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Evaluation of two‐dimensional electrophoresis and liquid chromatography – tandem mass spectrometry for tissue‐specific protein profiling of laser‐microdissected plant samples
Author(s) -
Schad Martina,
Lipton Mary S.,
Giavalisco Patrick,
Smith Richard D.,
Kehr Julia
Publication year - 2005
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200410399
Subject(s) - proteome , laser capture microdissection , chromatography , tandem mass spectrometry , mass spectrometry , chemistry , proteomics , sample preparation , liquid chromatography–mass spectrometry , homogeneous , protein purification , biochemistry , gene expression , physics , gene , thermodynamics
Laser microdissection (LM) allows the collection of homogeneous tissue‐ and cell‐specific plant samples. The employment of this technique with subsequent protein analysis has thus far not been reported for plant tissues, probably due to the difficulties associated with defining a reasonable cellular morphology and, in parallel, allowing efficient protein extraction from tissue samples. The relatively large sample amount needed for successful proteome analysis is an additional issue that complicates protein profiling on a tissue‐ or even cell‐specific level. In contrast to transcript profiling that can be performed from very small sample amounts due to efficient amplification strategies, there is as yet no amplification procedure for proteins available. In the current study, we compared different tissue preparation techniques prior to LM/laser pressure catapulting (LMPC) with respect to their suitability for protein retrieval. Cryo‐sectioning was identified as the best compromise between tissue morphology and effective protein extraction. After collection of vascular bundles from Arabidopsis thaliana stem tissue by LMPC, proteins were extracted and subjected to protein analysis, either by classical two‐dimensional gel electrophoresis (2‐DE), or by high‐efficiency liquid chromatography (LC) in conjunction with tandem mass spectrometry (MS/MS). Our results demonstrate that both methods can be used with LMPC collected plant material. But because of the significantly lower sample amount required for LC‐MS/MS than for 2‐DE, the combination of LMPC and LC‐MS/MS has a higher potential to promote comprehensive proteome analysis of specific plant tissues.