuritic plaques, and neuropil threads from autoptic brains of AD cases. Four mAbs recognized PHF-tau without significant cross-reactivity towards normal human tau, bovine tau, and dephosphorylated PHF-tau in ELISA and Western blot analysis. Thus, double phosphorylation is sufficient to distinguish PHF-tau from all other tau versions and there is no need to postulate any PHF-tau specific conformation for this region.

Neurofibrillary tangles as a neuropathological hallmark of Alzheimer's disease are mainly composed of abnormally phosphorylated microtubule-associated protein tau. The present work was primarily focused on the immunohistochemical characterization of recently developed monoclonal antibodies directed against disease-associated epitopes. Anti-phospho-threonine 212/phospho-serine 214 (HPT-1), anti-phospho-threonine 231/phospho-serine 235 (HPT-101) and their biotinylated derivatives were shown to be sensitive markers for the immunohistochemical detection of neuropathological alterations during Alzheimer's disease. Triple carbocyanine immunofluorescence labelling was based on digoxigenylated, fluoresceinated and biotinylated primary antibodies. AT8-immunolabelling of phospho-serine 202 and phospho-threonine 205 combined with HPT-1 and HPT-101-staining revealed similar distribution patterns of the three double-phosphorylated tau epitopes in the neocortex of patients with Alzheimer's disease.

We have identified a protein, FLJ12673 or FBXO11, that contains domains characteristically present in protein arginine methyltransferases (PRMTs). Immuno-purified protein expressed from one of the four splice variants in HeLa cells and in Escherichia coli exhibited methyltransferase activity. Monomethylarginine, symmetric, and asymmetric dimethylarginine (SDMA, ADMA) were formed on arginine residues. Accordingly, we have designated the protein PRMT9. PRMT9 is the third member of the PRMT family that forms SDMA modifications in proteins. Structurally, this protein is distinct from all other known PRMTs implying that convergent evolution allowed this protein to develop the ability to methylate arginine residues and evolved elements conserved in PRMTs to accomplish this.

The cDNA for PRMT7, a recently discovered human protein-arginine methyltransferase (PRMT), was cloned and expressed in Escherichia coli and mammalian cells. Immunopurified PRMT7 actively methylated histones, myelin basic protein, a fragment of human fibrillarin (GAR) and spliceosomal protein SmB. Amino acid analysis showed that the modifications produced were predominantly monomethylarginine and symmetric dimethylarginine (SDMA). Examination of PRMT7 expressed in E. coli demonstrated that peptides corresponding to sequences contained in histone H4, myelin basic protein, and SmD3 were methylated. Furthermore, analysis of the methylated proteins showed that symmetric dimethylarginine and relatively small amounts of monomethylarginine and asymmetric dimethylarginine were produced. SDMA was also formed when a GRG tripeptide was methylated by PRMT7, indicating that a GRG motif is by itself sufficient for symmetric dimethylation to occur. Symmetric dimethylation is reduced dramatically compared with monomethylation as the concentration of the substrate is increased. The data demonstrate that PRMT7 (like PRMT5) is a Type II methyltransferase capable of producing SDMA modifications in proteins.

Hyperphosphorylated tau (PHF-tau) is the major constituent of paired helical filaments (PHFs) from Alzheimer's disease (AD) brains. This conclusion has been based largely on the creation and characterization of monoclonal antibodies raised against PHFs, which can be classified in three categories: (a) those recognizing unmodified primary sequences of tau, (b) those recognizing phosphorylation-dependent epitopes on tau, and (c) those recognizing conformation-dependent epitopes on tau. Recent studies have suggested that the antibodies recognizing primary sequence and phosphorylation-dependent epitopes on tau are unable to distinguish between normal adult biopsy tau and PHF-tau. We now present evidence for a new fourth class of monoclonal antibodies recognizing conformation-dependent phosphoepitopes on tau, typified by TG-3, a monoclonal antibody raised to PHFs from AD brain homogenates. Studies using a series of deletional tau mutants, site-directed tau mutants, and synthetic peptides enable the precise epitope mapping of TG-3. Additional studies demonstrate that TG-3 reacts with neonatal mouse tau and PHF-tau but does not recognize adult mouse tau or tau derived from normal human autopsy or biopsy tissue. Further investigation reveals that TG-3 recognizes a unique conformation of tau found almost exclusively in PHFs from AD brains.

Salt-inducible kinase 2 (SIK2) is a multifunctional kinase of the AMPK family that plays a role in CREB1-mediated gene transcription and was recently reported to have therapeutic potential in ovarian cancer. The expression of this kinase was investigated in prostate cancer clinical specimens. Interestingly, auto-antibodies against SIK2 were increased in the plasma of patients with aggressive disease. Examination of SIK2 in prostate cancer cells found that it functions both as a positive regulator of cell-cycle progression and a negative regulator of CREB1 activity. Knockdown of SIK2 inhibited cell growth, delayed cell-cycle progression, induced cell death, and enhanced CREB1 activity. Expression of a kinase-dead mutant of SIK2 also inhibited cell growth, induced cell death, and enhanced CREB1 activity. Treatment with a small-molecule SIK2 inhibitor (ARN-3236), currently in preclinical development, also led to enhanced CREB1 activity in a dose- and time-dependent manner. Because CREB1 is a transcription factor and proto-oncogene, it was posited that the effects of SIK2 on cell proliferation and viability might be mediated by changes in gene expression. To test this, gene expression array profiling was performed and while SIK2 knockdown or overexpression of the kinase-dead mutant affected established CREB1 target genes; the overlap with transcripts regulated by forskolin (FSK), the adenylate cyclase/CREB1 pathway activator, was incomplete. Implications: This study demonstrates that targeting SIK2 genetically or therapeutically will have pleiotropic effects on cell-cycle progression and transcription factor activation, which should be accounted for when characterizing SIK2 inhibitors. Mol Cancer Res; 13(4); 620–35. ©2014 AACR.