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Based on a popular course at Cold Spring Harbor Laboratory, this new manual assembles cutting-edge protocols, helpful hints, and lecture notes to teach researchers from a wide variety of disciplines the essential methods of proteomics using state-of-the-art instrumentation. Detailed protocols involving protein microarrays, liquid chromatography, high-throughput cloning of expression constructs, IMAC, mass spectrometry, MALDI-TOF, and MudPIT are provided, along with well-illustrated descriptions of experimental procedures and lists of recommended Web sites and reading material. Proteomics: A Cold Spring Harbor Laboratory Course Manual can be used both as the basis for a course and as a detailed bench manual for those performing indispensable proteomic experiments. It is authored by Andrew J. Link and Joshua LaBaer, both leaders in their fields, who bring complementary expertise to the manual.
Preface
Introduction
Experiments
1. Analysis of Whole-cell Lysates by Two-dimensional Gel Electrophoresis and MALDI Mass Spectrometry
2. Purification of Protein Complexes for Mass Spectrometry Analysis
3. Qualitative and Quantitative Measurement of Peptides with MALDI TOF/TOF Mass Spectrometry
4. Analysis of Protein Complexes: High-sensitivity Liquid Chromatography Coupled with Tandem Mass Spectrometry
5. Phosphopeptide Analysis Using IMAC and Mass Spectrometry
6. Multidimensional Protein Identification Technology (MudPIT) Analysis of Whole-cell Lysates
7. Quantitative Mass Spectrometry Analysis of Whole-cell Extracts (iTRAQ)
8. Analysis and Validation of Tandem Mass Spectra
9. High-throughput Cloning of ORFs: Assembling Large Sets of Expression Constructs
10. Construction of Protein Microarrays Nucleic Acid Programmable Protein Array (NAPPA)
11. Using the Nucleic Acid Programmable Protein Array (NAPPA) for Identifying Protein–Protein Interactions
Appendices
1. Setup and Demonstration of a Nanoelectrospray Ionization (nanoESI) Source and Tandem Mass Spectrometry (MS/MS)
2. Solution Protein Digest
3. In-gel Trypsin Digest of Gel Fractionated Proteins
4. Trichloroacetic Acid (TCA) Precipitation of Proteins
5. Monoisotopic and Immonium Ion Masses of Amino Acids
6. Dipeptide Masses of Amino Acids
7. LTQ Instrument Methods
8. Off-line Desalting of Peptide Mixtures
9. Preparing Competent Cells
10. DNA Quantification
11. Cautions
Index
“[T]he book is very well presented, written in a logical and concise way, and unequivocally solid in terms of scientific content, with many ready-to-use, robust procedures tested and validated over the years by recognized experts. I warmly recommend this volume to anyone who wants to implement proteomics techniques in the laboratory, or who simply wishes to get a practical sense of these techniques, useful in correctly interpreting the significance of proteomic data in the scientific literature.”
- The Quarterly Review of Biology
