Protein and Peptide Analysis by LC-MS: Experimental Strategies (Rsc Chromatography Monographs, Band 15) - Hardcover

Hardcover

ISBN 10: 1849731829 ISBN 13: 9781849731829

Verlag: Royal Society of Chemistry, 2011

Inhaltsangabe

This book is the first example in presenting LC-MS strategies for the analysis of peptides and proteins with detailed information and hints about the needs and problems described from experts on-the-job. The best advantage is -for sure- the practical insight of experienced analysts into their novel protein analysis techniques. Readers starting in 'Proteomics' should be able to repeat each experiment with own equipment and own protein samples, like clean-up, direct protein analysis, after (online) digest, with modifications and others. Furthermore, the reader will learn more about strategies in protein analysis, like quantitative analysis, industrial standards, functional analysis and more.

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Über die Autorinnen und Autoren

Thomas Letzel is associate professor for analytical chemistry and bioanalysis at the Wissenschaftszentrum Weihenstephan (WZW) located at the TUM (Technische Universitõt M³nchen), Germany. He obtained his PhD in Chemistry with studies about soot aerosols and environmental analysis at the Chair for Analytical Chemistry of TUM. After a postdoc time in Amsterdam, The Netherlands, at the VU Amsterdam with topics in pharmaceutical and enzymatic analysis, he came back to TUM and was employed as a scientist, leading a new analytical research group with focus on bioanalysis. In latter position he got his Habilitation. He developed and established mass spectrometric techniques coupled with modern liquid phase solution techniques in enzymology, studying noncovalent complexes and observing molecular fingerprints. His research of novel mass spectrometric techniques comprises the identification and characterization of organic molecules in applications of biological, environmental, pharmaceutical, food, plant and nutrition sources.

Thomas Letzel is associate professor for analytical chemistry and bioanalysis at the Wissenschaftszentrum Weihenstephan (WZW) located at the TUM (Technische Universitõt M³nchen), Germany. He obtained his PhD in Chemistry with studies about soot aerosols and environmental analysis at the Chair for Analytical Chemistry of TUM. After a postdoc time in Amsterdam, The Netherlands, at the VU Amsterdam with topics in pharmaceutical and enzymatic analysis, he came back to TUM and was employed as a scientist, leading a new analytical research group with focus on bioanalysis. In latter position he got his Habilitation. He developed and established mass spectrometric techniques coupled with modern liquid phase solution techniques in enzymology, studying noncovalent complexes and observing molecular fingerprints. His research of novel mass spectrometric techniques comprises the identification and characterization of organic molecules in applications of biological, environmental, pharmaceutical, food, plant and nutrition sources.

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This book is the first example in presenting LC-MS strategies for the analysis of peptides and proteins with detailed information and hints about the needs and problems described from experts on-the-job. The best advantage is -for sure- the practical insight of experienced analysts into their novel protein analysis techniques. Readers starting in 'Proteomics' should be able to repeat each experiment with own equipment and own protein samples, like clean-up, direct protein analysis, after (online) digest, with modifications and others. Furthermore, the reader will learn more about strategies in protein analysis, like quantitative analysis, industrial standards, functional analysis and more.

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Protein and Peptide Analysis by LC–MS

Experimental Strategies

By Thomas Letzel

The Royal Society of Chemistry

Copyright © 2011 The Royal Society of Chemistry
All rights reserved.

ISBN: 978-1-84973-182-9

Contents

Contributors, xix, 
Chapter 1 Top Down and Bottom Up Analysis of Proteins (Focusing on Quantitative Aspects) Friedrich Lottspeich, 1, 
Chapter 2 How to Couple and Handle Liquid Chromatography with Mass Spectrometry Thomas Letzel, 11, 
Chapter 3 Expression and Purification of Bioactive Proteins/Peptides with Conventional Liquid Chromatography Takayuki Ohnuma and Tamo Fukamizo, 26, 
Chapter 4 Liquid Chromatography-Mass Spectrometry of Intact Proteins Nicolas L. Young and Benjamin A. Garcia, 38, 
Chapter 5 LC-MS(/MS) of Trypsin-Hydrolysed Proteins Seronei C. Cheison and Ulrich M. Kulozik, 56, 
Chapter 6 On-line Protein Digestion in Combination with Chromatographic Separation and Mass Spectrometric Detection S. Johannes Hoos and Wilfried M.A. Niessen, 71, 
Chapter 7 Bioinformatic Tools for the LC-MS/MS Analysis of Proteins and Peptides Christian Webhofer and Michael Schrader, 87, 
Chapter 8 Quantitative LC-MS of Proteins Gabriele Stöhr and Andreas Tebbe, 104, 
Chapter 9 LC-MS for the Identification of Post-Translational Modifications of Proteins Boris Macek, 123, 
Chapter 10 LC-MS for the Determination of the Enzymatic Activity of Proteins Romy K. Scheerle and Johanna Graßmann, 133, 
Chapter 11 Functional Analysis of Proteins, Including LC-MS and Special Freeware Michael Krappmann and Thomas Letzel, 142, 
Chapter 12 Industrial Standards and Strategies in LC-MS Analysis of Proteins Rene Wissiack, 156, 
Subject Index, 168, 

