Drug discovery and development are typically performed in the context of well-characterized model systems such as cell cultures and animals. Screening in such systems is likely to bias results given that the systems do not reflect the environment in which the drug will ultimately be used.
Human diseases are defined in a great many cases by cellular and, increasingly, molecular pathology. Of course, pathology defines normal biological states as well, which serves as a baseline for diagnosis and may be used to guide drug target discovery, and later on clinical treatment, in the context of the disease in the tissue microenvironment (TME).
The mainstays of molecular pathology such as immunohistochemistry (IHC) and fluorescent in-situ hybridization (FISH) are sensitive and capable of subcellular resolution. They are, however, not capable of the massively-multiplex assays routinely performed by mass spectrometry. Nor are they amenable to drug target discovery, in that the assay target must be known a priori and, in the case of IHC, have a validated, paraffin-compatible antibody. Such limitations have largely restricted these techniques to follow-on, validation roles in experimental workflows.
Two factors have inhibited omics-type investigations in samples traditionally used by and accessible to pathologists: formalin fixation of the sample and heterogeneity of the TME. Formalin fixation massively crosslinks cellular components making retrieval of individual molecular analytes essentially impossible.
Applications that use only short segments of a molecule existing between crosslinks can be performed however, witness IHC and FISH. The development of antigen retrieval in 1991 served to dramatically increase IHC sensitivity by partially reversing formaldehyde crosslinks, thereby making more of the protein retrievable. The same method significantly improves the quality of shotgun-proteomic analyses.
TME heterogeneity has similarly confounded proteomic analysis of tissue (Figure 1). To date, investigators have been confronted with two choices: analyze whole tissue or procure homogenous cellular populations from the TME via laser microdissection.
Analysis of whole tissue has not been palatable for the obvious reason that a multiplicity of cell types are typically present in the amount of tissue necessary for conventional proteomic analyses.
Therefore, the result will simply be an average of the proteomic states of the combined cell types. Comparison of such results across disease cases, in which the TME is often highly heterogeneous and often contains necrotic cells as well as vascular and inflammatory infiltration, is unlikely to result in meaningful insight into the disease system under study.
Alternatively, laser microdissection may be used to procure homogenous populations of cells, allowing for contextual analysis. The amount of protein obtainable, however, is typically well short of the quantity required for conventional shotgun-proteomic analyses. This has limited investigations using microdissected tissues to single dimension analysis by LC/MS, thus limiting proteome coverage, particularly of disease-relevant, low-abundance proteins.