Multi-Omic Analysis of Esophageal Adenocarcinoma Uncovers Candidate Therapeutic Targets and Cancer-Selective Posttranscriptional Regulation.

Efforts to address the poor prognosis associated with esophageal adenocarcinoma (EAC) have been hampered by a lack of biomarkers to identify early disease and therapeutic targets. Despite extensive efforts to understand the somatic mutations associated with EAC over the past decade, a gap remains in understanding how the atlas of genomic aberrations in this cancer impacts the proteome and which somatic variants are of importance for the disease phenotype. We performed a quantitative proteomic analysis of 23 EACs and matched adjacent normal esophageal and gastric tissues. We explored the correlation of transcript and protein abundance using tissue-matched RNA-seq and proteomic data from seven patients and further integrated these data with a cohort of EAC RNA-seq data (n = 264 patients), EAC whole-genome sequencing (n = 454 patients), and external published datasets. We quantified protein expression from 5879 genes in EAC and patient-matched normal tissues. Several biomarker candidates with EAC-selective expression were identified, including the transmembrane protein GPA33. We further verified the EAC-enriched expression of GPA33 in an external cohort of 115 patients and confirm this as an attractive diagnostic and therapeutic target. To further extend the insights gained from our proteomic data, an integrated analysis of protein and RNA expression in EAC and normal tissues revealed several genes with poorly correlated protein and RNA abundance, suggesting posttranscriptional regulation of protein expression. These outlier genes, including SLC25A30, TAOK2, and AGMAT, only rarely demonstrated somatic mutation, suggesting post-transcriptional drivers for this EAC-specific phenotype. AGMAT was demonstrated to be overexpressed at the protein level in EAC compared to adjacent normal tissues with an EAC-selective, post-transcriptional mechanism of regulation of protein abundance proposed. Integrated analysis of proteome, transcriptome, and genome in EAC has revealed several genes with tumor-selective, posttranscriptional regulation of protein expression, which may be an exploitable vulnerability.

A Practical and Analytical Comparative Study of Gel-Based Top-Down and Gel-Free Bottom-Up Proteomics Including Unbiased Proteoform Detection.

Proteomics is an indispensable analytical technique to study the dynamic functioning of biological systems via different proteins and their proteoforms. In recent years, bottom-up shotgun has become more popular than gel-based top-down proteomics. The current study examined the qualitative and quantitative performance of these two fundamentally different methodologies by the parallel measurement of six technical and three biological replicates of the human prostate carcinoma cell line DU145 using its two most common standard techniques, label-free shotgun and two-dimensional differential gel electrophoresis (2D-DIGE). The analytical strengths and limitations were explored, finally focusing on the unbiased detection of proteoforms, exemplified by discovering a prostate cancer-related cleavage product of pyruvate kinase M2. Label-free shotgun proteomics quickly yields an annotated proteome but with reduced robustness, as determined by three times higher technical variation compared to 2D-DIGE. At a glance, only 2D-DIGE top-down analysis provided valuable, direct stoichiometric qualitative and quantitative information from proteins to their proteoforms, even with unexpected post-translational modifications, such as proteolytic cleavage and phosphorylation. However, the 2D-DIGE technology required almost 20 times as much time per protein/proteoform characterization with more manual work. Ultimately, this work should expose both techniques' orthogonality with their different contents of data output to elucidate biological questions.

Individualization of Mycophenolic Acid Therapy through Pharmacogenetic, Pharmacokinetic and Pharmacodynamic Testing.

Mycophenolic acid (MPA) is a widely used immunosuppressive agent and exerts its effect by inhibiting inosine 5'-monophosphate dehydrogenase (IMPDH), the main regulating enzyme of purine metabolism. However, significant unexplained differences in the efficacy and tolerability of MPA therapy pose a clinical challenge. Therefore, broad pharmacogenetic, pharmacokinetic, and pharmacodynamic approaches are needed to individualize MPA therapy. In this prospective cohort study including 277 renal transplant recipients, IMPDH2 rs11706052 SNP status was assessed by genetic sequencing, and plasma MPA trough levels were determined by HPLC and IMPDH enzyme activity in peripheral blood mononuclear cells (PBMCs) by liquid chromatography-mass spectrometry. Among the 277 patients, 84 were identified with episodes of biopsy-proven rejection (BPR). No association was found between rs11706052 SNP status and graft rejection (OR 1.808, and 95% CI, 0.939 to 3.479; p = 0.076). Furthermore, there was no association between MPA plasma levels and BPR (p = 0.69). However, the patients with graft rejection had a significantly higher predose IMPDH activity in PBMCs compared to the controls without rejection at the time of biopsy (110.1 ± 50.2 vs. 95.2 ± 45.4 pmol/h; p = 0.001), and relative to the baseline IMPDH activity before transplantation (p = 0.042). Our results suggest that individualization of MPA therapy, particularly through pharmacodynamic monitoring of IMPDH activity in PBMCs, has the potential to improve the clinical outcomes of transplant patients.

