Laser capture sampling and analytical issues in proteomics

Abstract

Proteomics holds the promise of evaluating global changes in protein expression and posttranslational 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 (LCM) 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 LCM 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.

Enrichment of single neurons and defined brain regions from human brain tissue samples for subsequent proteome analysis

“…The BBBABSG collects brains from deceased individuals aged C50 years. The methods used by the BBBABSG have been previously reported (Grinberg et al 2007). …”

Proteomic Analyses of Cells Isolated by Laser Microdissection

“…It should be evident that sensitivity, resolution and speed in data capture are all significant problems with proteomics techniques. In order to circumvent these problems, methods have been developed to extract, separate, detect and identify a wide range of proteins from small sample amounts (Gutstein & Morris, 2007). Technical advances in mass spectometry have facilitated major progress in both the qualitative and quantitative analysis of proteins (Kaspar et al, 2010).…”

“…Obtaining a better understanding of tissue complexity could be accomplished by isolating specific cells and analyzing them through proteomic analyses, that could compliment mRNA studies. Over the last few years, the combined use of LM and proteomic analysis has been widely adopted in animal biology and significant progress has been made in adapting the technology to the study of plant cellular processes (Gutstein & Morris, 2007). A list of papers on the application of LM in proteomic and metabolomic studies in plant biology is showed in Table 2.…”

“…When using chemical methods, it is important to reduce the interactions between the proteins, as well as the interactions between the proteins and other substances, including nucleic acids and lipids. It is also important to remove contaminants and interfering substances, and prevent protein precipitation during the separation process (Rabilloud et al, 1996, Gutstein & Morris, 2007. Once the proteins have been extracted, the resultant complex mixture needs to be separated for the subsequent detection, abundance and differential expression analyses.…”

Analysis of mouse brain microvascular endothelium using immuno‐laser capture microdissection coupled to a hybrid linear ion trap with Fourier transform‐mass spectrometry proteomics platform

“…Laser capture microdissection (LCM) is a laser-based, microscope-aided method of tissue microdissection developed over the last decade [1], the applications of which have significantly broadened the opportunity to study protein expression of select cell populations in situ [2][3][4]. In allowing cells to be quickly plucked from their native tissue environments, LCM is less encumbered by the artifacts associated with other cell procurement techniques such as cell culture and bulk isolation protocols, which can suffer from phenotypic drift and anoxic damage, respectively.…”