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Protein profiling of single epidermal cell types from Arabidopsis thaliana by surface-enhanced laser desorption and ionization technology (CAT#: STEM-ST-0366-LJX)

Introduction

We use a novel approach for investigating differential protein expression within three epidermal cell types. In particular, 3000 single pavement, basal, and trichome cells from leaves of Arabidopsis thaliana were harvested by glass micro-capillaries. Subsequently, these single cell samples were joined to form pools of 100 individual cells and analyzed using the ProteinChip technology; SELDI: surface-enhanced laser desorption and ionization. As a result, numerous protein signals that were differentially expressed in the three epidermal cell types could be detected. One of these proteins was characterized by tryptical digestion and subsequent identification via tandem quadrupole-time of flight (Q-TOF) mass spectrometry. Down regulation of this sequenced small subunit precursor of ribulose-1,5 bisphosphate carboxylase(C) oxygenase(O) (RuBisCo) in trichome and basal cells indicates the sink status of these cell types that are located on the surface of A. thaliana source leaves. Based on the obtained protein profiles, we suggest a close functional relationship between basal and trichome cells at the protein level.




Principle

The surface enhanced laser desorption ionization technique belongs to laser desorption mass spectrometry (LDMS). It is different from ordinary LDMS in that the laser is not directly hit on the sample to desorption, but the sample is suspended in the matrix, the laser is hit on the matrix, the matrix absorbs and transmits the laser energy, so that the sample in the matrix desorption out. After desorption and ionization, the samples were examined in a time-flight mass spectrometer.

Applications

For protein analysis and measurement of molecular weight of complete proteins
For the diagnosis of a variety of diseases, especially cancer

Procedure

1. The surface of the protein chip is treated in a certain chemical or biochemical way (surface enhancement), so that it has the ability to bind specifically to a certain type of protein
2. The serum or protein extract is directly added to the surface of the chip, and the chip is washed after incubation. Specific proteins bind to the chip and are thus separated from the protein mixture
3. The chip then uses a "chip reader" (a kind of SELDI-TOF-MS) to obtain a mass spectrum of the protein bound to the chip
4. The SELDI protein chip system can be used to compare changes in the protein profile of any set of control samples or different disease states to identify biomarkers or disease-related targets

Materials

• Sample Type:
Arabidopsis thaliana

Notes

When operating, strictly follow the experimental steps.
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