Autoimmune Global Amnesia as Manifestation of AMPAR Encephalitis and Neuropathologic Findings.

OBJECTIVE: To report an unusual clinical phenotype of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) encephalitis and describe associated neuropathologic findings. METHODS: We retrospectively investigated 3 AMPAR encephalitis patients with autoimmune global hippocampal amnesia using comprehensive cognitive and neuropsychologic assessment, antibody testing by in-house tissue-based and cell-based assays, and neuropathologic analysis of brain autopsy tissue including histology and immunohistochemistry. RESULTS: Three patients presented with acute-to-subacute global amnesia without affection of cognitive performance, attention, concentration, or verbal function. None of the patients had epileptic seizures, change of behavior, personality changes, or psychiatric symptoms. The MRI was normal in 1 patient and showed increased fluid-attenuated inversion recovery/T2 signal in the hippocampus in the other 2 patients. Two patients showed complete remission after immunotherapy. The one patient who did not improve had an underlying adenocarcinoma of the lung and died 3.5 months after disease onset because of tumor progression. Neuropathologic analysis of the brain autopsy revealed unilateral hippocampal sclerosis accompanied by mild inflammatory infiltrates, predominantly composed of T lymphocytes, and decrease of AMPAR immunoreactivity. CONCLUSION: AMPAR antibodies usually associate with limbic encephalitis but may also present with immune responsive, acute-to-subacute, isolated hippocampal dysfunction without overt inflammatory CSF or MRI changes.

A novel proline substitution (Arg201Pro) in alpha helix 8 of TMEM98 causes autosomal dominant nanophthalmos-4, closed angle glaucoma and attenuated visual acuity.

Nanophthalmos-4 is a rare autosomal dominant disorder caused by two known variations in TMEM98. An Austrian Caucasian pedigree was identified suffering from nanophthalmos and late onset angle-closure glaucoma and premature loss of visual acuity. Whole exome sequencing identified segregation of a c.602G > C transversion in TMEM98 (p.Arg201Pro) as potentially causative. A protein homology model generated showed a TMEM98 structure comprising α4, α5/6, α7 and α8 antiparallel helix bundles and two predicted transmembrane domains in α1 and α7 that have been confirmed in vitro. Both p.Arg201Pro and the two missense variations representing proline insertions identified previously to cause nanophthalmos-4 (p.Ala193Pro and p.His196Pro) are located in the charge polarized helix α8 (p.183-p210). Stability of the C-terminal alpha helical structure of TMEM98 is therefore essential to prevent the development of human nanophthalmos-4. Precise molecular diagnosis could lead to the development of tailored therapies for patients with orphan ocular disease.

Pillar array columns for peptide separations in nanoscale reversed-phase chromatography.

Reversed-phase chromatography is the most common technique for separation of tryptic peptides. In this short communication, we describe the optimization of sample loading and separation parameters for a novel micromachined column and provide a detailed description on the performance and reproducibility of this separation system. Tryptic digest of a mixture of seven proteins with diverse mass and isoelectric point was used as a test sample. The methods developed and used are straight-forward; by using well-balanced, combined-step gradients an optimal distribution of peptides on the column could be achieved throughout the complete gradient window. The potential use of the column is exceptional due to the low back-pressure, better distribution of peptides over the separation window, enhanced stability and reproducibility of retention times, and the prolonged lifetime of columns compared to conventionally packed nano-HPLC column. The higher identification rates have been demonstrated through measurements of HeLa cell lysates under identical chromatographic conditions on the pillar array and packed-bed columns.

Quantitative proteome analysis of Merkel cell carcinoma cell lines using SILAC.

