Electron capture dissociation implementation progress in fourier transform ion cyclotron resonance mass spectrometry

Successful electron capture dissociation (ECD) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) applications to peptide and protein structural analysis have been enabled by constant progress in implementation of improved electron injection techniques. The rate of ECD product ion formation has been increased to match the liquid chromatography and capillary electrophoresis timescales, and ECD has been combined with infrared multiphoton dissociation in a single experimental configuration to provide simultaneous irradiation, fast switching between the two techniques, and good spatial overlap between ion, photon, and electron beams. Here we begin by describing advantages and disadvantages of the various existing electron injection techniques for ECD in FT-ICR MS. We next compare multiple-pass and single-pass ECD to provide better understanding of ECD efficiency at low and high negative cathode potentials. We introduce compressed hollow electron beam injection to optimize the overlap of ion, photon, and electron beams in the ICR ion trap. Finally, to overcome significant outgassing during operation of a powerful thermal cathode, we introduce nonthermal electron emitter-based electron injection. We describe the first results obtained with cold cathode ECD, and demonstrate a general way to obtain low-energy electrons in FT-ICR MS by use of multiple-pass EC

Periodic sequence distribution of product ion abundances in electron capture dissociation of amphipathic peptides and proteins

The rules for product ion formation in electron capture dissociation (ECD) mass spectrometry of peptides and proteins remain unclear. Random backbone cleavage probability and the nonspecific nature of ECD toward amino acid sequence have been reported, contrary to preferential channels of fragmentation in slow heating-based tandem mass spectrometry. Here we demonstrate that for amphipathic peptides and proteins, modulation of ECD product ion abundance (PIA) along the sequence is pronounced. Moreover, because of the specific primary (and presumably secondary) structure of amphipathic peptides, PIA in ECD demonstrates a clear and reproducible periodic sequence distribution. On the one hand, the period of ECD PIA corresponds to periodic distribution of spatially separated hydrophobic and hydrophilic domains within the peptide primary sequence. On the other hand, the same period correlates with secondary structure units, such as α-helical turns, known for solution-phase structure. Based on a number of examples, we formulate a set of characteristic features for ECD of amphipathic peptides and proteins: (1) periodic distribution of PIA is observed and is reproducible in a wide range of ECD parameters and on different experimental platforms; (2) local maxima of PIA are not necessarily located near the charged site; (3) ion activation before ECD not only extends product ion sequence coverage but also preserves ion yield modulation; (4) the most efficient cleavage (e.g. global maximum of ECD PIA distribution) can be remote from the charged site; (5) the number and location of PIA maxima correlate with amino acid hydrophobicity maxima generally to within a single amino acid displacement; and (6) preferential cleavage sites follow a selected hydrogen spine in an α-helical peptide segment. Presently proposed novel insights into ECD behavior are important for advancing understanding of the ECD mechanism, particularly the role of peptide sequence on PIA. An improved ECD model could facilitate protein sequencing and improve identification of unknown proteins in proteomics technologies. In structural biology, the periodic/preferential product ion yield in ECD of α-helical structures potentially opens the way toward de novo site-specific secondary structure determination of peptides and proteins in the gas phase and its correlation with solution-phase structur

Issues in Benchmarking Human Reliability Analysis Methods: A Literature Review.

There is a diversity of human reliability analysis (HRA) methods available for use in assessing human performance within probabilistic risk assessment (PRA). Due to the significant differences in the methods, including the scope, approach, and underlying models, there is a need for an empirical comparison investigating the validity and reliability of the methods. To accomplish this empirical comparison, a benchmarking study is currently underway that compares HRA methods with each other and against operator performance in simulator studies. In order to account for as many effects as possible in the construction of this benchmarking study, a literature review was conducted, reviewing past benchmarking studies in the areas of psychology and risk assessment. A number of lessons learned through these studies are presented in order to aid in the design of future HRA benchmarking endeavors

