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

BMC Biotechnol. 2004 Dec 3:4:30. doi: 10.1186/1472-6750-4-30.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biomarkers / metabolism
  • Chromatography, Affinity / methods
  • Electrophoresis, Polyacrylamide Gel / methods
  • Frozen Sections / methods
  • Lung / chemistry
  • Lung / cytology
  • Male
  • Mass Spectrometry / methods
  • Mice
  • Mice, Inbred BALB C
  • Microdissection / methods*
  • Peptide Fragments / chemistry
  • Peptide Fragments / metabolism
  • Peptide Mapping / methods
  • Protein Array Analysis / methods
  • Proteins / chemistry*
  • Pulmonary Alveoli / chemistry
  • Pulmonary Alveoli / cytology
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods*

Substances

  • Biomarkers
  • Peptide Fragments
  • Proteins