BACKGROUND: Merkel cell carcinoma (MCC) is an aggressive neuroendocrine tumour of the skin with growing incidence. To better understand the biology of this malignant disease, immortalized cell lines are used in research for in vitro experiments. However, a comprehensive quantitative proteome analysis of these cell lines has not been performed so far. METHODS: Stable isotope labelling by amino acids in cell culture (SILAC) was applied to six MCC cell lines (BroLi, MKL-1, MKL-2, PeTa, WaGa, and MCC13). Following tryptic digest of labelled proteins, peptides were analysed by mass spectrometry. Proteome patterns of MCC cell lines were compared to the proteome profile of an immortalized keratinocyte cell line (HaCaT). RESULTS: In total, 142 proteins were upregulated and 43 proteins were downregulated. Altered proteins included mitoferrin-1, histone H2A type 1-H, protein-arginine deiminase type-6, heterogeneous nuclear ribonucleoproteins A2/B1, protein SLX4IP and clathrin light chain B. Furthermore, several proteins of the histone family and their variants were highly abundant in MCC cell lines. CONCLUSIONS: The results of this study present a new protein map of MCC and provide deeper insights in the biology of MCC. Data are available via ProteomeXchange with identifier PXD008181.

Extracellular Purine Metabolism Is the Switchboard of Immunosuppressive Macrophages and a Novel Target to Treat Diseases With Macrophage Imbalances.

If misregulated, macrophage (Mϕ)-T cell interactions can drive chronic inflammation thereby causing diseases, such as rheumatoid arthritis (RA). We report that in a proinflammatory environment, granulocyte-Mϕ (GM-CSF)- and Mϕ colony-stimulating factor (M-CSF)-dependent Mϕs have dichotomous effects on T cell activity. While GM-CSF-dependent Mϕs show a highly stimulatory activity typical for M1 Mϕs, M-CSF-dependent Mϕs, marked by folate receptor ß (FRß), adopt an immunosuppressive M2 phenotype. We find the latter to be caused by the purinergic pathway that directs release of extracellular ATP and its conversion to immunosuppressive adenosine by co-expressed CD39 and CD73. Since we observed a misbalance between immunosuppressive and immunostimulatory Mϕs in human and murine arthritic joints, we devised a new strategy for RA treatment based on targeted delivery of a novel methotrexate (MTX) formulation to the immunosuppressive FRß+CD39+CD73+ Mϕs, which boosts adenosine production and curtails the dominance of proinflammatory Mϕs. In contrast to untargeted MTX, this approach leads to potent alleviation of inflammation in the murine arthritis model. In conclusion, we define the Mϕ extracellular purine metabolism as a novel checkpoint in Mϕ cell fate decision-making and an attractive target to control pathological Mϕs in immune-mediated diseases.

Impact of combined treatment with nimesulide and cisplatin on oral carcinoma cells.

BACKGROUND: Despite significant advances in diagnosis and therapy, the rate of survival of patients with oral cancers still remains poor as an appropriate treatment has not been found yet, due to side effects of chemo/radiotherapy. AIM: This study aimed to identify molecular mechanisms of cell death of oral cancer cells caused by treatment with a nonselective Cox-2 inhibitor in combination with a low-dose chemotherapeutic drug. METHODS: Squamous cell carcinoma (SCC) cells SCC9 and SCC25 were subjected to mono- and combination therapy with nimesulide and cisplatin. Fluorescence-activated cell sorting (FACS), immunohistochemistry, high-pressure liquid chromatography (HPLC), microarray gene chips, and isobaric tags for a relative and absolute quantitation (iTRAQ) system were used. RESULTS: Increased numbers of apoptotic and necrotic SCC9/SCC25 cells were detected after combined exposure. ATP levels and the energy charge of SCC9 cells were significantly decreased after both individual and combined treatment. We detected and quantified a responsible gene, keratin 6a, and 540 relevant proteins. In SCC25 cells, ATP levels significantly decreased only after combination therapy. After combined treatment of SCC9 cells, significant upregulation of Histon-H2A/H2B/H4 was found, with a local discovery false rate of 0.003 for Histon-H2A and 0.0027 for Histon-H2B, respectively. CONCLUSION: Compared to the single-drug treatment, combined treatment of the oral cancer cells with nimesulide and cisplatin increases and induces necrosis and apoptosis through different pathways. A significant effect of the cytoplasmic increase was also observed in histones of cell lines SCC9 and SCC25 that were previously treated with combined nimesulide and cisplatin therapy.

