Hewlett-Packard (Germany)

BACKGROUND: The integrity of RNA molecules is of paramount importance for experiments that try to reflect the snapshot of gene expression at the moment of RNA extraction. Until recently, there has been no reliable standard for estimating the integrity of RNA samples and the ratio of 28S:18S ribosomal RNA, the common measure for this purpose, has been shown to be inconsistent. The advent of microcapillary electrophoretic RNA separation provides the basis for an automated high-throughput approach, in order to estimate the integrity of RNA samples in an unambiguous way. METHODS: A method is introduced that automatically selects features from signal measurements and constructs regression models based on a Bayesian learning technique. Feature spaces of different dimensionality are compared in the Bayesian framework, which allows selecting a final feature combination corresponding to models with high posterior probability. RESULTS: This approach is applied to a large collection of electrophoretic RNA measurements recorded with an Agilent 2100 bioanalyzer to extract an algorithm that describes RNA integrity. The resulting algorithm is a user-independent, automated and reliable procedure for standardization of RNA quality control that allows the calculation of an RNA integrity number (RIN). CONCLUSION: Our results show the importance of taking characteristics of several regions of the recorded electropherogram into account in order to get a robust and reliable prediction of RNA integrity, especially if compared to traditional methods.

1. Introduction. 2. Analysis Models and Notations. 3. Process of Analysis. 4. Design. 5. Implementation. 6. Case Study. 7. Exercises. 8. Fusion and Other Methods. 9. Reuse. 10. Managing a Fusion Project. Appendix A: Fusion Process Summary. Appendix B: Fusion Notation Summary. Appendix C: Fusion Reference.

Less than half of patients with suspected genetic disease receive a molecular diagnosis. We have therefore integrated next-generation sequencing (NGS), bioinformatics, and clinical data into an effective diagnostic workflow. We used variants in the 2741 established Mendelian disease genes [the disease-associated genome (DAG)] to develop a targeted enrichment DAG panel (7.1 Mb), which achieves a coverage of 20-fold or better for 98% of bases. Furthermore, we established a computational method [Phenotypic Interpretation of eXomes (PhenIX)] that evaluated and ranked variants based on pathogenicity and semantic similarity of patients' phenotype described by Human Phenotype Ontology (HPO) terms to those of 3991 Mendelian diseases. In computer simulations, ranking genes based on the variant score put the true gene in first place less than 5% of the time; PhenIX placed the correct gene in first place more than 86% of the time. In a retrospective test of PhenIX on 52 patients with previously identified mutations and known diagnoses, the correct gene achieved a mean rank of 2.1. In a prospective study on 40 individuals without a diagnosis, PhenIX analysis enabled a diagnosis in 11 cases (28%, at a mean rank of 2.4). Thus, the NGS of the DAG followed by phenotype-driven bioinformatic analysis allows quick and effective differential diagnostics in medical genetics.

Allene oxide cyclase (EC ) catalyzes the stereospecific cyclization of an unstable allene oxide to (9S,13S)-12-oxo-(10,15Z)-phytodienoic acid, the ultimate precursor of jasmonic acid. This dimeric enzyme has previously been purified, and two almost identical N-terminal peptides were found, suggesting allene oxide cyclase to be a homodimeric protein. Furthermore, the native protein was N-terminally processed. Using degenerate primers, a polymerase chain reaction fragment could be generated from tomato, which was further used to isolate a full-length cDNA clone of 1 kilobase pair coding for a protein of 245 amino acids with a molecular mass of 26 kDa. Whereas expression of the whole coding region failed to detect allene oxide cyclase activity, a 5'-truncated protein showed high activity, suggesting that additional amino acids impair the enzymatic function. Steric analysis of the 12-oxophytodienoic acid formed by the recombinant enzyme revealed exclusive (>99%) formation of the 9S,13S enantiomer. Exclusive formation of this enantiomer was also found in wounded tomato leaves. Southern analysis and genetic mapping revealed the existence of a single gene for allene oxide cyclase located on chromosome 2 of tomato. Inspection of the N terminus revealed the presence of a chloroplastic transit peptide, and the location of allene oxide cyclase protein in that compartment could be shown by immunohistochemical methods. Concomitant with the jasmonate levels, the accumulation of allene oxide cyclase mRNA was transiently induced after wounding of tomato leaves.

