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Shotgun Proteomics: Methods and Protocols serves as a vital collection of protocols through which thousands of proteins can be simultaneously identified, quantified and characterized in a high throughput manner. Beginning with the history of proteomics centered on the vital role of mass spectrometry in its development, this detailed volume continues with chapters on sample pre-fractionation, in vivo and in vitro stable isotope labeling, label-free proteomics, informatics, protein-protein interactions, targeted proteomics and post-translational modifications. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols and tips on troubleshooting and avoiding known pitfalls.

Practical and comprehensive, Shotgun Proteomics: Methods and Protocols is an ideal and up-to-date guide for researchers seeking to understand the proteome of any given species.

Shotgun Proteomics: Methods and Protocols

Part I: Introduction

1. Survey of Shotgun Proteomics

Fabio Cesar Sousa Nogueira and Gilberto B. Domont

Part II: Sample Pre-Fractionation

2. LC-MALDI-TOF/TOF for Shotgun Proteomics

Patricia Fernández-Puente, Jesús Mateos, Francisco J. Blanco and Cristina Ruiz-Romero

3. Fully Automatable Multidimensional Reversed-Phase Liquid Chromatography with Online Tandem Mass Spectrometry

Maggie P.Y. Lam, Henry Law, Quan Quan, Yun Zhao, and Ivan K. Chu

4. GeLC-MS/MS Analysis of Complex Protein Mixtures

Monika Dzieciatkowska, Ryan Hill, and Kirk C. Hansen

5. IPG Strip-Based Peptide Fractionation for Shotgun Proteomics

Murat Eravci, Christian Sommer and Matthias Selbach

Part III: In Vivo Stable Isotope Labeling

6. SILAC Yeast: From Labeling to Comprehensive Proteome Quantification

Lyris M.F. de Godoy

7. Analysis of Proteome Dynamics in Mice by Isotopic Labeling

John C. Price and Sina Ghaemmaghami

8. Stable Isotope Labeling in Mammals (SILAM)

Daniel B. McClatchy and John R. Yates III

9. Analysis of Individual Protein Turnover in Live Animals on a Proteome-Wide Scale

Stefan Reckow and Christian Webhofer

Part IV: In Vitro Stable Isotope Labeling

10. Determining Protein Subcellular Localization in Mammalian Cell Culture with Biochemical Fractionation and iTRAQ 8-Plex Quantification

Andy Christoforou, Alfonso Martinez Arias and Kathryn S. Lilley

11. Brain Quantitative Proteomics Combining GeLC-MS and Isotope-Coded Protein Labeling (ICPL)

Giuseppina Maccarrone, Maria Lebar and Daniel Martins-de-Souza

12. Employing TMT Quantification in a Shotgun-MS Platform

Darragh P. O'Brien and John F. Timms

13. Employing TMT Quantification in Shotgun-MS Proteomic Analysis: A Focus on Skeletal Muscle

Bruno Menezes de Oliveira

Part V: Label-Free Proteomics

14. Spectral Counting Label-Free Proteomics

Liisa Arike and Lauri Peil

15. Quantification of Proteins by Label Free LC-MS^E

Alon Savidor and Yishai Levin

Part VI: Proteomics Informatics and Quantitation

16. Bioinformatics for Proteomics: Opportunities at the Interface Between the Scientists, Their Experiments and the Community

Marc Vaudel, Harald Barsnes, Lennart Martens and Frode S. Berven

Part VII: Protein-Protein Interactions

17. Identification of DNA Damage Checkpoint-Dependent Protein Interactions in Saccharomyces cerevisiae Using Quantitative Mass Spectrometry

Francisco M. Bastos de Oliveira and Marcus B. Smolka

18. Application of Shotgun Proteomics for Discovery-Driven Protein-Protein Interaction

Livia Goto-Silva, Zoltan Maliga, Mikolaj Slabicki, Jimmy Rodriguez Murillo and Magno Junqueira

19. Mapping Protein Complexes Using Covalently Linked Antibodies and Isobaric Mass Tags

Antje Dittmann, Sonja Ghidelli-Disse, Carsten Hopf and Marcus Bantscheff

Part VIII: Targeted Proteomics

20. Biomarker Verification Using Selected Reaction Monitoring and Shotgun Proteomics

Angel Mauricio Castro-Gamero, Clarice Izumi, and José César Rosa

21. Use of Universal Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-Based Selected Reaction Monitoring (SRM) Approach for Verification of Breast Cancer-Related Protein Markers

Ning Qing Liu, Lennard J.M. Dekker, Martijn M. Van Duijn, and Arzu Umar

22. The Secretome Analysis by High Throughput Proteomics and Multiple Reaction Monitoring (MRM)

Vitor M. Faça, Camila S. Palma, Daniele Albuquerque, Gabriela N. S. Canchaya, Mariana L. Grassi, Vera L. Epifânio and Eduardo B. de Oliveira

23. Preparation of Heteroelement-Incorporated and Stable Isotope-Labeled Protein Standards for Quantitative Proteomics

Anna Konopka, Nico Zinn, Christina Wild and Wolf D. Lehmann

Part IX: Post-Translational Modifications

24. One-Source Peptide/Phosphopeptide Ratio Standards for Accurate and Site-Specific Determination of the Degree of Phosphorylation

Martin E. Boehm, Bettina Hahn and Wolf D. Lehmann

25. Quantitative Glycoproteomics for N-Glycoproteome Profiling

Sheng Pan

26. A Practical Recipe to Survey Phosphoproteomes

William C. Edelman, Kelsey M. Haas, Joanne I. Hsu, Robert T. Lawrence and Judit Villén

27. Quantitation of the Phosphoproteome Using the Library Assisted eXtracted Ion Chromatogram (LAXIC) Strategy

Justine V. Arrington, Liang Xue and W. Andy Tao

28. Fast, Efficient, and Quality-Controlled Phosphopeptide Enrichment from Minute Sample Amounts Using Titanium Dioxide

Clarissa Dickhut, Sonja Radau and René P. Zahedi

29. Quantifying Small Molecule-Induced Changes in Cellular Protein Expression and Post-Translational Modifications Using Isobaric Mass Tags

Isabelle Becher, Maria Fälth Savitski, and Marcus Bantscheff

Part X: Mass Spectrometry for Protein Analysis

30. Analysis of Protein Structure by Crosslinking Combined with Mass Spectrometry

Evgeniy V. Petrotchenko, Karl A.T. Makepeace, Jason J. Serpa and Christoph H. Borchers

31. Top Down Proteomics by Means of Orbitrap Mass Spectrometry

Kai Scheffler