Electron capture dissociation implementation progress in Fourier transform ion cyclotron resonance mass spectrometry

Successful electron capture dissociation (ECD) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) applications to peptide and protein structural analysis have been enabled by constant progress in implementation of improved electron injection techniques. The rate of ECD product ion formation has been increased to match the liquid chromatography and capillary electrophoresis timescales, and ECD has been combined with infrared multiphoton dissociation in a single experimental configuration to provide simultaneous irradiation, fast switching between the two techniques, and good spatial overlap between ion, photon, and electron beams. Here we begin by describing advantages and disadvantages of the various existing electron injection techniques for ECD in FT-ICR MS. We next compare multiple-pass and single-pass ECD to provide better understanding of ECD efficiency at low and high negative cathode potentials. We introduce compressed hollow electron beam injection to optimize the overlap of ion, photon, and electron beams in the ICR ion trap. Finally, to overcome significant outgassing during operation of a powerful thermal cathode, we introduce nonthermal electron emitter-based electron injection. We describe the first results obtained with cold cathode ECD, and demonstrate a general way to obtain low-energy electrons in FT-ICR MS by use of multiple-pass ECD

Identification of Direct Target Engagement Biomarkers for Kinase-Targeted Therapeutics

Pharmacodynamic (PD) biomarkers are an increasingly valuable tool for decision-making and prioritization of lead compounds during preclinical and clinical studies as they link drug-target inhibition in cells with biological activity. They are of particular importance for novel, first-in-class mechanisms, where the ability of a targeted therapeutic to impact disease outcome is often unknown. By definition, proximal PD biomarkers aim to measure the interaction of a drug with its biological target. For kinase drug discovery, protein substrate phosphorylation sites represent candidate PD biomarkers. However, substrate phosphorylation is often controlled by input from multiple converging pathways complicating assessment of how potently a small molecule drug hits its target based on substrate phoshorylation measurements alone. Here, we report the use of quantitative, differential mass-spectrometry to identify and monitor novel drug-regulated phosphorylation sites on target kinases. Autophosphorylation sites constitute clinically validated biomarkers for select protein tyrosine kinase inhibitors. The present study extends this principle to phosphorylation sites in serine/threonine kinases looking beyond the T-loop autophosphorylation site. Specifically, for the 3′-phosphoinositide-dependent protein kinase 1 (PDK1), two phospho-residues p-PDK1Ser410 and p-PDK1Thr513 are modulated by small-molecule PDK1 inhibitors, and their degree of dephosphorylation correlates with inhibitor potency. We note that classical, ATP-competitive PDK1 inhibitors do not modulate PDK1 T-loop phosphorylation (p-PDK1Ser241), highlighting the value of an unbiased approach to identify drug target-regulated phosphorylation sites as these are complementary to pathway PD biomarkers. Finally, we extend our analysis to another protein Ser/Thr kinase, highlighting a broader utility of our approach for identification of kinase drug-target engagement biomarkers

The aggregatibacter actinomycetemcomitans heat shock protein GroEL interacts directly with human peripheral blood T cells

Heat shock family protein GroEL of Aggregatibacter actinomycetemcomitans (Aa) has antigenic properties. We previously demonstrated that A. actinomycetemcomitans GroEL-like protein affects human CD4 T cells by converting them into IL-10 and IFNg double cytokine producing Tbet+ Th1 cells. The objective of this study was to investigate whether or not AaGroEL communicates with T cells directly. To do this, sorted cells from peripheral blood mononuclear cells were stimulated with AaGroEL for 48 h. Flow cytometry was used to measure soluble and intracellular cytokine expression in the cell cultures and detect TLR2 expression on the surface of T cells. Expression of six different soluble cytokines was evaluated by CBA assay. To determine whether AaGroEL affects CD3+ T cells directly or not, purified CD3+ T cells or CD14+ cells were cultured with AaGroEL separately, and the quantity of soluble cytokine was measured. Results showed that sorted CD3+ cells produced soluble IL-6, TNFα-and IFNγ cytokines. Additionally, the intracellular cytokine staining data showed that AaGroEL-stimulated CD3+ cells were also TNFα-and IFNγ-positive. Moreover, AaGroEL-responsive T cells slightly increased their TLR2 expression. These findings suggest that CD3+ T cells produce cytokines in response to AaGroEL protein without requirements for other cells, such as CD14+ monocytes.Scientific and Technological Research Council of Turkey (TUBITAK 106T417

