Saturation labeling with cysteine-reactive cyanine fluorescent dyes provides increased sensitivity for protein expression profiling of laser-microdissected clinical specimens

Abstract

Laser capture microdissection (LCM) provides the capability to isolate and analyze small numbers of cells from a specific area of a histologic section. LCM has particular value for analysis of early stage tumors, which are often small and intermixed with non-tumor tissue. It has previously been shown that a new generation of cysteine-reactive cyanine dyes can, in principle, provide increased sensitivity for two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) profiling when sample quantitities are limiting. However, the comparative advantage of the new dyes in a clinical setting has not been established. Here, we report that cysteine-reactive dyes allowed the identification of more features than established, lysine-reactive dyes with a given number of cells. This was true both with extracts prepared from human papillomavirus E6 and E7-transduced human keratinocytes, a model for early-stage cervical cancer, and with LCM samples. In an experiment comparing LCM clinical samples of gastric adenocarcinoma versus precancerous, spasmolytic polypeptide expressing metaplasia (SPEM) from the same patient, cysteine labeling allowed the identification of more than 1000 discrete protein spots in samples containing 5000 cells. This is a 5- to 50-fold smaller sample than used in previous studies. Both labeling methods had a comparable success rate for protein identification by mass spectrometry (MS). The proteins associated with more than 40 differentially abundant spots in the clinical samples were identified by MS. In this exploratory analysis, changes in expression levels of cytoskeletal proteins, molecular chaperones, and cell-signaling proteins were seen. The identification of a number of proteins that are potentially relevant to tumor progression suggests that the method holds promise for biomarker discovery.