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

• Main Authors: Seeger Werner, Bohle Rainer M, Bogumil Ralf, Meyer Markus, Kwapiszewska Grazyna, Weissmann Norbert, Fink Ludger
• Format: Article
• Language: English
• Published: BMC 2004-12-01
• Series: BMC Biotechnology
• Online Access: http://www.biomedcentral.com/1472-6750/4/30

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p>