The sample fractionation steps conducted prior to mass detection are critically important for the comprehensive analysis of complex protein mixtures. This paper illustrates the effectiveness of OFFGEL electrophoresis with the Agilent 3100 OFFGEL Fractionator for the fractionation of peptides. An Escherichia coli tryptic digest was separated in 24 fractions, and peptides were identified by reversed-phase liquid chromatography on a microfluidic device with mass spectrometric detection. About 90% of the identified individual peptides were found in only one or two fractions. The distribution of the calculated isoelectric points for the peptides identified in each fraction was especially narrow in the acidic pH range. Standard deviations approached the size of the pH segment covered by the respective fraction. The experimental peptide isoelectric point measured by OFFGEL electrophoresis was used as an additional filter for validation of peptide identifications. The sample fractionation steps conducted prior to mass detection are critically important for the comprehensive analysis of complex protein mixtures. This paper illustrates the effectiveness of OFFGEL electrophoresis with the Agilent 3100 OFFGEL Fractionator for the fractionation of peptides. An Escherichia coli tryptic digest was separated in 24 fractions, and peptides were identified by reversed-phase liquid chromatography on a microfluidic device with mass spectrometric detection. About 90% of the identified individual peptides were found in only one or two fractions. The distribution of the calculated isoelectric points for the peptides identified in each fraction was especially narrow in the acidic pH range. Standard deviations approached the size of the pH segment covered by the respective fraction. The experimental peptide isoelectric point measured by OFFGEL electrophoresis was used as an additional filter for validation of peptide identifications. Comprehensive analysis of whole proteomes is an extraordinary challenge because of the complexity and wide range of protein concentrations. The success of proteome analysis projects is highly dependent on the quality of the sample fractionation employed prior to analysis by MS. Reducing sample complexity through efficient fractionation allows more complete in-depth analysis of the sample with MS/MS. For peptide-level analysis, cation-exchange (SCX) 1The abbreviations used are: SCX, strong cation-exchange chromatography; RP, reversed-phase chromatography; SPI, scored peak intensity; HPLC-Chip/MS, high performance liquid chromatography on a microfluidic device with mass spectrometric detection. and reversed-phase chromatography (RP) are typically combined (1Wolters D.A. Washburn M.P. Yates III, J.R. An automated multidimensional protein identification technology for shotgun proteomics.Anal. Chem. 2001; 73: 5683-5690Crossref PubMed Scopus (1572) Google Scholar). The use of IPG IEF instead of SCX has been described before (2Cargile B.J. Talley D.L. Stephenson Jr., J.L. Immobilized pH gradients as a first dimension in shotgun proteomics and analysis of the accuracy of pI predictability of peptides.Electrophoresis. 2004; 25: 936-945Crossref PubMed Scopus (136) Google Scholar, 3Essader A.S. Cargile B.J. Bundy J.L. Stephenson Jr., J.L. A comparison of immobilized pH gradient isoelectric focusing and strong-cation-exchange chromatography as a first dimension in shotgun proteomics.Proteomics. 2005; 5: 24-34Crossref PubMed Scopus (136) Google Scholar). Compared with cation-exchange chromatography, IPG IEF provides higher resolution separation and experimentally derived pI information, which can be used as a filter criterion for tandem mass spectral data validation (3Essader A.S. Cargile B.J. Bundy J.L. Stephenson Jr., J.L. A comparison of immobilized pH gradient isoelectric focusing and strong-cation-exchange chromatography as a first dimension in shotgun proteomics.Proteomics. 2005; 5: 24-34Crossref PubMed Scopus (136) Google Scholar, 4Cargile B.J. Bundy J.L. Stephenson Jr., J.L. Potential for false positive identifications from large databases through tandem mass spectrometry.J. Proteome Res. 2004; 3: 1082-1085Crossref PubMed Scopus (173) Google Scholar). However, a major limitation of this method is the tedious post-IEF sample processing that requires cutting the IPG gel strip into sections and then extracting and cleaning up peptides from the gel sections. In the present work, an Escherichia coli total protein digest was fractionated by OFFGEL electrophoresis with the Agilent 3100 OFFGEL Fractionator and by reversed-phase chromatography and then analyzed by mass detection (RP-LC/MS). OFFGEL electrophoresis is a recent advance in separation technology that fractionates proteins or peptides according to their pI (5Michel P.E. Reymond F. Arnaud I.L. Josserand J. Girault H.H. Rossier J.S. Protein fractionation in a multicompartment device using Off-Gel isoelectric focusing.Electrophoresis. 2003; 24: 3-11Crossref PubMed Scopus (138) Google Scholar, 6Heller M. Michel P.E. Morier P. Crettaz D. Wenz C. Tissot J.D. Reymond F. Rossier J.S. Two-stage Off-Gel isoelectric focusing: protein followed by peptide fractionation and application to proteome analysis of human plasma.Electrophoresis. 2005; 26: 1174-1188Crossref PubMed Scopus (102) Google Scholar, 7Heller M. Ye M. Michel P.E. Morier P. Stalder D. Junger M.A. Aebersold R. Reymond F. Rossier J.S. Added value for tandem mass spectrometry shotgun proteomics data validation through isoelectric focusing of peptides.J. Proteome Res. 2005; 4: 2273-2282Crossref PubMed Scopus (95) Google Scholar, 8Michel P.E. Crettaz D. Morier P. Heller M. Gallot D. Tissot J.D. Reymond F. Rossier J.S. Proteome analysis of human plasma and amniotic fluid by Off-Gel™ isoelectric focusing followed by nano-LC-MS/MS.Electrophoresis. 2006; 27: 1169-1181Crossref PubMed Scopus (100) Google Scholar). This technique achieves the same high resolution as IPG gels but recovers the sample in the liquid phase. Therefore, it fits nicely into the LC/MS workflow. After fractionation and acidification, a portion of the sample was injected directly onto a chip-based reversed-phase column without additional sample preparation, eliminating the need for tedious and error-prone peptide isolation from the IPG gel. The distribution of the calculated isoelectric points for the identified peptides in the respective fractions was especially narrow in the acidic pH range, with standard deviations in the range of the expected fraction width. About 90% of the identified peptides were found in only one or two fractions, demonstrating the resolution of the technique. The combination of peptide fractionation by OFFGEL electrophoresis and LC/MS data analysis with the Spectrum Mill software allowed the use of the experimental isoelectric points as an efficient additional filter for validation of peptide identifications. An E. coli total protein lysate from Bio-Rad was reduced and denatured using 50% 2,2,2-trifluoroethanol with 200 mm dithiothreitol at 95 °C for 20 min. This was followed by alkylation with iodoacetamide at room temperature for 1 h. The reduced and alkylated sample was diluted 1:10. Trypsin (Promega, Madison, WI) was added at 1:20 enzyme:substrate, and then the sample was incubated overnight at 37 °C. The digest was aliquoted, dried, and stored frozen until use. For pI-based peptide separation, the 3100 OFFGEL Fractionator and the OFFGEL Kit pH 3–10 (both Agilent Technologies) with a 24-well setup was used according to the protocol of the supplier. Ten min prior to sample loading, 24-cm-long IPG gel strips with a linear pH gradient ranging from 3 to 10 were rehydrated in the assembled device with 25 μl of focusing buffer per well. Two hundred μg of the E. coli tryptic digest was diluted in focusing buffer to a final volume of 3.6 ml, and 150 μl of sample was loaded in each well. The sample was focused with a maximum current of 50 μA and typical voltages ranging from 500 to 4000 V until 50 kVh was reached after 24 h. The recovered fractions (volumes between 100 and 150 μl) were acidified with 5 μl of formic acid. A 0.5-μl aliquot of each OFFGEL electrophoresis fraction was injected onto an LC/MS system consisting of an 1100 Series liquid chromatograph, HPLC-Chip Cube MS interface, and 1100 Series LC/MSD Trap XCT Ultra ion trap mass spectrometer (all Agilent Technologies). The system was equipped with an HPLC-Chip (Agilent Technologies) that incorporated a 40-nl enrichment column and a 43-mm × 75-μm analytical column packed with Zorbax 300SB-C18 5-μm particles. Peptides were loaded onto the enrichment column with 97% solvent A (water with 0.1% formic acid) and 3% B (90% acetonitrile with 0.1% formic acid) at 4 μl/min. They were then eluted with a gradient from 3% B to 45% B in 30 min, followed by a steep gradient to 80% B in 5 min at a flow rate of 0.3 μl/min. The total runtime, including column reconditioning, was 45 min. Molecular masses were recorded using data-dependent MS/MS acquisition. The MS and MS/MS conditions employed were: Drying gas flow: 4 liters/min, 300 °C; capillary voltage: 1800 V; skim 1: 30 V; capillary exit: 75 V; trap drive: 85; averages: 1; ion current control: on; maximum accumulation time: 150 ms; smart target: 500,000; MS scan range: 300–2000; ultra scan: on.MS/MS: number of parents: 5; averages: 1; fragmentation amplitude: 1.25 V; SmartFrag: on, 30–200%; active exclusion: on, 2 spectra, 1 min; prefer +2: on; exclude +1: on, MS/MS scan range: 100–2000; ultra scan: on; ion current control target: 500,000. The SwissProt database was searched with the restriction to E. coli, using the Agilent Spectrum Mill Server software (Rev A.03.02.) installed on a dual Xeon 2.4-GHz computer. Peak lists were created with the Spectrum Mill Data Extractor program with the following attributed: scans with the same precursor ± 1.4 m/z were merged within a time frame of ± 15 s. Precursor ions needed to have a minimum signal to noise value of 25. Charges up to a maximum of 7 were assigned to the precursor ion, and the 12C peak was determined by the Data Extractor. The SwissProt database (01/12/03), with 120,961 total entries and 4,830 for E. coli proteins, was searched for tryptic peptides with a mass tolerance of ± 2.5 Da for the precursor ions and a tolerance of ± 0.7 Da for the fragment ions. Two missed cleavages were allowed. A Spectrum Mill autovalidation was performed first in the protein details mode. Minimum scores, minimum scored peak intensity (SPI), forward minus reversed score threshold, and rank 1 minus rank 2 score threshold for peptides were dependent on the assigned precursor charge (see Table I).Table IDatabase search settingsPrecursor chargeMin. scoreMin. SPIMin. fwd-rev scoreMin. rank 1–2 score16.070%2226.060%2226.090%1138.070%2248.070%22 Open table in a new tab Then autovalidation in the peptide mode was performed using a score threshold of 13 and SPI of 70% for 1+, 3+, and 4+ and 11 and 60% for 2+ precursor ions. Forward minus reversed score threshold and rank 1 minus rank 2 score threshold were set to 2. All protein hits found in a distinct database search by Spectrum Mill are non-redundant. To eliminate redundancy, the Protein Summary Modes groups all proteins that have at least one common peptide, and only the highest scoring member of each protein group is shown and counted in the protein list. The recently developed OFFGEL electrophoresis method was used in this work to fractionate E. coli peptides in the pH range of 3–10 prior to mass analysis on a HPLC-Chip/MS system. The experimental setup employed for fractionation by OFFGEL electrophoresis is shown schematically in Fig. 1. Protein or peptide separation takes place in a two-phase system with an upper liquid phase that is divided in compartments and a lower phase that is a conventional rehydrated IPG gel strip. Typically, the sample is diluted in the focusing buffer and loaded into all wells. Because there is no fluidic connection between the wells, proteins or peptides are forced to migrate through the IPG gel where the actual separation takes place. After IEF, the proteins or peptides are present in the liquid phase and can be recovered conveniently from the wells for further processing. Tandem mass spectroscopic data were analyzed with the Spectrum Mill software