Sample Preparation for Protein Analysis Workflows with AFA Technology
Jessica R. Chapman PhD & Saeed Seyedmohammad, PhD
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Chapters
Scalable FFPE Sample Preparation Method for Clinical LC-MS Assays Introduction
About the Memorial Sloan Kettering Cancer Center
Clinical Proteomics Laboratory Overview
Pathology of Samples
Clinical Proteomics Laboratory Workflow
Tissue Processing Workflow
Current FFPE workflow
Homogenization of Tissue in LE220Rsc
AFA Digestion as Optimization Step
Digestion Monitoring
Digestion Monitoring with AFA testing
Optimized Workflow
Workflow Conclusions
Plasma Cell Neoplasm
Multiple Myeloma (Plasma Cell Myeloma)
Plasma Cell Neoplasm Diagnosis and Characterization
From Application to Assay
Ig Relative Abundance Assay
Ig Relative Abundance Assay Results
Monoclonal Protein Detection and Characterization in Serum
Ig Relative Abundance Assay Results Continued
Optimized Workflow Continued
Concordant Results
Case Example
Conclusions Continued
Cell Lysis Workflow
AFA Benefits Summary
Acknowledgements
High Throughput processing of Human Cardiomyocytes using Covaris AFA Technology Introduction
Presentation Agenda
Background
Experimental Methodology
Cell-Based Workflow for 96 Well-Based HTP Assay
Optimizing Experiments
Establishing Cell Line
Optimizing Protein Extraction
Assessing Suitability of Buffers
Optimizing Trypsin Digestion
Assessing Proteome Coverage on Different Acquisition Gradients
Assessing Proteome Coverage – Thermo Lumos Micro vs Exploris 480
Median Intensity Plot to Assess Proteome Coverage - Lumos Micro
Cell-Based Protein Extraction Workflow for 96 Well-Based HTP Assay – Pipelines
Proteome Dynamic Range – Median Intensity of Common Proteins in the Two Pipelines
Assessing Proteome Dynamic Range – Median Intensity of Unique Proteins
Biological Importance of the Cell-Based Workflow
Assessing Cell-Based Protein Extraction Workflow - Subcellular Localization
Future Directions
Proteome Coverage Using Panther GO Annotations
Protein Turnover in Cell Lysates of Cardiomyocytes from Covaris-Integrated Platform - Network Analysis (PINE) of Significant Differentially Expressed Proteins
Summary
Jenny Van Eyk Lab Collaboration with Covaris
Thank You!
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