Periodic Sequence Distribution of Product Ion Abundances in Electron Capture Dissociation of Amphipathic Peptides and Proteins

The rules for product ion formation in electron capture dissociation (ECD) mass spectrometry of peptides and proteins remain unclear. Random backbone cleavage probability and the nonspecific nature of ECD toward amino acid sequence have been reported, contrary to preferential channels of fragmentation in slow heating-based tandem mass spectrometry. Here we demonstrate that for amphipathic peptides and proteins, modulation of ECD product ion abundance (PIA) along the sequence is pronounced. Moreover, because of the specific primary (and presumably secondary) structure of amphipathic peptides, PIA in ECD demonstrates a clear and reproducible periodic sequence distribution. On the one hand, the period of ECD PIA corresponds to periodic distribution of spatially separated hydrophobic and hydrophilic domains within the peptide primary sequence. On the other hand, the same period correlates with secondary structure units, such as a-helical turns, known for solution-phase structure. Based on a number of examples, we formulate a set of characteristic features for ECD of amphipathic peptides and proteins: (1) periodic distribution of PIA is observed and is reproducible in a wide range of ECD parameters and on different experimental platforms; (2) local maxima of PIA are not necessarily located near the charged site; (3) ion activation before ECD not only extends product ion sequence coverage but also preserves ion yield modulation; (4) the most efficient cleavage (e.g. global maximum of ECD PIA distribution) can be remote from the charged site; (5) the number and location of PIA maxima correlate with amino acid hydrophobicity maxima generally to within a single amino acid displacement; and (6) preferential cleavage sites follow a selected hydrogen spine in an a-helical peptide segment. Presently proposed novel insights into ECD behavior are important for advancing understanding of the ECD mechanism, particularly the role of peptide sequence on PIA. An improved ECD model could facilitate protein sequencing and improve identification of unknown proteins in proteomics technologies. In structural biology, the periodic/preferential product ion yield in ECD of a-helical structures potentially opens the way toward de novo site-specific secondary structure determination of peptides and proteins in the gas phase and its correlation with solution-phase structure. (J Am Soc Mass Spectrom 2009, 20, 1182-1192) (C) 2009 Published by Elsevier Inc. on behalf of American Society for Mass Spectrometr

LDRD report : parallel repartitioning for optimal solver performance.

We have developed infrastructure, utilities and partitioning methods to improve data partitioning in linear solvers and preconditioners. Our efforts included incorporation of data repartitioning capabilities from the Zoltan toolkit into the Trilinos solver framework, (allowing dynamic repartitioning of Trilinos matrices); implementation of efficient distributed data directories and unstructured communication utilities in Zoltan and Trilinos; development of a new multi-constraint geometric partitioning algorithm (which can generate one decomposition that is good with respect to multiple criteria); and research into hypergraph partitioning algorithms (which provide up to 56% reduction of communication volume compared to graph partitioning for a number of emerging applications). This report includes descriptions of the infrastructure and algorithms developed, along with results demonstrating the effectiveness of our approaches