and the SwissProt database restricted to E. coli using autovalidation criteria as described above. In total, 3454 peptides and 670 proteins were identified. A list of all protein hits that gives information about the number of unique peptides, sequence coverage, and peptide scores, etc. is available as supplemental data 1. More detailed information regarding protein assignments based on single-peptide assignments is available as supplemental data 2. An in silico tryptic digest of the whole E. coli proteome shows that the pI values of E. coli peptides are unevenly distributed across the pH scale with gaps at pH 7–8 and around pH 5 and pH 9 and the majority of peptides clustering at pH 3.6–4.8 and pH 5.2–6.2 (9Cargile B.J. Bundy J.L. Freeman T.W. Stephenson Jr., J.L. Gel based isoelectric focusing of peptides and the utility of isoelectric point in protein identification.J. Proteome Res. 2004; 3: 112-119Crossref PubMed Scopus (120) Google Scholar). In agreement with these theoretical considerations, most peptides were identified in OFFGEL electrophoresis fractions 2–6 (covering on the IPG gel the pH range 3.6–4.9), 9–14 (pH range 5.7–7.0) and 21–24 (pH range 8.9–9.7) (Table II, Fig. 2a).Table IIResults of the analysis of E. coli peptides by OFFGEL electrophoresis and HPLC-Chip/MSFractionExpected pH rangeUnique/total peptidesAverage pI with standard deviation13.35–3.6143/973.85 ± 0.1923.61–3.88343/4733.99 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Open table in a new tab to the fractionation quality of this technique is to at the number of fractions in which each distinct peptide was shown in Fig. of the identified peptides are found in only one fraction and about 90% are found in one or two fractions. In agreement with (2Cargile B.J. Talley D.L. Stephenson Jr., J.L. Immobilized pH gradients as a first dimension in shotgun proteomics and analysis of the accuracy of pI predictability of peptides.Electrophoresis. 2004; 25: 936-945Crossref PubMed Scopus (136) Google most peptides unique to each fraction were found in the pH range from 3.6 to Fig. which this pH segment for a analysis on a narrow range IPG strip (3Essader A.S. Cargile B.J. Bundy J.L. Stephenson Jr., J.L. A comparison of immobilized pH gradient isoelectric focusing and strong-cation-exchange chromatography as a first dimension in shotgun proteomics.Proteomics. 2005; 5: 24-34Crossref PubMed Scopus (136) Google Scholar). The Spectrum Mill software the pI value for identified peptide according to the developed by C. F. The focusing of in immobilized pH gradients can be from their PubMed Scopus Google Scholar). these pI values with standard deviations were without data for all peptides identified in each fraction. The pI values with the expected pH range calculated according to the of the IPG gel strip deviations were only for fractions (pH range deviations from the number of peptides found in this pH range (see between expected and pI values were for conventional IPG IEF as (2Cargile B.J. Talley D.L. Stephenson Jr., J.L. Immobilized pH gradients as a first dimension in shotgun proteomics and analysis of the accuracy of pI predictability of peptides.Electrophoresis. 2004; 25: 936-945Crossref PubMed Scopus (136) Google Scholar, 3Essader A.S. Cargile B.J. Bundy J.L. Stephenson Jr., J.L. A comparison of immobilized pH gradient isoelectric focusing and strong-cation-exchange chromatography as a first dimension in shotgun proteomics.Proteomics. 2005; 5: 24-34Crossref PubMed Scopus (136) Google Scholar). Because IEF peptides according to their pI of the standard of the pI value distribution in each fraction an method to the of OFFGEL all fractions an standard of ± pI was However, the standard deviations between fractions. Two were fractions with a number of identified peptides high standard deviations Fig. a and and fractions in the and pH range higher standard deviations fractions in the acidic pH range fractions with a peptide peptide fractions and were the standard to ± pI standard deviations that the pH covered by a pH were for fractions with high peptide only in the acidic pH range (Table A distribution of standard deviations was for conventional IPG IEF as (2Cargile B.J. Talley D.L. Stephenson Jr., J.L. Immobilized pH gradients as a first dimension in shotgun proteomics and analysis of the accuracy of pI predictability of peptides.Electrophoresis. 2004; 25: 936-945Crossref PubMed Scopus (136) Google Scholar). An of the of the pI values of identified peptide from the pI calculated for each fraction is shown in Fig. 4 for as as for For the spectra, a narrow distribution around is with of the within the one of ± pI In to the spectra, the a distribution across the whole pI scale However, of these within one that the autovalidation criteria a number of peptide the experimentally determined pI be used as a filter to false of the of To this the quality of all peptide with ranging from 5 to 10 from fractions 3 and 4 were The majority of the peptide within ± pI of the fraction pI were quality 90% of the peptide with ranging from to 9 were as and in the score range from 9 to 10 only high quality were In to the quality of peptide with pI values ± pI of the fraction pI was lower data that an additional pI filter allows the validation criteria without a higher number of false However, the number of additional that be by this was or in the present the same data quality as only The success of proteomics has to an of the number of in the and is to a because of in separation the application of gel electrophoresis was by the of IPG gels that resolution and to the IEF E. R. focusing in immobilized pH and PubMed Scopus Google Scholar, J. electrophoresis with isoelectric focusing in immobilized pH gradients in the first dimension and to the Scopus (100) Google Scholar). Because gel electrophoresis is and to it be for high to IPG IEF technology with separation that are more to liquid However, the combination of conventional IPG IEF with liquid phase is by the that proteins or peptides have to be from the gel to liquid phase for further This is a tedious and error-prone that steps cutting the IPG strip after IEF, sample and (2Cargile B.J. Talley D.L. Stephenson Jr., J.L. Immobilized pH gradients as a first dimension in shotgun proteomics and analysis of the accuracy of pI predictability of peptides.Electrophoresis. 2004; 25: 936-945Crossref PubMed Scopus (136) Google Scholar, 3Essader A.S. Cargile B.J. Bundy J.L. Stephenson Jr., J.L. A comparison of immobilized pH gradient isoelectric focusing and strong-cation-exchange chromatography as a first dimension in shotgun proteomics.Proteomics. 2005; 5: 24-34Crossref PubMed Scopus (136) Google Scholar). An to this is by OFFGEL This technique takes of the separation of IPG gels but sample after IEF in the liquid phase. resolution protein and peptide can be using this method (5Michel P.E. Reymond F. Arnaud I.L. Josserand J. Girault H.H. Rossier J.S. Protein fractionation in a multicompartment device using Off-Gel isoelectric focusing.Electrophoresis. 2003; 24: 3-11Crossref PubMed Scopus (138) Google Scholar, 6Heller M. Michel P.E. Morier P. Crettaz D. Wenz C. Tissot J.D. Reymond F. Rossier J.S. Two-stage Off-Gel isoelectric focusing: protein followed by peptide fractionation and application to proteome analysis of human plasma.Electrophoresis. 2005; 26: 1174-1188Crossref PubMed Scopus (102) Google Scholar, 7Heller M. Ye M. Michel P.E. Morier P. Stalder D. Junger M.A. Aebersold R. Reymond F. Rossier J.S. Added value for tandem mass spectrometry shotgun proteomics data validation through isoelectric focusing of peptides.J. Proteome Res. 2005; 4: 2273-2282Crossref PubMed Scopus (95) Google Scholar, 8Michel P.E. Crettaz D. Morier P. Heller M. Gallot D. Tissot J.D. Reymond F. Rossier J.S. Proteome analysis of human plasma and amniotic fluid by Off-Gel™ isoelectric focusing followed by nano-LC-MS/MS.Electrophoresis. 2006; 27: 1169-1181Crossref PubMed Scopus (100) Google Scholar). the fractionation quality by OFFGEL electrophoresis by at the distribution of the identified peptides and the distribution of the calculated pI values of the identified peptides in each Fig. Fig. Table The most is the one because it on the accuracy of pI Because about 90% of the peptides were identified only in one or two fractions, that the fractionation quality was However, standard deviations of the pI values in each fraction that were lower ± pH and to the size of the pH segment covered by a in the respective setup pH were only in the acidic pH range. that for the higher standard deviations of fractions in the and pH range are on the one the pI values of peptides in the pH range and the focusing of and peptides M. Ye M. Michel P.E. Morier P. Stalder D. Junger M.A. Aebersold R. Reymond F. Rossier J.S. Added value for tandem mass spectrometry shotgun proteomics data validation through isoelectric focusing of peptides.J. Proteome Res. 2005; 4: 2273-2282Crossref PubMed Scopus (95) Google Scholar). the the lower of pI in the pH range that the accuracy of the pI is in the acidic pH range (2Cargile B.J. Talley D.L. Stephenson Jr., J.L. Immobilized pH gradients as a first dimension in shotgun proteomics and analysis of the accuracy of pI predictability of peptides.Electrophoresis. 2004; 25: 936-945Crossref PubMed Scopus (136) Google Scholar). that the accuracy of pI for a large portion of the standard in each fraction as (2Cargile B.J. Talley D.L. Stephenson Jr., J.L. Immobilized pH gradients as a first dimension in shotgun proteomics and analysis of the accuracy of pI predictability of peptides.Electrophoresis. 2004; 25: 936-945Crossref PubMed Scopus (136) Google Scholar). The of OFFGEL electrophoresis into the the pI of the peptides is this is information that is by peptides by SCX and F. A isoelectric focusing device for separation of protein PubMed Scopus Google proteins are available after isoelectric focusing in a liquid phase the of an automated with an the the method in a with fraction of 150 μl or and available IPG gel strips can be The use of IEF for peptide separation the to experimentally derived pI values to the of peptide identification (3Essader A.S. Cargile B.J. Bundy J.L. Stephenson Jr., J.L. A comparison of immobilized pH gradient isoelectric focusing and strong-cation-exchange chromatography as a first dimension in shotgun proteomics.Proteomics. 2005; 5: 24-34Crossref PubMed Scopus (136) Google Scholar, 4Cargile B.J. Bundy J.L. Stephenson Jr., J.L. Potential for false positive identifications from large databases through tandem mass spectrometry.J. Proteome Res. 2004; 3: 1082-1085Crossref PubMed Scopus (173) Google Scholar). the of an for the combination of peptide fractionation by OFFGEL electrophoresis and LC/MS data analysis with the Spectrum Mill The additional use of a pI filter allowed the of the peptide validation criteria without the false positive However, this in only a in the number of A further be by the pI filter with a filter on experimentally determined peptide the time on a column M. Ye M. Michel P.E. Morier P. Stalder D. Junger M.A. Aebersold R. Reymond F. Rossier J.S. Added value for tandem mass spectrometry shotgun proteomics data validation through isoelectric focusing of peptides.J. Proteome Res. 2005; 4: 2273-2282Crossref PubMed Scopus (95) Google Scholar). OFFGEL electrophoresis is a technique for the fractionation of peptides or proteins that can a in steps of multidimensional separation The of protein OFFGEL electrophoresis with peptide OFFGEL electrophoresis or protein OFFGEL electrophoresis using wide range IPG gel strips with protein OFFGEL electrophoresis using narrow range IPG gel strips has been shown M. Michel P.E. Morier P. Crettaz D. Wenz C. Tissot J.D. Reymond F. Rossier J.S. Two-stage Off-Gel isoelectric focusing: protein followed by peptide fractionation and application to proteome analysis of human plasma.Electrophoresis. 2005; 26: 1174-1188Crossref PubMed Scopus (102) Google Scholar). have peptide fractionation by OFFGEL electrophoresis with the Agilent 3100 OFFGEL Fractionator of the sample combined with resolution and higher identifications because of the of the experimentally derived pI in the validation of peptide identifications. for on OFFGEL electrophoresis as as all at Agilent in the OFFGEL for and for critically the with

