MALDI versus ESI: The Impact of the Ion Source on Peptide Identification.

For mass spectrometry-based proteomic analyses, electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) are the commonly used ionization techniques. To investigate the influence of the ion source on peptide detection in large-scale proteomics, an optimized GeLC/MS workflow was developed and applied either with ESI/MS or with MALDI/MS for the proteomic analysis of different human cell lines of pancreatic origin. Statistical analysis of the resulting data set with more than 72 000 peptides emphasized the complementary character of the two methods, as the percentage of peptides identified with both approaches was as low as 39%. Significant differences between the resulting peptide sets were observed with respect to amino acid composition, charge-related parameters, hydrophobicity, and modifications of the detected peptides and could be linked to factors governing the respective ion yields in ESI and MALDI.

Peptidomics-based phylogeny and biogeography of Mantophasmatodea (Hexapoda).

The insect order Mantophasmatodea was described in 2002. Prior to that time, several generations of entomologists had assumed that all major insect taxa were known; thus, its description was a sensation for zoologists. Since then, a surprising abundance and species diversity of this taxon have been found, particularly in the winter rainfall region of South Africa. To learn more about the evolutionary lineages, speciation, and biogeography of Mantophasmatodea, we applied an unusual peptidomics approach. We collected specimens of almost all known and novel taxa of these insects, developed methods for immediate sample preparation in the field, introduced peptide mass fingerprints for the unambiguous identification of taxa, and subsequently analyzed the most extensive data set on peptide hormones ever compiled for insect taxa. To account for intraspecific variation, we analyzed several individuals per putative species. Increased taxon sampling was preferred over a further increase in the number of characters to optimize the accuracy of phylogenetic analyses. The large data set made it possible to test the validity of using neuropeptide sequences, which coevolve with their respective receptors, to analyze phylogenetic relationships among closely related taxa. Altogether, the data from 71 populations of Mantophasmatodea were sufficient to clearly separate the major clades of Mantophasmatodea, including previously undescribed taxa such as Pachyphasma, Striatophasma, and Austrophasmatidae gen. et sp. nov. "RV." The data confirm the monophyly of Austrophasmatidae and show a relatively recent and extensive radiation in the winter rainfall region of South Africa but also suggest that the species-level diversification of Namibian Mantophasma is less marked than previously thought. We discuss the biogeographical and ecological factors that may have resulted in different regional patterns of endemism and species diversity in Mantophasmatodea. The unique development of the neuroendocrine capa-neurons in the ventral nervous system is described as synapomorphy of Mantophasmatodea + Grylloblattodea and is a further argument for a close relationship between these insect taxa.

New sensitive high-performance liquid chromatography-tandem mass spectrometry method for the detection of horse and pork in halal beef.

The accidental or fraudulent blending of meat from different species is a highly relevant aspect for food product quality control, especially for consumers with ethical concerns against species, such as horse or pork. In this study, we present a sensitive mass spectrometrical approach for the detection of trace contaminations of horse meat and pork and demonstrate the specificity of the identified biomarker peptides against chicken, lamb, and beef. Biomarker peptides were identified by a shotgun proteomic approach using tryptic digests of protein extracts and were verified by the analysis of 21 different meat samples from the 5 species included in this study. For the most sensitive peptides, a multiple reaction monitoring (MRM) method was developed that allows for the detection of 0.55% horse or pork in a beef matrix. To enhance sensitivity, we applied MRM(3) experiments and were able to detect down to 0.13% pork contamination in beef. To the best of our knowledge, we present here the first rapid and sensitive mass spectrometrical method for the detection of horse and pork by use of MRM and MRM(3).

Prevalidation of potential protein biomarkers in toxicology using iTRAQ reagent technology.

Today, toxicoproteomics still relies mainly on 2-DE followed by MS for detection and identification of proteins, which might characterize a certain state of disease, indicate toxicity or even predict carcinogenicity. We utilized the classical 2-DE/MS approach for the evaluation of early protein biomarkers which are predictive for chemically induced hepatocarcinogenesis in rats. We were able to identify statistically significantly deregulated proteins in N-nitrosomorpholine exposed rat liver tissue. Based on literature data, biological relevance in the early molecular process of hepatocarcinogenicity could be suggested for most of these potential biomarkers. However, in order to ensure reliable results and to create the prerequisites necessary for integration in routine toxicology studies in the future, these protein expression patterns need to be prevalidated using independent technology platforms. In the current study, we evaluated the usefulness of iTRAQ reagent technology (Applied Biosystems, Framingham, USA), a recently introduced MS-based protein quantitation method, for verification of the 2-DE/MS biomarkers. In summary, the regulation of 26 2-DE/MS derived protein biomarkers could be verified. Proteins like HSP 90-beta, annexin A5, ketohexokinase, N-hydroxyarylamine sulfotransferase, ornithine aminotransferase, and adenosine kinase showed highly comparable fold changes using both proteomic quantitation strategies. In addition, iTRAQ analysis delivered further potential biomarkers with biological relevance to the processes of hepatocarcinogenicity: e.g. placental form of glutathione S-transferase (GST-P), carbonic anhydrase, and aflatoxin B1 aldehyde reductase. Our results show both the usefulness of iTRAQ reagent technology for biomarker prevalidation as well as for identification of further potential marker proteins, which are indicative for liver hepatocarcinogenicity.

A second outer-core region in Klebsiella pneumoniae lipopolysaccharide.

