An approach to C- and N-terminal sequencing of protein: Mass spectrometry-based de novo amino acid sequencing with the combination of site-specific tris (2,4,6-trimethoxyphenyl) phosphonium-acetylation of alpha-amino group and selective recovery of terminal peptidesHiroki Kuyama, Osamu Nishimura, Susumu Tsunasawapp 145-177Abstract:Terminals of proteins play highly important roles in cellular functions, so it is crucial to determine the terminal structures of proteins. The amino acid sequences of mature proteins are usually altered from those expected from DNA sequences, due to editing and splicing of RNAs before translation and/or a variety of protein post-translational modifications (PTMs) to newly expressed proteins. Conventional mass spectrometry (MS)-based methods for determining the structure of protein (peptide mass fingerprinting (PMF) or peptide fragment fingerprinting (PFF)) use a genome and protein database. Hence, they do often not provide the exact covalent structures of proteins. Because of their importance, various analytical methods for determining N- and C-terminal structures have been developed. Several practical methods for N-terminal sequencing have been reported, but no satisfactory methods for C-terminal sequencing have been developed. Therefore, it has long been desired to develop a simple and efficient method for C-terminal sequencing by MS. Here we describe new approaches directed toward simple and highly successful isolation of C- and N-terminal peptides from proteins and their de novo sequence analyses by MALDI-MS. Also described is an application of arginine derivatization that seeks to improve fragmentation of arginine-containing, TMPP-Ac-modified peptides in tandem MS.Progress of proteomic analysis in silkworm Bombyx moriMuthukalingan Krishnan, Simone Königpp 179-187Abstract:The recent availability of silkworm sequence databases results in a rapidly increasing identification of biologically relevant proteins of the economically important model organism Bombyx mori. These investigations facilitate the functional characterization of the proteins to not only have the insights into complex cellular processes in silkworm but also to pave the way to strengthen transgenic silkworm technology for the production of recombinant proteins. This paper reviews the current state of knowledge in silkworm proteomics.


Allergenic proteins in settled and airborne dustWeiqun Wang, Doreen Ackermann, Lothar Grün, Simone Königpp 191-198Abstract:Many allergies in human are caused by protein contaminants in the ambient air. Prominent examples are pet allergies as response to animal hair or seasonal allergies to plants due to pollen. In the workplace baker´s asthma and farmer´s disease due to organic dust are acknowledged occupational diseases. The identification and quantification of protein allergens in breathing air, both in households and at the workplace, becomes more and more important, in particular, because toxicological limits of exposure have still not been defined. Instead, the precept of avoidance of the allergen is applied for sensitive workers. In order to evaluate modern proteomic technology for its use in the quantification of protein aeroallergens, both two-dimensional gel electrophoresis and label-free mass spectrometry were employed to investigate household dust. The first technique is a valuable visualization methodology of the protein content but it is often hampered by sample components such as soot in dust from various sources which cause streaking and smearing on the gels. Chromatography coupled to mass spectrometry is not as sensitive to such contaminants and can be used to absolutely quantify protein components in mixtures. We find that not only keratin is abundant in household dust, but also a number of other mammalian proteins associated with skin cells. Allergens from animal dander such as Fel d 1, the cat allergen, are readily detected in high quantities.Urine profiling using ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry and pattern analysisArabi Chandrakumaran, Zeineba Hortebusch, Simone Königpp 199-210Abstract:Reproducible reversed-phase chromatography coupled to quadrupole time-of-flight mass spectrometry, and in a limited way also ion trap mass spectrometry, provides the means to profile urine in differential experiments. Only one microliter of centrifuged and filtered urine is needed to measure hundreds of abundant biomolecules separated on C18 material. Analysis of three or more biological replicate profiles using statistics approaches such as principal component analysis allows to distinguish individual profiles of humans and to find biomarkers responsible for sample grouping. Samples of morning urine of healthy females and males can be differentiated. Medication also changes urine profiles and so do hormones as for instance in the female cycle. The potential of the methodology lies in the monitoring of hundreds molecular factors at the same time with very little sample treatment.Compilation of fragmentation data of single amino acids prepared by electrospraySimone König, Henry M. Falespp 211-220Abstract:Fragmentation spectra of all the common naturally occurring as well as some less common amino acids were measured using electrospray mass spectrometry (ion trap and triple-stage quadrupole). Since the electrospray process prepares (M+H)+ ions, the MS/MS spectra of the amino acids were expected to resemble those obtained by chemical ionization where the input of energy is provided by interaction with a protonating source such as CH5+ or NH4+ and, in general, this was found to be true. Common to all amino acids is the early loss of the elements of formic acid HCOOH. Detailed fragmentation pathways are discussed for the different amino acid groups.


Software tool PLGS2Excel for data reduction in complex sample sets such as LC-MS/MS experiments for downstream processingAlexander Pirkl, Simone Königpp 223-228Abstract:Data processing software tool PLGS2Excel, capable of combining and preparing data from several mass spectrometric experiments for further statistical analysis, is described. Methods to filter and condense the dataset are presented. In general, this software can be used to combine any data from files of comma-separated values (CSV), which are used in many fields of scientific research to store simple-structured data, to a data matrix. The program can be employed in any context where pairs of values stored in two columns of a CSV-table have to be combined with further files, having the same basic structure, to a data matrix. The software, programmed in Borland Delphi, can be adapted with respect to file size and number of columns and rows in the final data matrix depending on the memory of the computer. The software is freely available at