BIRD's eye view: Adding periodic BIRD J-refocusing (BIRD=bilinear rotation decoupling) to data acquisition in an HSQC experiment causes broadband homonuclear decoupling, giving a single signal for each proton chemical shift. This pure shift method improves both resolution and signal-to-noise ratio, without the need for special data processing. A method for acquiring pure shift heteronuclear single quantum correlation (HSQC) NMR spectra in real time is described. A windowed acquisition scheme consisting of trains of bilinear rotation decoupling (BIRD)1, 2 refocusing elements is used to acquire chunks of data with refocused JHH modulation while suppressing JXH with broadband heteronuclear decoupling. The resultant spectra show both enhanced resolution in F2 and enhanced signal-to-noise ratio. Scalar spin–spin (J) coupling provides valuable information for molecular structure elucidation, but the multiplet structure it causes is very expensive in terms of spectral resolution. In 1H NMR spectroscopy, multiplets are often many times the width of a single line. It is routine to suppress heteronuclear couplings (JXH) by broadband decoupling,3–7 but only recently have experimental methods for homonuclear broadband decoupling become practical. These “pure shift” or “chemical-shift resolved” or “δ-resolved” methods8–19 can give resolution improvements approaching an order of magnitude, far in excess of any gains to be realistically expected from increases in the static magnetic field. However, all of these methods suffer to a greater or lesser extent from reduced sensitivity compared to conventional measurements. Here we describe an experimental method for obtaining pure shift heteronuclear single quantum correlation (HSQC) spectra, in which real-time homodecoupling using the BIRD pulse sequence element1 leads to the first simultaneous resolution and signal enhancement in the directly detected (1H) dimension. (Homodecoupling has previously been described for the HSQC experiment, but only in the indirect (13C) dimension.20) The HSQC experiment is the most widely used NMR method for correlating the chemical shifts of directly-bonded 13C–1H pairs. In its conventional21 form, it shows proton multiplet structure in F2, which limits resolution in the spectra of complex species. It has recently been shown17, 22, 23 that it is possible to extend the pure shift methods currently used, which rely on stitching together separate measurements of short periods of decoupled signal, to real-time acquisition, in which homonuclear couplings are periodically refocused, by applying appropriate spin manipulations during the acquisition of a single free-induction decay. Such J-refocusing sequence elements are generally designed to be broadband, as distinct from classical selective24, 25 or band-selective26 homodecoupling; in the case of HSQC, J-refocusing uses a BIRD pulse sequence element and a hard (nonselective) 180° pulse. The BIRD sequence element,1 which, as its name suggests, was originally intended for broadband homonuclear decoupling, has, until recently,12 been used almost exclusively for decoupling in the indirect dimension of heteronuclear 2D experiments.27 Here, the combined effect of the BIRD sequence and the hard 180° pulse is to invert only those protons not directly coupled to 13C, thus refocusing the effects of couplings between the latter protons and protons that are directly coupled (bonded) to 13C and whose signals are recorded in HSQC. The great advantage of the BIRD method here is that, in contrast to Zangger–Sterk type methods,8, 9, 22, 23 it incurs no extra sensitivity penalty; indeed, the sensitivity is generally increased. The BIRD sequence element has already been very effectively used to obtain pure shift 1H-13C HSQC spectra,16 and pure shift 1D proton spectra of strongly coupled species.12 In both cases, the pure shift dimension was constructed from multiple separate acquisitions of short chunks of data, requiring ancillary software for the generation of decoupled spectra. Here we demonstrate how pure shift HSQC data with comparable resolution may be obtained much more quickly (to the point where a pure shift spectrum can require less time to acquire than a conventional spectrum) and without the need for any extra data processing. The one restriction is that the nucleus observed indirectly, generally 13C, should not itself show homonuclear coupling; thus, for example, the proposed sequence is not suitable for fully 13C-labeled compounds. The pulse sequence used is shown in Figure 1. The initial part of the sequence is a conventional gHSQC,21 with the double insensitive nuclei enhanced by polarization transfer (INEPT) followed by a windowed data acquisition, in which the effects of homonuclear coupling are periodically refocused. Applying n BIRD/180° J-refocusing elements during the acquisition time (at) results in a free induction decay built up of an initial chunk of data of duration at/2n, (n−1) chunks of duration at/n, and a final chunk of at/2n. Provided that n≫(at×JHH), evolution under the homonuclear scalar coupling can be neglected, although care is needed to ensure that chemical shift evolution is accurately refocused during the J-refocusing element. More frequent J-refocusing gives cleaner spectra, but at the expense of some extra line broadening owing to imperfect refocusing and T2 relaxation. The BIRD real-time acquisition scheme differs slightly in timing from that previously proposed,17 requiring fewer J-refocusing elements for a given spectral quality. Heteronuclear couplings are suppressed as usual by broadband irradiation (denoted CPD in Figure 1); the intermittent nature of the decoupling limits the types of modulation favored. Because BIRD selects protons directly bonded to 13C, one class of coupling is not refocused, that between geminal protons. Spectra thus show singlet signals for all 1H sites except for nonequivalent methylene protons, for which doublet signals are seen (full details of the sequence are given in the Supporting Information). Pulse sequence for real-time pure shift gHSQC using BIRD. Narrow rectangles are 90° RF pulses, wide are 180° pulses, and wide with a diagonal line are either hard 180° pulses or composite 180° pulses. Gradient pulses G1−G4 follow the normal pattern for gHSQC, and τ=1/(41JXH). The dotted proton RF pulse (0–2 times the duration of 90° pulse) centered between δ1 delays is for multiplicity editing; for edited spectra this pulse is 180° and δ1=2τ, which causes methylene protons to appear with opposite phase to methine and methyl; for unedited spectra this pulse is removed and δ1 is set to δ3 plus associated stabilization delay. The second δ1 delay precedes a delay equivalent to a hard proton 180° pulse, which compensates for the evolution during the 180° pulse in middle of the t1 evolution. Each BIRD/180° J-refocusing block consists of a BIRD element, a hard 180° pulse, and a data acquisition window, with small delays (ca. 20 μs) flanking the hard 180° proton pulse set to refocus the chemical shift. The first and last chunks are half in size (at/2n) relative to the rest of the chunks (at/n). Phase cycling: ϕ1=[1 3]4, ϕ2=[0 2], ϕ3=[0 2]8, ϕ4=[0 2]16, ϕ5=[0 1]2, ϕ6=[1 2]2, ϕ7=[2 3]2, ϕR={1 3 1 3 (3 1 3 1)2 1 3 1 3 3 1 3 1 (1 3 1 3)2 3 1 3 1}, all other pulses are of phase 0 (for the explicit phase table, see Table S1). Figure 2 illustrates the application of the new real-time pure shift method to 1H-13C correlated spectra. The conventional gHSQC spectrum (Figure 2 a) of D(+)-fucose shows multiplet structure in the 1H frequency (F2) dimension; the structure is collapsed to singlets in the pure shift spectrum (Figure 2 b) obtained using the real-time pure shift gHSQC sequence of Figure 1. The 1D projections onto the 1H (F2) axis show, as expected, that the singlets in the pure shift spectrum are more intense than the corresponding multiplets in the conventional HSQC. Peak heights increase by an average factor of 1.7 for doublets and 2.9 for multiplets. Linewidths in the pure shift spectrum are very similar to those in the conventional spectrum; although signal losses from imperfect pulses, mismatch between τ and 1JCH, and transverse relaxation should, in principle, lead to wider lines in the pure shift spectrum, for this example the degradation is negligible. Similar results were obtained for quinine (Supporting Information, Figure S3); in this case the wider range of 13C chemical shifts means that some degradation in performance is seen at the edges of the spectrum. Any discontinuities in the decoupled signal, such as those caused by T2 relaxation during the BIRD sequence element, mismatch between the BIRD timing and 1JCH, or a breakdown of the condition n≫(at×JHH), will lead to small F2 sidebands at multiples of n/at. In the current work, the level of these sideband artifacts is typically around 1 % (Figure S5). Selected regions (Indicated with dashed lines in the full spectra of Figure S1) of 1H-13C HSQC spectra of D(+)-fucose in D2O with TSP as internal reference: a) conventional gHSQC; b) real-time pure shift gHSQC. 1D traces are integral projections onto the F2 (1H) axis. Data were acquired, processed, and plotted with equivalent parameters, to allow quantitative comparison. The proposed method is also applicable to 1H-15N correlation, either at natural abundance or in labeled systems where the labels are too far apart for 15N-15N coupling to be significant (as is generally the case in peptides and proteins). Figure 3 compares conventional and real-time pure shift HSQC spectra for 15N-labeled beta-amyloid peptide 1-42 (Aβ). The shaded region in the conventional HSQC spectrum (Figure 3 a) shows doublet resonances, which are collapsed to singlets in the pure shift HSQC spectrum (Figure 3 b). As shown in the spectra, this collapsing of multiplets again improves both the resolution and sensitivity of the signals. Overcrowding in the shaded region is reduced; for example, with overlap between the signals of isoleucines 32 and 41 much reduced in the pure shift spectrum. 1H-15N HSQC spectra of 15N-labeled Aβ in [D6]dimethylsulfoxide containing H2O (5 %): a) conventional gHSQC; b) real-time pure shift gHSQC. 1D spectra are corresponding 1H traces at δ15N of 119.7 ppm. All data were acquired, processed, and plotted with equivalent parameters, to allow quantitative comparison. Expansions from shaded regions are shown in Figure S6. In conclusion, the pure shift gHSQC method described here leads to complete collapse of multiplet resonances into singlets (except for nonequivalent methylene signals, which collapse to doublets). This homonuclear decoupling produces signals with increased intensity and better resolution, lowering detection limits, speeding up experiments, and improving the ability to distinguish between signals in complex spectra. This method is potentially well-suited to automated spectral analysis, as a single signal is seen for each distinct chemical site or correlation. All experimental data were obtained using a Varian VNMRS 500 MHz spectrometer equipped with a triple resonance (1H/13C/15N) triple axis gradient probe of maximum z gradient 68.5 G cm−1, using GARP5 heteronuclear decoupling (γB2/2π=4.2 kHz for 13C, 1.3 kHz for 15N) during data acquisition and BIP28 composite pulses. The spectra in Figure 2 were acquired at 20 °C using a 100 mM sample of D(+)-fucose in deuterium oxide, containing trimethylsilyl propanoic acid (TSP) as internal reference. The unusually high concentration was used in order to confirm that clean results are obtainable, with artifact signals at around the 1 % level. The following experimental and processing parameters were used: a hard 90° 1H pulse of duration 10.9 μs, a hard 13C 900 pulse of duration 15.2 μs, a BIP composite 180° pulse (for Figure 2 b) of duration 125 μs and bandwidth 25 kHz; INEPT transfer delays τ=1.66 ms and BIRD delays 2τ=3.31 ms (equivalent to 1JCH=151 Hz); homospoil gradient pulses of 23.0 G cm−1 (G1) and 13.8 G cm−1 (G3) of durations 4.0 ms (δ2) and 2.4 ms (δ4), respectively; and coherence selection (CTP) gradients of 33.4 G cm−1 (G2) and 16.8 G cm−1 (G4) of durations 2.0 ms (δ3) and 1.0 ms (δ5), respectively; 1H spectral width (sw) was 3592.0 Hz; 4 transients were averaged for each of 2×512 free induction decays in which t1 was incremented to provide a 13C spectral width of 11 467.9 Hz (sw1) in the F1 dimension; total number of points (np) stored per FID was 4104, and for Figure 2 b n was 27. Data were zero filled to 16 384×8192, and Gaussian weighting was applied before double Fourier transformation. The total experiment times were 4.2 h for Figure 2 a and 4.4 h for Figure 2 b, the slightly greater duration for the latter arising from the 27 extra BIRD/180° elements in each FID. For Figure 3, data were acquired at 25 °C using a solution of 15N-labeled Aβ in [D6]dimethylsulfoxide containing H2O (5 %). Experimental and processing parameters were: a hard 90° 1H pulse of duration 12.8 μs, a hard 15N 90° pulse of duration 44 μs, a BIP composite 180° pulse (for Figure 3 b) of duration 400 μs and bandwidth 6.5 kHz; INEPT transfer delays τ=2.78 ms and BIRD delays 2τ=5.56 ms (equivalent to 1JNH=90 Hz); homospoil gradient pulses of 23.0 G cm−1 (G1) and 13.8 G cm−1 (G3) of durations 4.0 ms (δ2) and 2.4 ms (δ4), respectively; and coherence selection (CTP) gradients of 33.4 G cm−1 (G2) and 16.9 G cm−1 (G4) of durations 2.0 ms (δ3) and 0.4 ms (δ5), respectively; 1H spectral width (sw) was 10.0 kHz; 32 transients were averaged for each of 2×64 free induction decays in which t1 was incremented to provide a 15N spectral width of 3.0 kHz (sw1) in the F1 dimension; number of points (np) sampled per FID was 4096, and for Figure 3 b n was 8. Data were zero filled to 16 384×512 and then Fourier transformed without weighting. The total experiment time was approximately 2.7 h in each case. 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Aims to provide an overview of major work that has been carried out in the area of strategy development in the past and outlines how it will develop in the future. Presents an analysis of the term “strategy”. Discusses the evolution of different conceptual frameworks over time together with their impact on today′s understanding of strategy formulation and implementation. Concludes by presenting the requirements for a dynamic approach to strategy development together with the way in which it can be realized.