Hyperoxia Induces Inflammation and Cytotoxicity in Human Adult Cardiac Myocytes.

Supplemental oxygen (O2) is used as adjunct therapy in anesthesia, emergency, and intensive care medicine. We hypothesized that excessive O2 levels (hyperoxia) can directly injure human adult cardiac myocytes (HACMs). HACMs obtained from the explanted hearts of transplantation patients were exposed to constant hyperoxia (95% O2), intermittent hyperoxia (alternating 10 min exposures to 5% and 95% O2), constant normoxia (21% O2), or constant mild hypoxia (5% O2) using a bioreactor. Changes in cell morphology, viability as assessed by lactate dehydrogenase (LDH) release and trypan blue (TB) staining, and secretion of vascular endothelial growth factor (VEGF), macrophage migration inhibitory factor (MIF), and various pro-inflammatory cytokines (interleukin, IL; chemokine C-X-C motif ligand, CXC; granulocyte-colony stimulating factor, G-CSF; intercellular adhesion molecule, ICAM; chemokine C-C motif ligand, CCL) were compared among treatment groups at baseline (0 h) and after 8, 24, and 72 h of treatment. Changes in HACM protein expression were determined by quantitative proteomic analysis after 48 h of exposure. Compared with constant normoxia and mild hypoxia, constant hyperoxia resulted in a higher TB-positive cell count, greater release of LDH, and elevated secretion of VEGF, MIF, IL-1ß, IL-6, IL-8, CXCL-1, CXCL-10, G-CSF, ICAM-1, CCL-3, and CCL-5. Cellular inflammation and cytotoxicity gradually increased and was highest after 72 h of constant and intermittent hyperoxia. Quantitative proteomic analysis revealed that hypoxic and hyperoxic O2 exposure differently altered the expression levels of proteins involved in cell-cycle regulation, energy metabolism, and cell signaling. In conclusion, constant and intermittent hyperoxia induced inflammation and cytotoxicity in HACMs. Cell injury occurred earliest and was greatest after constant hyperoxia, but even relatively brief repeating hyperoxic episodes induced a substantial inflammatory response.

Folate Receptor ß Regulates Integrin CD11b/CD18 Adhesion of a Macrophage Subset to Collagen.

Folate, also known as vitamin B9, is necessary for essential cellular functions such as DNA synthesis, repair, and methylation. It is supplied to the cell via several transporters and receptors, including folate receptor (FR) ß, a GPI-anchored protein belonging to the folate receptor family. As FRß shows a restricted expression to cells of myeloid origin and only a subset of activated macrophages and placental cells have been shown to express functional FRß, it represents a promising target for future therapeutic strategies. In this study, we performed affinity purification and mass spectrometric analysis of the protein microenvironment of FRß in the plasma membrane of human FRß(+) macrophages and FRß-transduced monocytic THP-1 cells. In this manner, we identified a novel role of FRß: that is, we report functional interactions of FRß with receptors mediating cellular adhesion, in particular the CD11b/CD18 ß2 integrin heterodimer complement receptor type 3/Mac-1. This interaction results in impeded adhesion of FRß(+) human primary macrophages and THP-1 cells to collagen in comparison with their FRß(-) counterparts. We further show that FRß is only expressed by human macrophages when differentiated with M-CSF. These findings thus identify FRß as a novel CD11b/CD18 regulator for trafficking and homing of a subset of macrophages on collagen.

Association of CD147 and Calcium Exporter PMCA4 Uncouples IL-2 Expression from Early TCR Signaling.

The Ig superfamily member CD147 is upregulated following T cell activation and was shown to serve as a negative regulator of T cell proliferation. Thus, Abs targeting CD147 are being tested as new treatment strategies for cancer and autoimmune diseases. How CD147 mediates immunosuppression and whether association with other coreceptor complexes is needed have remained unknown. In the current study, we show that silencing of CD147 in human T cells increases IL-2 production without affecting the TCR proximal signaling components. We mapped the immunosuppressive moieties of CD147 to its transmembrane domain and Ig-like domain II. Using affinity purification combined with mass spectrometry, we determined the domain specificity of CD147 interaction partners and identified the calcium exporter plasma membrane calcium ATPase isoform 4 (PMCA4) as the interaction partner of the immunosuppressive moieties of CD147. CD147 does not control the proper membrane localization of PMCA4, but PMCA4 is essential for the CD147-dependent inhibition of IL-2 expression via a calcium-independent mechanism. In summary, our data show that CD147 interacts via its immunomodulatory domains with PMCA4 to bypass TCR proximal signaling and inhibit IL-2 expression.

