Cold Spring Harbor Laboratory Press Proteomics: A Cold Spring Harbor Laboratory Course Manual

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, Philip C. Andrews, and Joshua LaBaer, all leaders in their fields, who bring complementary expertise to the manual. Contents: 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. In-Gel Trypsin Digest of Gel Fractionated Proteins 3. Solution Protein Digest 4. TCA Precipitation of Proteins 5. Average and Monoisotopic Masses of Amino Acids 6. Dipeptide Masses of Amino Acids 7. LTQ Instrument Methods 8. Offline Desalting of Peptide Mixtures 9. Preparing Competent Cells 10. DNA Quantification 11. Cautions Index