Two-dimensional liquid chromatography (2D-LC) is increasingly being viewed as a viable tool for solving difficult separation problems, ranging from targeted separations of structurally similar molecules to untargeted separations of highly complex mixtures. In spite of this performance potential, though, many users find method development challenging and most frequently cite the “incompatibility” between the solvent systems used in the first and second dimensions as a major obstacle. This solvent strength related incompatibility can lead to severe peak distortion and loss of resolution and sensitivity in the second dimension. In this paper, we describe a novel approach to address the incompatibility problem, which we refer to as Active Solvent Modulation (ASM). This valve-based approach enables dilution of 1D effluent with weak solvent prior to transfer to the 2D column but without the need for additional instrument hardware. ASM is related to the concept we refer to as Fixed Solvent Modulation (FSM), with the important difference being that ASM allows toggling of the diluent stream during each 2D separation cycle. In this work, we show that ASM eliminates the major drawbacks of FSM including complex elution solvent profiles, baseline disturbances, and slow 2D re-equilibration and demonstrate improvements in 2D separation quality using both simple small molecule probes and degradants of heat-treated bovine insulin as case studies. We believe that ASM will significantly ease method development for 2D-LC, providing a path to practical methods that involve both highly complementary 1D and 2D separations and sensitive detection.

A multi-target method for the determination of 191 fungal metabolites in almonds, hazelnuts, peanuts and pistachios was developed. The method includes all mycotoxins regulated in the European Union and mycotoxins regularly found in food. After extraction with an acidified acetonitrile water mixture, the raw extract was diluted and injected directly into the UHPLC-MS/MS system. In two chromatographic runs, analysis was performed in positive and in negative ionisation mode. The method was in-house validated for the most important 65 analytes in these four commodities. Apparent recoveries between 80 and 120% were obtained for about half of the analyte-matrix combinations. Good repeatabilities (standard deviations < 10%) were achieved for the vast majority (83%) of all cases. Only in 6% of all combinations did the standard deviations exceed 15%. Matrix effects, arising during electrospray ionisation, significantly influenced the determination. For instance, signal suppression was observed for several early-eluting analytes and also signal enhancement up to 295% for physcion in peanuts was determined. Concerning extraction recovery, 94% of the analyte-matrix combinations showed values higher than 50%. Lower limits of quantification ranged between 0.04 μg kg(-1) for enniatin B3 in peanuts and 500 μg kg(-1) for HC toxin in hazelnuts. Additionally, the applicability of the developed method was demonstrated through the analysis of 53 naturally contaminated nut samples from Austria and Turkey. Overall, 40 toxins were quantified; the most frequently found mycotoxins were beauvericin (79%), enniatin B (62%) and macrosporin (57%). In the most contaminated hazelnut sample, 26 different fungal metabolites were detected.

