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

2004; BioMed Central; Volume: 4; Issue: 1 Linguagem: Inglês

10.1186/1472-6750-4-30

1472-6750

Grażyna Kwapiszewska, Markus R. Meyer, Ralf Bogumil, Rainer M. Bohle, Werner Seeger, Norbert Weißmann, Ludger Fink,

Mass Spectrometry Techniques and Applications

Abstract 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.