SILAC: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Dynamic SILAC to Determine Protein Turnover in Neurons and Glia
1 Introduction
2 Materials
2.1 General Labware
2.2 Cell Culture
2.3 Cell Harvest and Lysis
2.4 Sample Preparation for LC-MS Analysis
2.5 LC-MS Analysis
3 Methods
3.1 Preparation and Maintenance of Primary Neuron and Glia Cultures
3.1.1 Primary ``Mixed´´ Neuron-Glia Cultures
3.1.2 Neuron-Enriched Cultures
3.1.3 Glia-Enriched Cultures
3.1.4 Preparation of Fully Semi-heavy Labeled Primary Cultures as Internal Standard (Optional)
3.2 Dynamic SILAC Labeling in Cultured Primary Neurons and Glia Cells
3.3 Cell Harvest
3.4 Sample Preparation for LC-MS Analysis
3.4.1 Cell Lysis and Measurement of Protein Concentration
3.4.2 Optional Internal Standard Spike-In
3.4.3 Protein Reduction, Alkylation, and Digest
3.4.4 Optional Sample Pre-fractionation
3.4.5 Sample Clean-Up
3.5 LC-MS Analysis
3.6 Database Search for Peptide and Protein Identification and Quantification
3.7 Data Processing/Analysis
3.7.1 Correction Factor for (Re-)incorporation of Light Arg/Lys into Nascent Proteins
3.7.2 Determination of Protein Half-Lives
3.7.3 Analysis of Changes in Protein Synthesis and Degradation
4 Notes
References
Chapter 2: SILAC-Based Proteomic Analysis of Meiosis in the Fission Yeast Schizosaccharomyces pombe
1 Introduction
2 Materials
2.1 Cell Culture and SILAC Labeling
2.2 Cell Lysis by Cryogenic Grinding and Protein Extraction
2.3 Protein Digestion and Desalting
2.4 Peptide Fractionation Based on Strong Cation Exchange (SCX)
2.5 LC-MS/MS Analysis
2.6 Data Analysis
3 Methods
3.1 SILAC Labeling
3.2 Harvesting the Cells
3.3 Protein Extraction
3.4 Protein Digestion
3.5 Off-Line Fractionation by Strong Cation Exchange (SCX) Chromatography
3.6 LC-MS/MS Analysis
3.7 Data Analysis
4 Notes
References
Chapter 3: Identification of Protein Interaction Partners in Bacteria Using Affinity Purification and SILAC Quantitative Prote...
1 Introduction
2 Materials
2.1 Bacterial Strains
2.2 Bacterial Growth Media
2.3 Bacterial Lysis Buffer
2.4 Strep-Tag Purification
2.5 In-Solution Protein Digestion
2.6 Ethyl-Acetate Extraction
2.7 Reversed Phase Solid Phase Extraction
2.8 Mass Spectrometry
3 Methods
3.1 Bacterial Cultivation and Disruption
3.2 Strep-Tag Purification
3.3 In-Solution Digestion
3.4 Removal of SDC by Ethyl Acetate Extraction
3.5 Reversed Phase Solid Phase Extraction
3.6 Mass Spectrometry and Data Analysis
4 Notes
References
Chapter 4: pSILAC-Based Determination of Cellular Protein Sorting into Extracellular Vesicles
1 Introduction
2 Materials
2.1 Cell Culture
2.2 EVs Isolation by Differential Centrifugations
2.3 EVs Characterization
2.4 EVs Proteomic Sample Preparation Using In-Gel Digestion
2.5 Cellular Proteomic Sample Preparation Using In-Solution Digestion
2.6 LC-MS/MS Proteomics Analysis
2.7 Quantitative Proteomic Data Analysis (See Note 14)
2.8 Interpretation of the pSILAC Results
3 Methods
3.1 Cell Culture and Pulsed SILAC Treatment
3.2 Isolation of EVs
3.3 Characterization of EVs by Nanoparticle Tracking Analysis and TEM Imaging
3.3.1 Nanoparticle Tracking Analysis
3.3.2 Transmission Electron Microscopy (TEM) Imaging
3.4 EVs Proteomic Sample Preparation (See Note 18)
3.5 Cellular Proteomic Sample Preparation
3.6 LC-MS/MS Analysis
3.7 Protein Identification and Quantitation
3.8 Interpretation of the pSILAC Results
4 Notes
References
Chapter 5: Quantification of Protein Palmitoylation by Cysteine-SILAC
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Protein Extraction
2.3 SDS Polyacrylamide Gel (See Note 3)
2.4 In-Gel Digestion
2.5 Enrichment of Palmitoylated Proteins/Peptides
3 Methods
3.1 Cell Culture
3.2 Checking the Cysteine-SILAC Labeling Efficiency