CHAPTER 1

Top Down and Bottom Up Analysis of Proteins (Focusing on Quantitative Aspects)

FRIEDRICH LOTTSPEICH

Max Planck Institute of Biochemistry, Protein Analysis, Am Klopferspitz 18, 82152 Martinsried, Germany

1.1 Introduction

One key focal point in proteome research is the determination of changes in protein expression and their modifications. In the early years of proteomics the field was dominated by protein chemists and the main approach was 2D-PAGE where differential maps revealed protein pattern differences. The detailed analysis of the different protein spots only became feasible after the introduction of mass spectrometry. However, 2D-PAGE was difficult to reproduce, was not automated and had several limitations with important subsets of proteins (e.g. hydrophobic, very basic, very large or very small proteins). Furthermore, the quantification of the proteins was usually performed by image analysis following several staining methods, which exhibit different signal intensities with different proteins. The dynamic range of detection spans only about 2–3 orders of magnitude, resulting in the visualization of only relatively highly abundant proteins. Additionally, image analysis in principle cannot deal with protein mixtures in a single spot, which, due to the complexity of a proteome, is the common case. Finally, enzymatic cleavage of the protein in the gel matrix suffered from low peptide recovery.

All these limitations encouraged mass spectrometric experts to develop alternative strategies for proteome analyses. Mass spectrometry was used to work with small molecules and therefore it was tempting to cleave the very heterogeneous and unpleasant protein complexity of a proteome enzymatically into small peptides (see also Chapter 5) with much more favourable properties concerning hydrophobicity, diversity and accessibility for multidimensional chromatographic separations and mass spectrometry (see also Chapter 6). Soon mass spectrometry together with informatics and protein databases were developed and optimized to handle complex peptide mixtures, allowing peptide identification by high-throughput MS-MS (see also Chapter 7). The mainstream of proteome research followed this track, called 'bottom up' or 'shotgun' proteomics (Figure 1.1).

Unfortunately, the bottom up approach also has several severe and fundamental limitations. First, by cleaving the proteome into peptides the complexity increases by a factor of about 40, producing hundreds of thousands of peptides. This is a number that swamps even the most modern mass spectrometers. In consequence, only a fraction of these peptides can be analysed in detail ('under-sampling' effect), and it is difficult to assure that identical peptides are analysed from each sample, which is essential to unravel a quantitative fluctuation in the amounts of certain proteins. Second, and even more serious, the context of a protein and the derived peptides is destroyed. A certain peptide may be derived from different proteins or from different forms of a certain gene product, such as post-translational modified, processed or truncated protein species, or from proteins having in common major amino acid sequence stretches like splicing variants or protein isoforms. A single gene will almost always produce an unpredictable multiplicity (tens or even hundreds) of different protein species, which are composed predominantly of identical peptides. Consequently, the quantitative analysis of a peptide monitors only the sum of all proteins that contain this particular peptide. Unfortunately, usually it is not known which protein species are expressed at a certain proteome state. Since in proteome analysis the sequence coverage usually is far less than 50%, the probability of missing the nature of the diversity or the modifications is rather high. In conclusion, the quantity of a peptide determined by a bottom up approach does not necessarily reflect the quantity of a protein of interest. This is completely different in a protein-based, i.e. 'top down', approach (see also Chapter 4). The molecular structure and the nature of an intact protein are well defined by molecular properties like the molecular mass and the position in a separation space (isoelectric point, chromatographic position, etc.). If, for example, one compares two proteome states, where a single protein is processed by a cleavage after a lysine, this is easily seen on a protein-based (top down) analysis, like 2D-PAGE or even 1D-PAGE. However, with a bottom up approach, in the two situations all the peptides appear exactly the same and the biological difference will not be detectable (Figure 1.2).

Despite these obvious limitations, bottom up approaches are widely used. The relative technical simplicity and the enormous instrumental development tailor-made towards peptide-based approaches, on the separation as well as on the mass spectrometry side, forced the proteomics field to go mainly with bottom up strategies. However, in recent years the importance of the protein diversity caused by post-translational modifications (PTM; see also Chapter 9), degradations and processing events has become evident to proteome scientists. Still, major technical hurdles have to be vanquished but the awareness of the potential and the advantages of top down proteomic approaches is significantly increasing.

1.2 Quantitative Proteomics

1.2.1 Quantitative Proteomics by Label-Free Techniques

Mass spectrometry per se is not an absolutely quantitative technique. Sequence-dependent peptide ionization efficiencies and suppression of neighbouring signals by dominant peptides results in a low correlation between peptide mass signal intensity and the amount of the peptide (see also Chapter 2). Especially with highly complex mixtures, as commonly achieved in...

Details zur Ausgabe

• Verlag: Royal Society of Chemistry
• Erscheinungsdatum: 2011
• Sprache: Englisch
• ISBN 10: 1849731829
• ISBN 13: 9781849731829
• Einband: Gebundene Ausgabe
• Auflage: 1
• Anzahl der Seiten: 194
• Herausgeber: Letzel Thomas
• Kontakt zum Hersteller: Nicht verfügbar
• Verantwortliche Person: Buchpark GmbH; gpsr@buchpark.de; ; Krügerweg 1; Trebbin; Brandenburg; 14959; Deutschland