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


CONCLUSIONS
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.
Kwapiszewska
Grazyna
G
Department of Pathology, Justus-Liebig University Giessen, Langhansstr,10, 35392 Giessen, Germany. Grazyna.Kwapiszewska@patho.med.uni-giessen.de <Grazyna.Kwapiszewska@patho.med.uni-giessen.de>
Meyer
Markus
M
Bogumil
Ralf
R
Bohle
Rainer M
RM
Seeger
Werner
W
Weissmann
Norbert
N
Fink
Ludger
L
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005629:Frozen Sections / Q000379:methods; D008168:Lung / Q000737:chemistry / Q000166:cytology; D008297:Male; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008807:Mice, Inbred BALB C; D042282:Microdissection / Q000379:methods*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping / Q000379:methods; D040081:Protein Array Analysis / Q000379:methods; D011506:Proteins / Q000737:chemistry*; D011650:Pulmonary Alveoli / Q000737:chemistry / Q000166:cytology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D015415:Biomarkers; D010446:Peptide Fragments; D011506:Proteins