3.3 Isolation of Palmitoylated Peptides and Preparation for Proteomic Analysis
3.4 Identifying and Quantifying Peptides from Cysteine-SILAC Samples Using LC-MS/MS Analysis
4 Notes
References
Chapter 6: Comparative SUMO Proteome Analysis Using Stable Isotopic Labeling by Amino Acids (SILAC)
1 Introduction
2 Materials
2.1 Differential Labeling of HeLa Cells by Isotopic Amino Acids
2.2 Preparation of Protein G Beads Conjugated with SUMO Antibodies
2.3 Preparation of HeLa Lysates
2.4 Immunoprecipitation of Endogenous Sumoylated Proteins
2.5 Special Equipment
3 Methods
3.1 Differential Labelling of HeLa Cells by Isotopic Amino Acids (See Note 6)
3.2 Preparation of Protein G Beads Conjugated with SUMO Antibodies
3.3 Preparation of Cell Lysates
3.4 Immunoprecipitation of Endogenous Sumoylated Proteins
3.5 Verification of Efficient SUMO-Target Enrichment in Western Blotting (WB)
3.6 MS Analysis
3.7 Analysis of MS Data/Normalization-SUMO Specificity
3.8 Verification of Sumoylation Status of Target Proteins by WB
4 Notes
References
Chapter 7: A Super-SILAC Approach for Profiling Histone Posttranslational Modifications
1 Introduction
2 Materials
2.1 Standard Cell Culture
2.2 SILAC Labeling
2.3 Histone Enrichment
2.4 SDS-Page
2.5 Arg-C-like In-Gel Digestion
2.6 High-Performance Liquid Chromatography-Mass Spectrometry
2.7 Data Acquisition and Analysis
3 Methods
3.1 SILAC Labeling
3.2 Evaluation of Labeling Efficiency
3.3 Histone Purification
3.4 Super-SILAC Mix Assembly
3.5 ``Arg-C-like´´ Digestion
3.6 LC/MS Analysis
3.7 Data Analysis
4 Notes
References
Chapter 8: SILAC-Based Quantitative Phosphoproteomics in Yeast
1 Introduction
2 Materials
2.1 SILAC Labeling
2.2 Cell Lysis
2.3 Protein Precipitation
2.4 In Solution Protein Digestion
2.5 Phosphopeptides Enrichment
2.6 Desalting and Concentrating Peptides on StageTips
2.7 Analysis of Peptides on Q-Exactive
2.8 Softwares
3 Methods (See Note 1)
3.1 Preparation of Culture Media
3.2 Inoculation of Culture Media and Growth (See Note 5)
3.3 Cellular Lysis and Yeast Protein Extracts Preparation (See Note 8)
3.4 Protein Precipitation
3.5 In Solution Digestion
3.6 Enrichment of Phosphopeptides in Batch
3.7 Analysis in LC-MS/MS
3.8 Identification of Phosphopeptides
3.9 Relative Quantification of Phosphopeptides Using Proteome Discoverer 2.4 Software
4 Notes
References
Chapter 9: Phosphotyrosine Profiling Using SILAC
1 Introduction
2 Materials
2.1 SILAC Labeling
2.2 Protein Extraction and Estimation
2.3 In-Solution Digestion
2.4 Reverse Phase Clean Up
2.5 Phosphotyrosine Enrichment
2.6 LC-MS/MS Analysis
3 Methods
3.1 SILAC Labeling
3.2 Cell Lysis and Protein Extraction
3.3 In-Solution Protein Digestion
3.4 Reverse Phase Clean-Up
3.5 Phosphotyrosine Enrichment
3.6 Desalting
3.7 Mass Spectrometry Data Acquisition
3.8 Data Analysis
4 Notes
References
Chapter 10: Chemoproteomic Profiling of Geranyl Pyrophosphate-Binding Proteins
1 Introduction
2 Materials
2.1 Probe Synthesis
2.2 Cell Lysate Labeling with the GPP Probe
2.3 LC-MS/MS Sample Preparation
2.4 LC-MS/MS Analysis
2.5 Quantitative Analysis with SILAC
3 Methods
3.1 Synthesis and Purification of the Desthiobiotin-GPP Affinity Probe
3.2 Cell Lysate Preparation and Labeling with the GPP Probe
3.3 LC-MS/MS Sample Preparation
3.4 LC-MS/MS Analysis
3.5 Quantitative Analysis with SILAC
4 Notes
References
Chapter 11: Quantitative Analysis of the Endoplasmic Reticulum-Associated Proteins Using ER-Localizable Reactive Molecules
1 Introduction
2 Materials
2.1 General Biological Instruments
2.2 LC-MS/MS Setup
2.3 SILAC-Based MS Analysis
3 Methods
3.1 Sample Preparation for SILAC-Based Quantitative Analysis of Proteins Labeled with ERM in Tunicamycin-Treated and Non-treat...