Based on protein sequence data and RT-PCR, a full length cDNA encoding betanidin 5-O-glucosyltransferase (5-GT) was obtained from a cDNA library of Dorotheanthus bellidiformis (Burm.f.) N.E.Br. (Aizoaceae). 5-GT catalyses the transfer of glucose from UDP-glucose to the 5-hydroxyl group of the chromogenic betanidin. Betanidin and its conjugates, referred to as betacyanins, are characteristic fruit and flower pigments in most members of the Caryophyllales, which fail to synthesise anthocyanins. The 5-GT cDNA displayed homology to previously published glucosyltransferase sequences and exhibited high identity to sequences of several inducible glucosyltransferases of tobacco and tomato (Solanaceae). The open reading frame encodes a polypeptide of 489 amino acids with a calculated molecular mass of 55.24 kDa. The corresponding cDNA was expressed in Escherichia coli. The recombinant protein displayed identical substrate specificity compared to the native enzyme purified from D. bellidiformis cell suspension cultures. In addition to the natural substrate betanidin, ortho-dihydroxylated flavonols and flavones were glycosylated preferentially at the B-ring 4'-hydroxyl group. 5-GT is the first enzyme of betalain biosynthesis in plants, of which the corresponding cDNA has been cloned and expressed. The results are discussed in relation to molecular evolution of plant glucosyl- transferases.

