1. Chapter 9 PART II: Concepts in Molecular Biology and Genetics The Human Proteome: Implications for the Understanding of Human Disease Companion site for Molecular Pathology Author: William B. Coleman and Gregory J. Tsongalis

2. Companion site for Molecular Pathology Copyright © 2009 by Academic Press. All rights reserved. 2 Laser capture microdissection (LCM). LCM is a technology for procuring pure cell populations from a stained tissue section under direct microscopic visualization. Tissues contain heterogeneous cellular populations (e.g., epithelium, cancer cells, fibroblasts, endothelium, and immune cells). The diseased cellular population of interest usually comprises only a small percentage of the tissue volume. LCM directly procures the subpopulation of cells selected for study, while leaving behind all of the contaminating cells. A stained section of the heterogeneous tissue is mounted on a glass microscope slide and viewed under high magnification. The experimenter selects the individual cell(s) to be studied using a joystick or via a computer screen. The chosen cells are lifted out of the tissue by the action of a laser pulse. The infrared laser, mounted in the optical axis of the microscope, locally expands a thermoplastic polymer to reach down and capture the cell beneath the laser pulse (insert). When the film is lifted from the tissue section, only the pure cells for study are excised from the heterogeneous cellular population. The DNA, RNA, and proteins of the captured cells remain intact and unperturbed. Using LCM, one to several thousand tissue cells can be captured in less than 5 minutes. Using appropriate buffers, the cellular constituents are solubilized and subjected to microanalysis methods. Proteins from all compartments of the cell can be readily procured. Protein conformation and enzymatic activity are retained if the tissues are frozen or fixed in ethanol before sectioning. The extracted proteins can be analyzed by any method that has sufficient sensitivity. FIGURE 9.1

3. Companion site for Molecular Pathology Copyright © 2009 by Academic Press. All rights reserved. 3 A roadmap for individualized cancer therapy. Following biopsy or needle aspiration, and laser microdissection, signal pathway analysis is performed using protein microarrays for phosphoproteomic analysis, and RNA transcript arrays.The specific signaling portrait becomes the basis of a patient-tailored therapeutic regime. Therapeutic assesment is obtained by follow-up biopsy, and the molecular portrait of signaling events is reassessed to determine if therapeutic selection should be modified futher. FIGURE 9.2

4. Companion site for Molecular Pathology Copyright © 2009 by Academic Press. All rights reserved. 4 Pre-analytical variables during tissue acquisition. Excision delay time, processing delay time, and the type of preservation chemistry selected will affect the quality of molecular analyses. FIGURE 9.3

5. Companion site for Molecular Pathology Copyright © 2009 by Academic Press. All rights reserved. 5 Molecular stages and timeline of tissue cell death. Post tissue excision, cascades of cellular kinases are activated and deactivated as tissue reacts to wounding, ischemia, inflammation, environmental stresses, hypoxia, and nutrient depletion (Adapted from Espina et al. Molecular and Cellular Proteomics, October 2008). FIGURE 9.4

6. Companion site for Molecular Pathology Copyright © 2009 by Academic Press. All rights reserved. 6 The peptidome hypothesis: Circulating peptides and protein fragments are shed from all cell types in the tissue microenvironment. Proteolytic cascades within the tissue generate fragments that diffuse into the circulation. The identity and cleavage pattern of the peptides provide two dimensions of diagnostic information. FIGURE 9.5

7. Companion site for Molecular Pathology Copyright © 2009 by Academic Press. All rights reserved. 7 Proteomics mass spectrometry analytical workflow. FIGURE 9.6

8. Companion site for Molecular Pathology Copyright © 2009 by Academic Press. All rights reserved. 8 Mass spectrometry multiple reaction monitoring (MRM) is an emerging technology for quantifying and specifically identifying protein analytes based on highly accurate mass profiling of a protease peptide fragment. This technology has the potential to validate discovered biomarker candidates without the requirement for a specific antibody. FIGURE 9.7

9. Companion site for Molecular Pathology Copyright © 2009 by Academic Press. All rights reserved. 9 Schematic representation of particle structure and function. Particles are constituted by a bait containing core, surrounded by a sieving shell. When introduced into a complex solution, such as serum, core-shell particles remove low molecular weight proteins from carrier albumin with affinity capture and perform molecular weight sieving with total exclusion of high molecular weight proteins. Depending on the starting volume of the fluid sample, the particles can effectively amplify low abundance biomarkers 100- to 1000-fold. FIGURE 9.8