Laser capture sampling and analytical issues in proteomics

Abstract

Proteomics holds the promise of evaluating global changes in protein expression and post-translational modification in response to environmental stimuli. However, difficulties in achieving cellular anatomic resolution and extracting specific types of proteins from cells have limited the efficacy of these techniques. Laser capture microdissection has provided a solution to the problem of anatomical resolution in tissues. New extraction methodologies have expanded the range of proteins identified in subsequent analyses. This review will examine the application of laser capture microdissection to proteomic tissue sampling, and subsequent extraction of these samples for differential expression analysis. Statistical and other quantitative issues important for the analysis of the highly complex datasets generated are also reviewed.

Figure 1. LCM workflow

Samples are generally fresh-frozen and sectioned using a cryostat to 5-8 μm thickness. This figures shows a dissection of a brain region known as the striatum. A cap that fits on an eppendorf tube is placed over the sample. A thin film that is melted by the heat of the laser is premounted on the cap. The laser is pulsed over areas of interest, causing the film to melt and cells of interest to adhere to the film. The cap is then lifted from the section, and captured cells subjected to further processing.

Figure 2. An example of a laser capture microscope

The Arcturus XT is pictured. This instrument is based on the Nikon TE2000U inverted microscope, and has both UV and IR lasers to enable both cutting and adherent LCM. From http://www.moleculardevices.com/pages/instruments/arcturusXT.html

Figure 3. Representative Average Gel

This average gel was created by taking the pixel-wise average over a series of 28 gels previously reported by Nishihara and Champion [116] with feature alignment performed in our laboratory using the TT900 program (Nonlinear Dynamics, Newcastle, UK). “Hotter” colors indicate regions of higher intensity, while “cooler” colors indicate lower intensities. The units of the x and y axes are pixel distance from the origin (upper left corner of the image). White “x's” mark the 1380 spots detected using the Pinnacle method. These represent local maxima in both the x- and y-directions with intensities greater than the 75^th percentile intensity on the average gel. Figure from [83].