Navigated laser capture microdissection as an alternative to direct histological staining for proteomic analysis of brain samples

Abstract

Proteomic analysis of the brain is complicated by the need to obtain cells from specific anatomical regions, or nuclei. Laser capture microdissection (LCM) is a technique that is precise enough to dissect single cells within a tissue section, and thus could be useful for isolating specific brain nuclei for analysis. However, we and others have previously demonstrated that histological staining protocols used to guide LCM have detrimental effects on protein separation by two-dimensional electrophoresis (2-DE). Here we describe a new LCM method called navigated LCM. This microdissection method uses fixed but unstained tissue as starting material and thus enables us to avoid artifacts induced by tissue staining. By comparing 2-DE results obtained from fixed, unstained LCM brain tissue samples to those obtained from manually dissected samples, we demonstrated that this microdissection process gave similar protein recovery rates and similar resolution of protein spots on 2-DE gels. Moreover, matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis of selected spots from gels derived from control and fixed, LCM samples revealed that the fixation-LCM process had no effect on protein identification. Navigated LCM of tissue sections is therefore a practical and powerful method for performing proteomic studies in specifically defined brain regions.

Evaluation of two‐dimensional electrophoresis and liquid chromatography – tandem mass spectrometry for tissue‐specific protein profiling of laser‐microdissected plant samples

“…The application of LM in combination with tissue-specific protein profiling was so far the exception and only applied to animal tissue [13,14,20]. For this reason, we started to optimize tissue pretreatment procedures for Arabidopsis stems in order to allow LMPC collection of vascular bundles and efficient protein retrieval from such samples.…”

“…Although 2-DE has been greatly improved by advances in gel quality and refinement of separation methodologies, the biggest drawbacks in its utilization for cell-specific proteomics are the low dynamic range of the available staining techniques [18] and, more importantly, the relatively high amount of starting material needed. This issue is apparent in several publications on human tissue where 2-DE gels resulting from laser-microdissected tissue led only to a limited number of visible, well separated spots [13,14,19,20]. …”

“…Thus, high precision and touch-free, nondestructive laser-based techniques provide the better option for microdissection [11]. Laser microdissection (LM) has been developed and routinely used in mammalian systems [5,[12][13][14] and only recently this technique has been introduced successfully into plant sciences [3,15]. In the LM version developed by P.A.L.M.…”

Neuroproteomics of the Synapse and Drug Addiction

“…One technique that can be used to increase the precision of neuroproteomic analyses is laser capture microdissection (Emmert-Buck et al, 1996). With this dissection technique, it is possible to target specific brain nuclei, subpopulations of neurons, and even individual neuron types from fixed or frozen tissue, with a minimal effect on gene and protein expression profiles (Mouledous et al, 2003). Another strategy is to establish subcellular proteomes, taking advantage of the compartmentalization of cells and the assembly of functional units, such as protein complexes.…”

Intact‐protein based sample preparation strategies for proteome analysis in combination with mass spectrometry

“…Moulédous et al applied this approach to unstained rat brain tissues to precisely dissect nuclei from specifically defined brain regions. Proteins from the captured tissue cells were extracted and separated by 2D PAGE, and selected spots were identified by Bio-MS (Moulédous et al, 2003).…”