Identification of proteins in laser-microdissected small cell numbers by SELDI-TOF and Tandem MS

BMC Biotechnology

Ludger Fink¹  Norbert Weissmann³  Werner Seeger¹  Rainer M Bohle¹  Ralf Bogumil²  Markus Meyer²  Grazyna Kwapiszewska¹ 

[1]Department of Pathology, Justus-Liebig University Giessen, Langhansstr.10, 35392 Giessen, Germany

[2]Ciphergen Biosystems GmbH, Göttingen, Germany

[3]Department of Internal Medicine II, Justus-Liebig University Giessen, 35392 Giessen, Germany

Others  :  1154462
DOI  :  10.1186/1472-6750-4-30

 received in 2004-07-13, accepted in 2004-12-03,  发布年份 2004

【 摘 要 】

Background

Laser microdissection allows precise isolation of specific cell types and compartments from complex tissues. To analyse proteins from small cell numbers, we combine laser-microdissection and manipulation (LMM) with mass spectrometry techniques.

Results

Hemalaun stained mouse lung sections were used to isolate 500–2,000 cells, enough material for complex protein profiles by SELDI-TOF MS (surface enhanced laser desorption and ionization/time of flight mass spectrometry), employing different chromatographic ProteinChip^® Arrays. Initially, to establish the principle, we identified specific protein peaks from 20,000 laser-microdissected cells, combining column chromatography, SDS-PAGE, tryptic digestion, SELDI technology and Tandem MS/MS using a ProteinChip^® Tandem MS Interface. Secondly, our aim was to reduce the labour requirements of microdissecting several thousand cells. Therefore, we first defined target proteins in a few microdissected cells, then recovered in whole tissue section homogenates from the same lung and applied to these analytical techniques. Both approaches resulted in a successful identification of the selected peaks.

Conclusion

Laser-microdissection may thus be combined with SELDI-TOF MS for generation of protein marker profiles in a cell-type- or compartment-specific manner in complex tissues, linked with mass fingerprinting and peptide sequencing by Tandem MS/MS for definite characterization.

【 授权许可】

   
2004 Kwapiszewska et al; licensee BioMed Central Ltd.

【 图 表 】

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