Investigation of a calmodulin/peptide complex by chemical cross-linking and high-resolution mass spectrometrySabine Schaks, Daniel Maucher, Christian H. Ihling, Andrea Sinzpp 249-260Abstract:The combination of chemical cross-linking and high-resolution mass spectrometry has matured into an alternative technique for structural analysis of proteins and protein complexes. This strategy relies on determining distance constrains by introducing covalent bonds between functional groups of distinct amino acids within one molecule (intramolecular) or between different molecules (intermolecular). The resulting distance constrains allow to create low-resolution structures of proteins and protein complexes by molecular modeling approaches or serve as basis to confirm or disprove an existing structural model. In this chapter we exemplify the chemical cross-linking/mass spectrometry approach for deriving structural information of a complex between the 17 kDa-calcium binding protein calmodulin with a target peptide derived from skeletal myosin light chain kinase. We describe the use of two cross-linking reagents with different reactivities yielding complementary structural information. This report is intended to be a practical guideline for beginners as well as for those who are already working in the field of protein mass spectrometry.Avoiding pitfalls in proteomics sample preparationRobert L. J. Graham, Anastasia Kalli, Geoffrey T. Smith, Michael J. Sweredoski, Sonja Hesspp 261-271Abstract:The purpose of this chapter is to focus on areas that are often underestimated in their importance for optimized results in mass spectrometric analyses, namely sample preparation. While there is no doubt that the advancements in technology led us to where we are now – it is also clear that a deep understanding of appropriate sample preparation techniques can be key to success of a proteomics experiment. Equally, lack of such knowledge can be the source of avoidable, failed experiments. Here, we will share our experience and discuss common contaminants such as buffers, solubilization reagents and those contaminants that are introduced through the use of specific biological protocols. The chapter will outline how best to deal with these common problems in order to maximize the returns from a proteomics experiment. We will also briefly discuss the importance of liquid chromatography and mass spectrometry optimization as well as bioinformatics tools to increase the amount of information that can be retrieved from such experiments.Hands-on phosphoproteomicsFilip Sucharski, Jerzy Silberringpp 273-283Abstract:Ever since it was known how complex a challenge is phosphoprotein analysis, much effort have been spent on the development of analytical methods. The major difficulty in phosphoproteome analysis is the effective isolation of phosphoproteins from whole cell lysate. Main objectives of phosphoproteomics are detection, identification, quantitation of phosphoproteins, and mapping phosphorylation sites. In the past decade numerous methods for phosphoproteome analysis were developed. Nowadays, the identification of proteins is performed by mass spectrometry. In this chapter we describe various approaches and methods used for effective phosphoprotein isolation and analysis including practical information, which can be transferred directly to the bench.Using native gel electrophoresis or isoelectric focussing as experimental "clock" for the (iso)aspartate formation process of tTR-NGR fusion proteinsChristian Schwöppe, Heike Hintelmann, Rolf M. Mesters, Wolfgang E. Berdel, Simone Königpp 285-293Abstract:The amino acid asparagine, especially if followed by a glycine, is known to spontaneously deamidate to (iso)aspartate in a non-enzymatic reaction. In particular, this is interesting for NGR-binding-motif-bearing proteins and peptides, which target aminopeptidase N (CD13) overexpressed in tumor vasculature and which represent promising therapy substances. After deamidation to isoDGR, these proteins/peptides may act as ligands for the integrin avß3 (CD51/CD61) which remarkably also is overexpressed in tumor vasculature.
Recently, we have constructed a recombinant-expressed fusion protein consisting of the coagulation-inducing protein tissue factor (truncated tissue factor, tTF) and peptide GNGRAHA (tTF-NGR). This protein activates the blood coagulation within tumor vessels by selectively binding to CD13. For us, it is important to determine the degradation grade of the tTF-NGR motif over time and temperature for quality control purposes of the purification process, especially if it is kept in mind that in contrast to the deamidated variant tTF-isoDGR, tTF-DGR is not capable to bind to integrin avß3. To that end, we chose the two simple and convenient electrophoresis techniques native (blue) polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF) to separate the tTF-NGR protein variants according to their different isoelectric points (pI). With respect to monitoring deamidation progress, and here for the grade of (iso)aspartate formation in tTF-NGR proteins, these techniques serve as a sensitive experimental “clock”.ETD reactions in peptide sequencingPiotr Suder, Anna Drabik, Anna Bodzon-Kulakowskarzkapp 295-303Abstract:Electron Transfer Dissociation (ETD) is a novel fragmentation method applied in the field of peptides and protein identification in the life sciences area. ETD allows for the more random disruption of the covalent bonds which is especially useful for peptide/protein sequencing. The technique seems to be also one of the most promising methods for identification of posttranslational modifications within protein sequences. Additionally, thanks to its properties, ETD usually allows for a more confident sequence determination than other techniques used up to date. In this chapter the principles of the methodology, necessary equipment, basic settings of the instruments, and selected applications are described.Peptide cleavage and oxidation using ROXY EC system with on-line mass spectrometry detectionPrzemyslaw Mielczarek, Hana Raoof, Jerzy Silberringpp 304-309Abstract:The electrochemical conversion of many target compounds with mass spectrometry (MS) detection has been utilized for a wide range of applications. It can serve as a model for metabolites development, signal enhance,emt in MS and finally, for the electrochemical cleavage of proteins and peptides, which is shown in this chapter. The electrochemical system for on-line electrochemistry (EC)-MS was used to obtain cleavage and oxidation of peptides containing tyrosine or tryptophan. Such approach can be a new, complementary technique to standard peptide and protein cleavage using proteolytic enzymes.


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