Melatonin: Free Radicals and Metabolites Resulting by Emission and Consumption of Solvated Electrons (eaq-): Reaction Mechanisms.

BACKGROUND/AIM: Melatonin not only regulates circadian rhythm, but also induces apoptosis in tumor cells. Hence, elucidation of the basic reaction mechanisms of melatonin and its metabolites is a matter of interest. MATERIAL AND METHODS: Melatonin dissolved in a mixture of water/ethanol=40/60 form associates (unstable complexes). For simulation of biological processes, melatonin was excited by UV light into the singlet state. RESULTS: By using monochromatic UV light (λ=254 nm) melatonin ejects solvated electrons (eaq (-)), a part of which is scavenged by melatonin in ground state contained in the associates. Consequently, with increase of melatonin concentration a decrease of the determined quantum yield of emitted eaq (-), Q(eaq (-)), is obtained. The complex molecular structure of melatonin contains functional groups which can emit eaq (-), as well such consuming eaq (-). As a succession of these processes various types of metabolites are generated, as well as degradation products, with lower molecular weight, are formed. CONCLUSION: Not melatonin per se, but the ejected eaq (-) and thereby resulting various metabolites are responsible for different biological properties of melatonin.

Differential Potential of Pharmacological PARP Inhibitors for Inhibiting Cell Proliferation and Inducing Apoptosis in Human Breast Cancer Cells.

BRCA1/2-mutant cells are hypersensitive to inactivation of poly(ADP-ribose) polymerase 1 (PARP-1). We recently showed that inhibition of PARP-1 by NU1025 is strongly cytotoxic for BRCA1-positive BT-20 cells, but not BRCA1-deficient SKBr-3 cells. These results raised the possibility that other PARP-1 inhibitors, particularly those tested in clinical trials, may be more efficacious against BRCA1-deficient SKBr-3 breast cancer cells than NU1025. Thus, in the presented study the cytotoxicity of four PARP inhibitors under clinical evaluation (olaparib, rucaparib, iniparib and AZD2461) was examined and compared to that of NU1025. The sensitivity of breast cancer cells to the PARP-1 inhibition strongly varied. Remarkably, BRCA-1-deficient SKBr-3 cells were almost completely insensitive to NU1025, olaparib and rucaparib, whereas BRCA1-expressing BT-20 cells were strongly affected by NU1025 even at low doses. In contrast, iniparib and AZD2461 were cytotoxic for both BT-20 and SKBr-3 cells. Of the four tested PARP-1 inhibitors only AZD2461 strongly affected cell cycle progression. Interestingly, the anti-proliferative and pro-apoptotic potential of the tested PARP-1 inhibitors clearly correlated with their capacity to damage DNA. Further analyses revealed that proteomic signatures of the two studied breast cancer cell lines strongly differ, and a set of 197 proteins was differentially expressed in NU1025-treated BT-20 cancer cells. These results indicate that BT-20 cells may harbor an unknown defect in DNA repair pathway(s) rendering them sensitive to PARP-1 inhibition. They also imply that therapeutic applicability of PARP-1 inhibitors is not limited to BRCA mutation carriers but can be extended to patients harboring deficiencies in other components of the pathway(s).

Empirical multidimensional space for scoring peptide spectrum matches in shotgun proteomics.

