Front Cover -- Proteomics Mass Spectrometry Methods -- Front Matter -- Copyright -- Contents -- Contributors -- I -- Introduction -- 1 -- Book introduction -- References -- 2 -- Introduction to sample preparation for proteomics and mass spectrometry -- 1. Introduction -- 2. Sample preparation for proteomic analysis -- 2.1 Sample fractionation -- 2.1.1 Immunodepletion for clinical proteomics -- 2.1.2 Subcellular fractionation and enrichment -- 2.2 Proteolytic digestion for proteomic analysis -- 3. Application of mass spectrometry for the analysis of proteins and peptides
3.1 Bottom up/shotgun proteomics -- 3.1.1 Data-dependent acquisition -- 3.1.2 Data-independent acquisition -- 3.2 MS/MS fragmentation strategies -- 3.2.1 Collision-induced dissociation -- 3.2.2 High collision dissociation -- 3.2.3 Neutral loss MS3 -- 3.2.4 Electron transfer dissociation -- 3.2.5 Multistage activation -- 3.3 Identifying peptides from mass spectra -- 3.4 Sequence databases -- 3.5 Mass spectrometry methods for differential proteomics -- 3.5.1 Label-free differential expression proteomics -- 3.5.2 Stable isotopes tags/isobaric mass tags
3.6 Mass spectrometry for quantitative targeted proteomics -- 4. Bioinformatic analysis of proteomic data -- 5. Conclusions -- References -- II -- Sample preparation -- 3 -- Sample preparation for proteomics and mass spectrometry from mammalian cell lines -- 1. Introduction -- 2. Materials and equipment -- 3. Before you begin -- 3.1 Filter-aided sample preparation -- 3.2 PreOmics iST kit -- 3.3 Membrane fractionation -- 3.4 High pH reversed-phase fractionation -- 3.5 Peptide purification and fractionation: strong cation exchange -- 3.6 Tandem Mass Tag (TMT) labeling
4. Step-by-step method details -- 4.1 Cell culture and collection of cell pellets for proteomics -- 4.2 Filter-aided sample preparation -- 4.3 PreOmics iST protocol -- 4.4 Upstream and downstream applications -- 4.4.1 Membrane fractionation -- 4.4.2 High pH reversed-phase fractionation -- 4.4.3 Peptide purification and fractionation: strong cation exchange -- 4.4.4 Chemical labeling of digested protein samples using TMT kit -- 5. Strengths and limitations of FASP and PreOmics iST protocol -- 6. Optimization and troubleshooting/notes -- 7. Alternative procedures and protocols
8. Safety considerations and standards -- 9. Summary -- References -- 4 -- Sample preparation for proteomics and mass spectrometry from clinical tissue -- 1. Before you begin -- 1.1 Timing: 3-4h -- 2. Key resources table -- 3. Materials and equipment -- 4. Step-by-step method details -- 4.1 Preparation of biopsy specimens -- 4.1.1 Timing: 1-2h -- 4.2 Homogenization of tissue material -- 4.2.1 Timing: 2-3h -- 4.3 Determination of protein concentration -- 4.3.1 Timing: 1-2h -- 4.4 Digestion of protein sample -- 4.4.1 Timing: 10-24h -- 4.5 Removal of interfering chemicals from peptide fraction
