Can museum egg specimens be used for proteomic analyses?

Extent: 5p.Background: Mass spectrometry and proteomic analyses have become powerful tools for the analysis of proteins and peptides. Investigation of proteins contained in the various layers of the avian eggshell has focused entirely on domesticated species. It has been widely assumed that this existing research can inform the study of wild bird species despite the fact that the vast majority of the diversity in avian species (~95%) exists outside the Orders to which domestic and poultry species belong. Museum collections offer a potentially valuable source of material for studying composition of wild avian eggshell matrix proteins. We used museum and fresh eggshells of common quails Coturnix coturnix to compare the protein composition of their organic matrices. Four eggs of domestic chickens were analysed simultaneously as a control for comparison to the fresh and museum quail eggs. The determination of the proteins was carried out using enzymatic cleavage followed by high-performance mass spectrometry. Results: We found that some of the expected key eggshell proteins (3 out of 11) were not present in the samples of museum quail egg. These proteins were either entirely absent from the museum eggs or the technique was unable to detect them. There was no pattern in the absent proteins in the sense of protein function or where they are located within the eggshell. Conclusion: We conclude it is likely that such studies on museum specimens using a proteomic approach will be limited in coverage of proteins and may, therefore, be misleading.Steven J Portugal, Helen J Cooper, Cleidiane G Zampronio, Laine L Wallace, Phillip Casse

SELDI-TOF-MS determination of hepcidin in clinical samples using stable isotope labelled hepcidin as an internal standard

<p>Abstract</p> <p>Background</p> <p>Hepcidin is a 25-residue peptide hormone crucial to iron homeostasis. It is essential to measure the concentration of hepcidin in cells, tissues and body fluids to understand its mechanisms and roles in physiology and pathophysiology. With a mass of 2791 Da hepcidin is readily detectable by mass spectrometry and LC-ESI, MALDI and SELDI have been used to estimate systemic hepcidin concentrations by analysing serum or urine. However, peak heights in mass spectra may not always reflect concentrations in samples due to competition during binding steps and variations in ionisation efficiency. Thus the purpose of this study was to develop a robust assay for measuring hepcidin using a stable isotope labelled hepcidin spiking approach in conjunction with SELDI-TOF-MS.</p> <p>Results</p> <p>We synthesised and re-folded hepcidin labelled with ¹³C/¹⁵N phenylalanine at position 9 to generate an internal standard for mass spectrometry experiments. This labelled hepcidin is 10 Daltons heavier than the endogenous peptides and does not overlap with the isotopic envelope of the endogenous hepcidin or other common peaks in human serum or urine mass spectra and can be distinguished in low resolution mass spectrometers. We report the validation of adding labelled hepcidin into serum followed by SELDI analysis to generate an improved assay for hepcidin.</p> <p>Conclusion</p> <p>We demonstrate that without utilising a spiking approach the hepcidin peak height in SELDI spectra gives a good indication of hepcidin concentration. However, a stable isotope labelled hepcidin spiking approach provides a more robust assay, measures the absolute concentration of hepcidin and should facilitate inter-laboratory hepcidin comparisons.</p

Novel glycosylation sites localized in Campylobacter jejuni flagellin FlaA by liquid chromatography electron capture dissociation tandem mass spectrometry

[Image: see text] Glycosylation of flagellin in Campylobacter jejuni is essential for motility and virulence. It is well-known that flagellin from C. jejuni 81−176 is glycosylated by pseudaminic acid and its acetamidino derivative, and that Campylobactor coli VC167 flagellin is glycosylated by legionaminic acid and its derivatives. Recently, it was shown, by use of a metabolomics approach, that C. jejuni 11168 is glycosylated by dimethyl glyceric acid derivatives of pseudaminic acid, but the sites of glycosylation were not confirmed. Here, we apply an online liquid chromatography electron capture dissociation (ECD) tandem mass spectrometry approach to localize sites of glycosylation in flagellin from C. jejuni 11168. Flagellin A is glycosylated by a dimethyl glyceric acid derivative of pseudaminic acid at Ser181, Ser207 and either Thr464 or Thr 465; and by a dimethyl glyceric acid derivative of acetamidino pseudaminic acid at Ser181 and Ser207. For comparison, on-line liquid chromatography collision-induced dissociation of the tryptic digests was performed, but it was not possible to assign sites of glycosylation by that method

Quantitation Of Isomeric Ethyl Pyridine Mixtures By Multivariate Calibration Applied To Ion-molecule Reaction/collision-induced Dissociation Triple-stage Mass Spectra.

In reactions of the distonic ion (+)CH(2)OCH(2) with the three isomeric ethyl pyridines, ionized methylene transfer occurs readily yielding distonic N-methylene-ethylpyridinium ions. On-line mass selection and 10 eV collision-induced dissociation (CID) of the CH(2)(+) transfer products yields characteristic fragment ions, which are formed via processes greatly influenced by the ortho, meta or para location of the ethyl substituent in the pyridine ring. Quantitation of mixtures of isomeric 2-, 3-, and 4-ethyl pyridines of varying compositions was then performed by multivariate calibration in the form of the partial least square (PLS) model applied to both single-stage (MS) 70 eV electron ionization (EI) and pentaquadrupole triple-stage sequential ion-molecule reaction/CID product ion mass spectra. The results exemplify the superior ability of combined chemometric analysis and sequential mass spectrometric techniques, which benefits from both characteristic ion chemical reactivity and dissociation behavior, for rapid and accurate quantitation of complex isomeric mixtures.6037-4