The tubulin monomers of brain microtubules reassembled in vitro are arranged on a 3-start helix, irrespective of whether the number of protofilaments is 13 or 14. The dimer packing is that of the B-lattice described for flagellar microtubules. This implies that the tubulin core of microtubules contains at least one helical discontinuity. Neither 5-start nor 8-start helices have a physical significance and thus cannot be implicated in models of microtubule elongation, but the structure is compatible with elongation of protofilaments by dimers or protofilamentous oligomers. The inner and outer surfaces of the microtubule wall can be visualized by propane jet freezing, freeze fracturing, and metal replication, at a resolution of at least 4 nm. The 3-start helix is left-handed, in contrast to a previous study based on negative staining and shadowing. The reasons for this discrepancy are discussed.

Metabolism in cells adapts quickly to changes in nutrient availability and cellular differentiation status, including growth conditions in cell culture settings. The last decade saw a vast increase in three-dimensional (3D) cell culture techniques, engendering spheroids and organoids. These methods were established to improve comparability to in vivo situations, differentiation processes and growth modalities. How far spheroids mimic in vivo metabolism, however, remains enigmatic. Here, to our knowledge, we compare for the first time metabolic fingerprints between cells grown as a single layer or as spheroids with freshly isolated in situ tissue. While conventionally grown cells express elevated levels of glycolysis intermediates, amino acids and lipids, these levels were significantly lower in spheroids and freshly isolated primary tissues. Furthermore, spheroids differentiate and start to produce metabolites typical for their tissue of origin. 3D grown cells bear many metabolic similarities to the original tissue, recommending animal testing to be replaced by 3D culture techniques.

Today, it is necessary to identify relevant compounds appearing in discovery and development of new drug substances in the pharmaceutical industry. For that purpose, the measurement of accurate molecular mass and empirical formula calculation is very important for structure elucidation in addition to other available analytical methods. In this work, the identification and confirmation of degradation products in a finished dosage form of the antibiotic drug amoxicillin obtained under stress conditions will be demonstrated. Structure elucidation is performed utilizing liquid chromatography (LC) ion trap MS/MS and MS3 together with accurate mass measurement of the molecular ions and of the collision induced dissociation (CID) fragments by liquid chromatography electro spray ionization time-of-flight mass spectrometry (LC/ESI-TOF).

A fast, easy-to-handle and cost-effective analytical method for 11 mycotoxins currently regulated in maize and other cereal-based food products in Europe was developed and validated for maize. The method is based on two extraction steps using different acidified acetonitrile–water mixtures. Separation is achieved using ultrahigh-performance liquid chromatography (UHPLC) by a linear water–methanol gradient. After electrospray ionisation, tandem mass spectrometric detection is performed in dynamic multiple reaction monitoring mode. Since accurate mass spectrometric quantification is hampered by matrix effects, uniformly [(13)C]-labelled mycotoxins for each of the 11 compounds were added to the sample extracts prior to UHPLC-MS/MS analysis. Method performance parameters were obtained by spiking blank maize samples with mycotoxins before as well as after extraction on six levels in triplicates. The twofold extraction led to total recoveries of the extraction steps between 97% and 111% for all target analytes, including fumonisins. The [(13)C]-labelled internal standards efficiently compensated all matrix effects in electrospray ionisation, leading to apparent recoveries between 88% and 105% with reasonable additional costs. The relative standard deviations of the whole method were between 4% and 11% for all analytes. The trueness of the method was verified by the measurement of several maize test materials with well-characterized concentrations. In conclusion, the developed method is capable of determining all regulated mycotoxins in maize and presuming similar matrix effects and extraction recovery also in other cereal-based foods.

Substitution of the asparagine-linked GlcNAc by alpha1,3-linked fucose is a widespread feature of plant as well as of insect glycoproteins, which renders the N-glycan immunogenic. We have purified from mung bean seedlings the GDP-L-Fuc:Asn-linked GlcNAc alpha1,3-fucosyltransferase (core alpha1,3-fucosyltransferase) that is responsible for the synthesis of this linkage. The major isoform had an apparent mass of 54 kDa and isoelectric points ranging from 6. 8 to 8.2. From that protein, four tryptic peptides were isolated and sequenced. Based on an approach involving reverse transcriptase-polymerase chain reaction with degenerate primers and rapid amplification of cDNA ends, core alpha1,3-fucosyltransferase cDNA was cloned from mung bean mRNA. The 2200-base pair cDNA contained an open reading frame of 1530 base pairs that encoded a 510-amino acid protein with a predicted molecular mass of 56.8 kDa. Analysis of cDNA derived from genomic DNA revealed the presence of three introns within the open reading frame. Remarkably, from the four exons, only exon II exhibited significant homology to animal and bacterial alpha1,3/4-fucosyltransferases which, though, are responsible for the biosynthesis of Lewis determinants. The recombinant fucosyltransferase was expressed in Sf21 insect cells using a baculovirus vector. The enzyme acted on glycopeptides having the glycan structures GlcNAcbeta1-2Manalpha1-3(GlcNAcbeta1-2Manalpha1- 6)Manbeta1-4GlcNAcbet a1-4GlcNAcbeta1-Asn, GlcNAcbeta1-2Manalpha1-3(GlcNAcbeta1-2Manalpha1- 6)Manbeta1-4GlcNAcbet a1-4(Fucalpha1-6)GlcNAcbeta1-Asn, and GlcNAcbeta1-2Manalpha1-3[Manalpha1-3(Manalpha1-6 )Manalpha1-6]Manbeta1 -4GlcNAcbeta1-4GlcNAcbeta1-Asn but not on, e.g. N-acetyllactosamine. The structure of the core alpha1,3-fucosylated product was verified by high performance liquid chromatography of the pyridylaminated glycan and by its insensitivity to N-glycosidase F as revealed by matrix-assisted laser desorption/ionization time of flight mass spectrometry.

Hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT; EC 2.3.1.110) catalyzes the transfer of hydroxycinnamic acids from the respective CoA esters to tyramine and other amines in the formation of N-(hydroxycinnamoyl)amines. Expression of THT is induced by Phytophthora infestans, the causative agent of late blight disease in potato. The amino acid sequences of nine endopeptidase LysC-liberated peptides from purified potato THT were determined. Using degenerate primers, a THT-specific fragment was obtained by reverse transcription-polymerase chain reaction, and THT cDNA clones were isolated from a library constructed from RNA of elicitor-treated potato cells. The open reading frame encoding a protein of 248 amino acids was expressed in Escherichia coli. Recombinant THT exhibited a broad substrate specificity, similar to that of native potato THT, accepting cinnamoyl-, 4-coumaroyl-, caffeoyl-, feruloyl- and sinapoyl-CoA as acyl donors and tyramine, octopamine, and noradrenalin as acceptors tested. Elicitor-induced THT transcript accumulation in cultured potato cells peaked 5 h after initiation of treatment, whereas enzyme activity was highest from 5 to 30 h after elicitation. In soil-grown potato plants, THT mRNA was most abundant in roots. Genomic Southern analyses indicate that, in potato, THT is encoded by a multigene family.

The pea chloroplastic outer envelope protein OEP24 can function as a general solute channel. OEP24 is present in chloroplasts, etioplasts, and non-green root plastids. The heterologously expressed protein forms a voltage-dependent, high-conductance (Lambda = 1.3 nS in 1 M KCl), and slightly cation-selective ion channel in reconstituted proteoliposomes. The highest open probability (P open approximately 0. 8) is at 0 mV, which is consistent with the absence of a transmembrane potential across the chloroplastic outer envelope. The OEP24 channels allow the flux of triosephosphate, dicarboxylic acids, positively or negatively charged amino acids, sugars, ATP, and Pi. Structure prediction algorithms and circular dichroism spectra indicate that OEP24 contains seven amphiphilic beta strands. The primary structure of OEP24 shows no homologies to mitochondrial or bacterial porins on a primary sequence basis, and OEP24 is functionally not inhibited by cadaverine, which is a potent inhibitor of bacterial porins. We conclude that OEP24 represents a new type of solute channel in the plastidic outer envelope.

Proteasomes are involved in the proteolytic generation of major histocompatibility complex (MHC) class I epitopes but their exact role has not been elucidated. We used highly purified murine 20S proteasomes for digestion of synthetic 22-mer and 41/44-mer ovalbumin partial sequences encompassing either an immunodominant or a marginally immunogenic epitope. At various times, digests were analyzed by pool sequencing and by semiquantitative electrospray ionization mass spectrometry. Most dual cleavage fragments derived from 22-mer peptides were 7-10 amino acids long, with octa- and nonamers predominating. Digestion of 41/44-mer peptides initially revealed major cleavage sites spaced by two size ranges, 8 or 9 amino acids and 14 or 15 amino acids, followed by further degradation of the latter as well as of larger single cleavage fragments. The final size distribution was slightly broader than that of fragments derived from 22-mer peptides. The majority of peptide bonds were cleaved, albeit with vastly different efficiencies. This resulted in multiple overlapping proteolytic fragments including a limited number of abundant peptides. The immunodominant epitope was generated abundantly whereas only small amounts of the marginally immunogenic epitope were detected. The frequency distributions of amino acids flanking proteasomal cleavage sites are correlated to that reported for corresponding positions of MHC class I binding peptides. The results suggest that proteasomal degradation products may include fragments with structural properties similar to MHC class I binding peptides. Proteasomes may thus be involved in the final stages of proteolytic epitope generation, often without the need for downstream proteolytic events.

Mugwort and birch pollen allergy are frequently associated with IgE-mediated hypersensitivity to celery and spices. We analyzed 22 sera from patients with the mugwort-birch-celery-spice syndrome for IgE binding to the spices pepper and paprika by immunoblotting. Immunoblot results revealed two major allergens of 28 and 60 kDa in pepper and a 23-kDa allergen together with allergens of higher molecular weight in paprika. In immunoblot-inhibition studies, crude mugwort, birch pollen, and celery extracts significantly reduced the IgE binding to pepper and paprika allergens. However, no inhibition was achieved with rBet v 1 and rBet v 2, suggesting that no homologs of these birch proteins act as allergens in pepper or paprika extracts. N-terminal sequence analysis of the 14- and 28-kDa pepper and 23-kDa paprika allergens revealed no homology to known allergens. The 28-kDa pepper allergen showed homology to a wheat germin protein, and the 23-kDa paprika allergen was identified as a homolog of a osmotin-like or pathogenesis-related protein in tomato. Therefore, we conclude that the IgE cross-reactivity in the mugwort-birch-celery-spice syndrome to the spices pepper and paprika is not caused by homologs of Bet v 1 and profilin. N-terminal amino acid sequence analysis of the main allergens in pepper and paprika indicate a relation to frequently occurring plant proteins.

BACKGROUND: Little is known about the role of bell peppers in food allergy. We collected sera from 11 patients with food allergy to bell peppers to analyze bell pepper extracts for allergen composition. METHODS: Proteins of mature fruits of eight horticultural strains of bell peppers were extracted and tested with patients' sera for IgE binding and with monoclonal and polyclonal antibodies in immunoblot. RESULTS: Profilin was detected in bell pepper extracts by an anti-celery profilin antibody. It showed high IgE binding activity in all extracts, which could be inhibited by recombinant birch pollen profilin. Anti-birch pollen monoclonal antibody BIP3, directed against birch pollen proteins between 30 and 69 kD, bound to bell pepper antigens of comparable molecular weights. A homologue of the major birch pollen allergen Bet v 1 was detected in four of eight horticultural strains of bell peppers, and was shown to bind IgE in 1 of the 11 patients. A 23-kD allergen of bell peppers was shown to correspond to the 23-kD major paprika allergen by IgE absorption experiments. Its N-terminal sequence showed 100% identity to P23 from tomatoes. CONCLUSION: The appearance of profilin in all and Bet v 1 in 50% of the tested horticultural strains indicates that bell peppers have to be considered potentially dangerous for Bet v 1- and profilin-sensitized patients. Moreover, in 4 of 8 horticultural strains of bell peppers a homologue of the osmotin-like protein P23 from tomatoes is responsible for substantial IgE binding. Contact with Bet v 1 and P23 homologues in bell peppers can therefore be minimized by avoidance of the respective horticultural strains.