Up to now only one major type of core oligosaccharide has been found in the lipopolysaccharide of all Klebsiella pneumoniae strains analyzed. Applying a different screening approach, we identified a novel Klebsiella pneumoniae core (type 2). Both Klebsiella core types share the same inner core and the outer-core-proximal disaccharide, GlcN-(1,4)-GalA, but they differ in the GlcN substituents. In core type 2, the GlcpN residue is substituted at the O-4 position by the disaccharide beta-Glcp(1-6)-alpha-Glcp(1, while in core type 1 the GlcpN residue is substituted at the O-6 position by either the disaccharide alpha-Hep(1-4)-alpha-Kdo(2 or a Kdo residue (Kdo is 3-deoxy-D-manno-octulosonic acid). This difference correlates with the presence of a three-gene region in the corresponding core biosynthetic clusters. Engineering of both core types by interchanging this specific region allowed studying the effect on virulence. The replacement of Klebsiella core type 1 in a highly type 2 virulent strain (52145) induces lower virulence than core type 2 in a murine infection model.

Separation and first structure elucidation of Cremophor EL-components by hyphenated capillary electrophoresis and delayed extraction-matrix assisted laser desorption/ionization-time of flight-mass spectrometry.

The polyethoxylated heterogeneous components of the so far poorly characterized nonionic emulsifier Cremophor EL (polyoxyl 35 castor oil) (CrEL) were fractionated by cyclodextrin-modified micellar electrokinetic capillary chromatography (CD-MEKC). Due to the low UV absorbance of most of the CrEL-components an indirect UV detection was used with phenobarbital-sodium as background absorber. For a precise assignment of the resulting peaks to the corresponding components capillary electrophoresis (CE) had to be combined with delayed extraction-matrix assisted laser desorption/ionization-time of flight-mass spectrometry (DE-MALDI-TOF-MS) as detection system. For this purpose, the fractionating robot Probot was employed which enables both the on-line fractionation of the CE eluate on a MALDI target during the electrophoretic separation and the simultaneous dosage of the MALDI matrix solution. The applied CrEL amount was optimized by varying the CE injection parameters time, pressure and concentration of the sample in order to obtain homologue peak series of sufficient intensity without decreasing the separation efficiency. Evaluation of the mass spectra was performed by comparing the residue masses of the homologue peak series with the calculated residue masses of potential CrEL-components. However, the high number of polyethoxylated components leads to overlapping of homologue peak series with isobaric residue masses. These isobaric interferences were detected by a high mass accuracy of the measurements (obtained by internal calibration with polyethylene glycol (PEG) 1000 and by means of the residue mass plot, the newly developed evaluation method. The combination of these techniques allowed the first detailed structure analysis of the CrEL-components showing glycerol polyoxyethylene (POE) monoricinoleate and POE monoricinoleate to be the two main components of the emulsifier. Furthermore, the coupling of CE with DE-MALDI-TOF-MS is generally applicable to the fractionation and identification of polymers.

Deamidation of labile asparagine residues in the autoregulatory sequence of human phenylalanine hydroxylase.

Two dimensional electrophoresis has revealed a microheterogeneity in the recombinant human phenylalanine hydroxylase (hPAH) protomer, that is the result of spontaneous nonenzymatic deamidations of labile asparagine (Asn) residues [Solstad, T. and Flatmark, T. (2000) Eur. J. Biochem.267, 6302-6310]. Using of a computer algorithm, the relative deamidation rates of all Asn residues in hPAH have been predicted, and we here verify that Asn32, followed by a glycine residue, as well as Asn28 and Asn30 in a loop region of the N-terminal autoregulatory sequence (residues 19-33) of wt-hPAH, are among the susceptible residues. First, on MALDI-TOF mass spectrometry of the 24 h expressed enzyme, the E. coli 28-residue peptide, L15-K42 (containing three Asn residues), was recovered with four monoisotopic mass numbers (i.e., m/z of 3106.455, 3107.470, 3108.474 and 3109.476, of decreasing intensity) that differed by 1 Da. Secondly, by reverse-phase chromatography, isoaspartyl (isoAsp) was demonstrated in this 28-residue peptide by its methylation by protein-l-isoaspartic acid O-methyltransferase (PIMT; EC 2.1.1.77). Thirdly, on incubation at pH 7.0 and 37 degrees C of the phosphorylated form (at Ser16) of this 28-residue peptide, a time-dependent mobility shift from tR approximately 34 min to approximately 31 min (i.e., to a more hydrophilic position) was observed on reverse-phase chromatography, and the recovery of the tR approximately 34 min species decreased with a biphasic time-course with t0.5-values of 1.9 and 6.2 days. The fastest rate is compatible with the rate determined for the sequence-controlled deamidation of Asn32 (in a pentapeptide without 3D structural interference), i.e., a deamidation half-time of approximately 1.5 days in 150 mm Tris/HCl, pH 7.0 at 37 degrees C. Asn32 is located in a cluster of three Asn residues (Asn28, Asn30 and Asn32) of a loop structure stabilized by a hydrogen-bond network. Deamidation of Asn32 introduces a negative charge and a partial beta-isomerization (isoAsp), which is predicted to result in a change in the backbone conformation of the loop structure and a repositioning of the autoregulatory sequence and thus affect its regulatory properties. The functional implications of this deamidation was further studied by site-directed mutagenesis, and the mutant form (Asn32-->Asp) revealed a 1.7-fold increase in the catalytic efficiency, an increased affinity and positive cooperativity of L-Phe binding as well as substrate inhibition.

New fragmentation mechanisms in matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry of carbohydrates.

The spectra recorded by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS/MS) of complex carbohydrates from human milk are presented. Besides ions originating from glycosidic cleavages and from sugar ring fragmentations, these spectra show intense peaks that may be assigned to ions produced by three new fragmentation pathways involving a six-atom rearrangement. These ions, together with the A fragments from sugar ring fragmentations, open the possibility of obtaining a complete mapping of the linkage positions present in the carbohydrates investigated by MALDI-TOF/TOF.