Data-dependent tandem mass spectrometry (MS/MS) is one of the main techniques for protein identification in shotgun proteomics. In a typical LC-MS/MS workflow, peptide product ion mass spectra (MS/MS spectra) are compared with those derived theoretically from a protein sequence database. Scoring of these matches results in peptide identifications. A set of peptide identifications is characterized by false discovery rate (FDR), which determines the fraction of false identifications in the set. The total number of peptides targeted for fragmentation is in the range of 10,000 to 20,000 for a several-hour LC-MS/MS run. Typically, <50% of these MS/MS spectra result in peptide-spectrum matches (PSMs). A small fraction of PSMs pass the preset FDR level (commonly 1%) giving a list of identified proteins, yet a large number of correct PSMs corresponding to the peptides originally present in the sample are left behind in the "grey area" below the identity threshold. Following the numerous efforts to recover these correct PSMs, here we investigate the utility of a scoring scheme based on the multiple PSM descriptors available from the experimental data. These descriptors include retention time, deviation between experimental and theoretical mass, number of missed cleavages upon in-solution protein digestion, precursor ion fraction (PIF), PSM count per sequence, potential modifications, median fragment mass error, (13)C isotope mass difference, charge states, and number of PSMs per protein. The proposed scheme utilizes a set of metrics obtained for the corresponding distributions of each of the descriptors. We found that the proposed PSM scoring algorithm differentiates equally or more efficiently between correct and incorrect identifications compared with existing postsearch validation approaches.

Identification and validation of platelet low biological variation proteins, superior to GAPDH, actin and tubulin, as tools in clinical proteomics.

Accurate biomarker quantification requires carefully chosen normalisation procedures. When single proteins are used as loading controls (LCs), it is crucial that their expressional stability must be known. Platelets are an important biomarker source, especially for neurological diseases. We performed a systematical analysis of the platelet proteome to identify proteins suitable as LCs, using the 2-D DIGE system. We first screened a healthy population (n=137), aged between 18 and 104years, to find proteins with small coefficients of total variation (CVtot), herein termed low biological variation proteins (LBVP). Thereafter, expressional stability was verified in 101 patients suffering from Alzheimer's- (AD), Parkinson's- disease, vascular dementia or schizophrenia. Interestingly, traditional LCs such as tubulin beta-1 and GAPDH, were not found amongst LBVP. The least variable protein, calculated over all 238 individuals, was 14-3-3 gamma, with a CVtot of 9.3%, showing no gender, age or disease dependency. The normalisation capability of 14-3-3 gamma was superior to traditional LC in quantifying Western blot signals of the platelet AD-biomarker Monoamine Oxidase B of patient versus controls. Similar results were obtained with HepG2 cells, treated in vitro with DNA-methyltransferase inhibitor 5-aza-2'deoxicytidine. Finally, we provide a list of alternative normalisation candidates for accurate biomarker quantification. BIOLOGICAL SIGNIFICANCE: This paper suggests a considerable list of platelet proteins with a lower biological variation than well known "housekeeping" proteins like GAPDH and tubulin. Spot abundances of found proteins are middle ranged and unaffected by gender, age and certain diseases. Hence, listed proteins might be valuable normalisation candidates used additionally or alternatively. Platelet's least variable protein 14-3-3 gamma is validated as normalisation protein in platelet biomarker quantification. Furthermore 14-3-3 gamma is demonstrated to be also stable expressed by in HepG2, cells others than platelets, when treated by DNA methylation inhibitor.

Peptide orientation affects selectivity in ion-exchange chromatography.

Here we demonstrate that separation of proteolytic peptides, having the same net charge and one basic residue, is affected by their specific orientation toward the stationary phase in ion-exchange chromatography. In electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) with an anion-exchange material, the C-terminus of the peptides is, on average, oriented toward the stationary phase. In cation exchange, the average peptide orientation is the opposite. Data with synthetic peptides, serving as orientation probes, indicate that in tryptic/Lys-C peptides the C-terminal carboxyl group appears to be in a zwitterionic bond with the side chain of the C-terminal Lys/Arg residue. In effect, the side chain is then less basic than the N-terminus, accounting for the specific orientation of tryptic and Lys-C peptides. Analyses of larger sets of peptides, generated from lysates by either Lys-N, Lys-C, or trypsin, reveal that specific peptide orientation affects the ability of charged side chains, such as phosphate residues, to influence retention. Phosphorylated residues that are remote in the sequence from the binding site affect retention less than those that are closer. When a peptide contains multiple charged sites, then orientation is observed to be less rigid and retention tends to be governed by the peptide's net charge rather than its sequence. These general observations could be of value in confirming a peptide's identification and, in particular, phosphosite assignments in proteomics analyses. More generally, orientation accounts for the ability of chromatography to separate peptides of the same composition but different sequence.