Structure and mechanism of human DNA polymerase η

The variant form of the human syndrome xeroderma pigmentosum (XPV) is caused by a deficiency in DNA polymerase eta (Pol eta), a DNA polymerase that enables replication through ultraviolet-induced pyrimidine dimers. Here we report high-resolution crystal structures of human Pol eta at four consecutive steps during DNA synthesis through cis-syn cyclobutane thymine dimers. Pol eta acts like a 'molecular splint' to stabilize damaged DNA in a normal B-form conformation. An enlarged active site accommodates the thymine dimer with excellent stereochemistry for two-metal ion catalysis. Two residues conserved among Pol eta orthologues form specific hydrogen bonds with the lesion and the incoming nucleotide to assist translesion synthesis. On the basis of the structures, eight Pol eta missense mutations causing XPV can be rationalized as undermining the molecular splint or perturbing the active-site alignment. The structures also provide an insight into the role of Pol eta in replicating through D loop and DNA fragile sites

Increasing boys' and girls' intention to avoid teenage pregnancy: a cluster randomised control feasibility trial of an interactive video drama based intervention in post-primary schools in Northern Ireland

Background: Adolescent men have a vital yet neglected role in reducing unintended teenage pregnancy (UTP). There is a need for gender-sensitive educational interventions. Objectives: To determine the value and feasibility of conducting an effectiveness trial of the If I Were Jack Relationship and Sexuality Education (RSE) intervention in a convenience quota sample of post-primary schools in Northern Ireland. Secondary objectives were to assess acceptability to schools, pupils (male/female, aged 14–15 years) and parents/guardians; to identify optimal delivery structures and systems; to establish participation rates and reach, including equality of engagement of different socioeconomic and religious types; to assess trial recruitment and retention rates; to assess variation in normal RSE practice; to refine survey instruments; to assess differences in outcomes for male and female pupils; to identify potential effect sizes that might be detected in an effectiveness trial and estimate appropriate sample size for that trial; and to identify costs of delivery and pilot methods for assessing cost-effectiveness. Design: Cluster randomised Phase II feasibility trial with an embedded process and economic evaluation. Intervention: A teacher-delivered classroom-based RSE resource – an interactive video drama (IVD) with classroom materials, teacher training and an information session for parents – to immerse young people in a hypothetical scenario of Jack, a teenager whose girlfriend is unintentionally pregnant. It addresses gender inequalities in RSE by focusing on young men and is designed to increase intentions to avoid UTP by encouraging young people to delay sexual intercourse and to use contraception consistently in sexual relationships. Main outcome measures: Abstinence from sexual intercourse (delaying initiation of sex or returning to abstinence) or avoidance of unprotected sexual intercourse (consistent correct use of contraception). Secondary outcomes included Knowledge, Attitudes, Skills and Intentions. Results: The intervention proved acceptable to schools, pupils and parents, as evidenced through positive process evaluation. One minor refinement to the parental component was required, namely the replacement of the teacher-led face-to-face information session for parents by online videos designed to deliver the intervention to parents/guardians into their home. School recruitment was successful (target 25%, achieved 38%). No school dropped out. Pupil retention was successful (target 85%, achieved 93%). The between-group difference in incidence of unprotected sex of 1.3% (95% confidence interval 0.55% to 2.2%) by 9 months demonstrated an effect size consistent with those reported to have had meaningful impact on UTP rates (resulting in an achievable sample size of 66 schools at Phase III). Survey instruments showed high acceptability and reliability of measures (Cronbach’s alpha: 0.5–0.7). Economic evaluation at Phase III is feasible because it was possible to (1) identify costs of delivering If I Were Jack (mean cost per pupil, including training of teachers, was calculated as £13.66); and (2) develop a framework for assessing cost-effectiveness. Conclusion: Trial methods were appropriate, and recruitment and retention of schools and pupils was satisfactory, successfully demonstrating all criteria for progression to a main trial. The perceived value of culture- and gender-sensitive public health interventions has been highlighted. Future work: Progression to a Phase III effectiveness trial. Trial registration: Current Controlled Trials ISRCTN99459996. Funding: This project was funded by the NIHR Public Health Research programme and will be published in full in Public Health Research; Vol. 5, No. 1. See the NIHR Journals Library website for further project information