Microscale protein expression profiling during disease evolvement.
Advances in technology, such as laser capture microdissection (LCM), have allowed for the specific sampling of cells within their natural functional micro-environment. In model systems using LCM, we have studied the global protein expression profiles of airway epithelial cells during a response to allergen provocation. Bronchial epithelial cells were first identified and phenotyped histologically in snap frozen lung samples of experimentally sensitised mice. Consecutive thin sections of whole lung were then sampled using preparative LCM procedures. Lysates of the captured epithelium (7500 shots) or whole lung were prepared for two-dimensional gel electrophoretic separation and 1400 protein spots were annotated by image analysis. Protein identities were established by matching peptide masses detected using matrix-assisted laser desorption ionization time-of-flight MS as well as electrospray ionization MS-MS sequencing. Using the Mascot database of protein/peptide identities high significance scores in terms of sequence coverage (range 22-70%) and number of peptides (range 7-22 peptides/protein) were obtained for approximately 500 proteins, with examples listed in Table 1. In quantitative terms, the LCM procedure allows the statistical sampling of singular populations of cells distributed throughout tissues and organs. The absolute number of cells required for "entry level" measurements of protein profiles will vary over an order of magnitude depending on the physical size and frequency of the cells being studied within each biological compartment as well as the dynamic range of the proteins being measured, and the absolute limits of detection within the technologies being employed.
Marko-Varga
György A
GA
Department of Biological Sciences, AstraZeneca R&D, Respiratory and Inflammation, S-221 87 Lund, Sweden. gyorgy.marko-varga@astrazeneca.com
Fehniger
Thomas E
TE
D000818:Animals; D005260:Female; D013058:Mass Spectrometry; D051379:Mice; D008807:Mice, Inbred BALB C; D010336:Pathology; D011506:Proteins / Q000737:chemistry*
D011506:Proteins