3.2 Preparation of NanoLC-MS/MS Samples
4 Notes
References
Chapter 12: Workflow for Quantitative Proteomic Analysis of Intestinal Organoids Using SILAC
1 Introduction
2 Materials
2.1 SILAC Noggin and R-Spondin 1 Conditioned Media (See Note 1)
2.2 Crypt Extraction from 2-Month-Old Murine Jejunum
2.3 Organoid Culture in SILAC Media
2.4 Sample Collection, In-Solution, In-Gel Digestion, and Mass Spectrometry
3 Methods
3.1 Production of SILAC Noggin and R-Spondin 1 Conditioned Media
3.2 Crypt Extraction and Enteroid Culture
3.3 Organoid Passaging and Freezing
3.3.1 Passaging
3.3.2 Freezing
3.4 Organoid Collection and In-Solution or In-Gel Digestion (After 25-30 Days of SILAC Isotope Incorporation) (See Notes 5-7)
3.4.1 Organoid Collection
3.4.2 In-Solution Digestion
3.4.3 Gel Electrophoresis and In-Gel Digestion (Optional) (See Note 6)
3.5 Sample Cleaning Using ZipTips for Tryptic Digested Peptides (from Subheadings 3.3 or 3.4)
3.6 Liquid Chromatography-Tandem MS (LC-MS/MS), Quantification, and Bioinformatic Analysis
4 Notes
5 Relevance
References
Chapter 13: Stable Isotope Labeling by Amino Acids and Bioorthogonal Noncanonical Amino Acid Tagging in Cultured Primary Neuro...
1 Introduction
2 Materials
2.1 Equipment and Labware
2.2 Animal and Reagents
2.3 SILAC Media and Other Solutions
3 Methods
3.1 Dissection
3.2 SILAC Cell Culture
3.3 BONLAC Cell Culture
4 Notes
References
Chapter 14: Heavy Methyl SILAC Metabolic Labeling of Human Cell Lines for High-Confidence Identification of R/K-Methylated Pep...
1 Introduction
2 Materials
2.1 Metabolic Labeling of Human Cells with hmSILAC
2.2 Cell lysis and Protein Extraction and Digestion
2.3 Assessment of Protein Digestion
2.4 Proteolytic Peptide Desalting and Concentration
2.5 HPLC-MS/MS Analysis
2.6 MS Data Analysis
3 Methods
3.1 hmSILAC Labeling of Selected Cell Line
3.2 Protein Extraction
3.3 In Solution Tryptic Digestion of Proteins
3.4 Quality Control of Protein Digestion by SDS-PAGE
3.5 Desalting and Concentration of Peptides by C18 RP Microcolumns
3.6 UHPLC-MS/MS Analysis
3.7 Heavy Methionine Incorporation Test
3.8 Peptide Identification from MS-RAW Data by MaxQuant
3.9 Running hmSEEKER for High-Confidence Identification of In Vivo Methylation Sites
4 Notes
References
Chapter 15: SILAC-Based Quantitative Proteomic Analysis of Drosophila Embryos
1 Introduction
2 Materials
3 Methods
3.1 SILAC Labeling of Saccharomyces cerevisiae
3.1.1 Growing SILAC Yeast
3.1.2 Label Test for SILAC Yeast
3.2 SILAC Labeling of Drosophila melanogaster
3.2.1 Growing SILAC Flies
3.2.2 Embryo Collection and Lysis
3.2.3 In-Gel Proteolysis with Lys-C Endopeptidase
3.2.4 LC-MS/MS Analysis
3.2.5 Data Analysis
4 Notes
References
Chapter 16: Super-SILAC Quantitative Proteome Profiling of Zebrafish Larvae
1 Introduction
2 Materials
2.1 Equipment and Labware
2.2 Zebrafish Larvae and Cells Culture
2.3 Sample Preparation
3 Methods
3.1 Preparation of the Super-SILAC Mix
3.2 Zebrafish Exposure and Sampling
3.3 Protein Extraction from Zebrafish Larvae
3.4 SDS-PAGE and In-Gel digestion (See Note 11)
3.5 nanoLC-MS Analysis and Data Treatment
4 Notes
References
Chapter 17: Use of Nuclear and Chromatin Enrichment Procedures for Quantitation of Yeast DNA Replication Proteins Using SILAC
1 Introduction
2 Materials
2.1 Strains
2.2 SILAC Labeling and Culture Medium
2.3 Chromatin Preparation
2.4 SDS-PAGE and Gel Staining
3 Methods
3.1 SILAC Labeling and Cell-Cycle Synchronization
3.2 Chromatin Preparation
3.3 Estimate Protein Concentration
3.4 Peptide Preparation, Mass Spectrometry, and Data Processing
4 Notes
References
Chapter 18: Combination of Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and Substrate Trapping for the Detec...