A new acid mix enhances phosphopeptide enrichment on titanium- and zirconium dioxide for mapping of phosphorylation sites on protein complexes.

The selective enrichment of phosphorylated peptides prior to reversed-phase separation and mass spectrometric detection significantly improves the analytical results in terms of higher number of detected phosphorylation sites and spectra of higher quality. Metal oxide chromatography (MOC) has been recently described for selective phosphopeptide enrichment (Pinkse et al., 2004; Larsen et al., 2005; Kweon and Hakansson, 2006; Cantin et al., 2007; Collins et al., 2007). In the present work we have tested the effect of a modified loading solvent containing a novel acid mix and optimized wash conditions on the efficiency of TiO(2)-based phosphopeptide enrichment in order to improve our previously published method (Mazanek et al., 2007). Applied to a test mixture of synthetic and BSA-derived peptides, the new method showed improved selectivity for phosphopeptides, whilst retaining a high recovery rate. Application of the new enrichment method to digested purified protein complexes resulted in the identification of a significantly higher number of phosphopeptides as compared to the previous method. Additionally, we have compared the performance of TiO(2) and ZrO(2) columns for the isolation and identification of phosphopeptides from purified protein complexes and found that for our test set, both media performed comparably well. In summary, our improved method is highly effective for the enrichment of phosphopeptides from purified protein complexes prior to mass spectrometry, and is suitable for large-scale phosphoproteomic projects that aim to elucidate phosphorylation-dependent cellular processes.

Preventing carryover of peptides and proteins in nano LC-MS separations.

Sample carryover is a significant problem that occurs in high-performance liquid chromatography (HPLC) analysis. Carryover effects cannot be tolerated in any high-performance liquid chromatography-mass spectroscopy (HPLC-MS) separation system, and proteomics analysis must be performed in a separation system with virtually no carryover. Several procedures have been tested for effective and fast removal of interfering peptides and proteins originating from previous analyses in the HPLC system. We have developed and optimized a cleaning method for eliminating carryover caused by the autosampler and the trap column. The new washing method uses an injection of trifluoroethanol into the injection path and onto the trap column to remove strongly bound peptides and proteins, and it includes trifluoroethanol as an additional solvent in the chromatographic mobile phase for enhanced cleaning of the separation column. By application of this method, a significant reduction in carryover was achieved without any loss in the amount of proteins and peptides identified by MS.

Contribution of nicotine to acute endothelial dysfunction in long-term smokers.

OBJECTIVES: The aim of this study was to determine whether nicotine, a constituent of cigarette smoke, contributes to acute endothelial dysfunction after smoking one cigarette. BACKGROUND: Animal studies suggest that nicotine might cause an impairment of endothelium-dependent vasodilation via an increase in oxidative stress. METHODS: Sixteen healthy smokers were entered into a randomized, observer-blinded crossover study comparing the effects of nicotine nasal spray (1-mg nicotine) and cigarette smoke (1-mg nicotine, 12 mg tar) on vascular reactivity in the brachial artery. Using high-resolution ultrasound, flow-mediated dilation (FMD) and endothelium-independent, nitroglycerin-induced dilation were assessed at baseline and 20 min after the administration of nicotine (spray or cigarette). RESULTS: In response to similar increases in nicotine serum levels, FMD values declined from 10.2 +/- 4.4% to 6.7 +/- 4.0% after the spray (mean difference: -3.6 +/- 2.0%, 95% confidence interval: -4.6; -2.5, p < 0.0001) and from 9.4 +/- 3.8% to 4.3 +/- 2.8% after the cigarette (-5.1 +/- 2.6%, -6.5; -3.7, p < 0.0001). Nitroglycerin-induced dilation remained similar within both periods. Performing a period effect analysis of variance, a significant influence on FMD was found for the mode of administration (p = 0.017) and the baseline value (p = 0.021). The effect on FMD was more pronounced after the cigarette than after the spray (estimated average effect difference: 1.9% FMD). Oxidation parameters did not increase significantly after nicotine spray or tobacco exposure. CONCLUSIONS: These results demonstrate that nicotine alone causes acute endothelial dysfunction, although to a lesser extent than smoking a cigarette of the same nicotine yield. However, the precise mechanisms by which nicotine leads to this altered vascular reactivity remain unclear.