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Cell Lysis
2.3 Immunoprecipitation (IP)
2.4 Total Protein Quantitation, SDS-PAGE, and In-Gel Digestion
2.5 Desalting Using C18 Stop and Go Extraction (STAGE) Tips
2.6 Immunoblot Analysis
2.7 Antibodies (See Note 2)
2.8 LC-MS/MS
2.9 Database Searching/Protein Identification
3 Methods
3.1 Establishing Tetracycline-Inducible Cell Line
3.2 SILAC-Based Cell Culture
3.3 Cell Lysis
3.4 Determination of Protein Concentration by BCA Protein Assay
3.5 Immunoprecipitation (IP)
3.6 In-Gel Digestion
3.7 Desalting Extracted Peptides
3.8 Immunoblot Analysis
3.9 LC-MS/MS
3.10 SILAC-Labeled Protein Identification and Quantitation
4 Notes
References
Chapter 19: Profiling of Secreted Proteins in Serum-Containing Media Using BONCAT and Pulsed SILAC
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Secretome Preparation
2.3 Isolation of AHA-Labeled and Secreted Proteins by CuAAC
2.4 LC-MS/MS and Data Processing
3 Methods
3.1 Cell Culture with BONCAT and Pulsed-SILAC Labeling
3.2 Preparation of Secretome
3.3 Isolation of Secreted Proteins by CuAAC and On-Bead Digestion
3.4 Parameter Setting for Mass Spectrometric Data Processing
4 Notes
References
Chapter 20: Using SILAC to Develop Quantitative Data-Independent Acquisition (DIA) Proteomic Methods
1 Introduction
2 Materials
2.1 SILAC Cell Labeling
2.2 Sample Preparation
2.3 Serial Dilutions
2.4 HPLC and Mass Spectrometry
2.5 Peptide Searching and Quantitation
3 Methods
3.1 Cell Culture
3.1.1 SILAC Cell Labeling
3.1.2 Assessing Isotopic Incorporation in Cell Culture
3.1.3 Cell Culture Expansion
3.2 Sample Preparation
3.2.1 Cell Lysis
3.2.2 Reduction/Alkylation/Digestion
3.2.3 Desalting
3.3 Serial Dilutions
3.4 HPLC and Mass Spectrometry
3.5 Peptide Searching and Quantitation
4 Notes
References
Chapter 21: SILAC-IodoTMT for Assessment of the Cellular Proteome and Its Redox Status
1 Introduction
2 Materials
2.1 SILAC
2.2 IodoTMT Cysteine Labeling and Protein Digestion
2.3 SDC Extraction and Peptide Desalting
3 Methods
3.1 SILAC
3.2 IodoTMT Labeling of Cysteines
3.3 Protein Digestion and Peptide Desalting
3.4 Mass Spectrometry Analysis
3.5 Data Processing
3.6 Data Processing
4 Notes
References
Chapter 22: Targeted Absolute Protein Quantification Using SILAC Internal Standard and Full-Length Protein Calibrators (TAQSI)
1 Introduction
2 Materials
2.1 SILAC Cell Culture (See Note 1)
2.2 Standard Calibrator, Proteomics Reagent Preparation and Materials
3 Methods
3.1 Incorporation of Heavy Stable Isotope-Labeled Amino Acids
3.2 Protein Extraction
3.3 Calibration Standards and Quality Control (QC) Samples
3.4 Proteomics Sample Preparation
3.5 LC-MS/MS Assay
3.6 Data Analysis
4 Notes
References
Index