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Clinical Chemistry 49: 552-561, 2003; 10.1373/49.4.552
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(Clinical Chemistry. 2003;49:552-561.)
© 2003 American Association for Clinical Chemistry, Inc.

Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry-based Detection of Microsatellite Instabilities in Coding DNA Sequences: A Novel Approach to Identify DNA-Mismatch Repair-deficient Cancer Cells

Thomas Bonk1,2, Andreas Humeny1,2, Johannes Gebert2,2, Christian Sutter2, Magnus von Knebel Doeberitz2 and Cord-Michael Becker1,a

1 Institut für Biochemie, Emil-Fischer-Zentrum, Friedrich-Alexander Universität Erlangen-Nürnberg, Fahrstrasse 17, D-91054 Erlangen, Germany.

2 Abteilung für Molekulare Pathologie, Pathologisches Institut, Universitätsklinikum Heidelberg, Im Neuenheimer Feld 220, D-69120 Heidelberg, Germany.

aAuthor for correspondence. Fax 49-9131-85-22485; e-mail cmb{at}biochem.uni-erlangen.de.

Background: Inherited defects in the DNA mismatch repair system lead to increased loss or gain of repeat units in microsatellites, commonly referred to as microsatellite instability (MSI). MSIs in coding regions of critical genes contribute to the pathogenesis of DNA-mismatch repair-deficient cancers, particularly those associated with the hereditary nonpolyposis colorectal cancer syndrome (HNPCC). MSI typing is therefore increasingly used to guide the molecular diagnosis of HNPCC.

Methods: We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to identify MSIs in mononucleotide repeats within the coding sequences of genes relevant to the pathogenesis of MSI+ neoplastic lesions. After a primer extension reaction of PCR products encompassing the microsatellites, the molecular masses of the extension products were determined by MALDI-TOF-MS.

Results: MSIs were detected by MALDI-TOF-MS in the GART, AC1, TGFBR2, MSH3, and MSH6 genes in neoplastic tissues and MSI+ colorectal cancer cell lines but not in MSI- control tissues. The analysis of peak-integral ratios in a single spectrum of the peaks representing insertions or deletions compared with the full-length microsatellites allowed relative quantification of MSIs. MALDI-TOF-MS-based genotyping results were confirmed by conventional DNA sequencing and electrophoresis.

Conclusions: Because of its reliability, short run times, and low costs, this semiquantitative procedure represents an effective alternative, in particular for diagnostic high-throughput typing of MSIs in neoplastic lesions.




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Detection of Loss of Heterozygosity by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry-Based Analysis of Single-Nucleotide Polymorphisms
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D. Seichter, S. Krebs, and M. Forster
Rapid and accurate characterisation of short tandem repeats by MALDI-TOF analysis of endonuclease cleaved RNA transcripts
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E. F. Petricoin and L. A. Liotta
Mass Spectrometry-based Diagnostics: The Upcoming Revolution in Disease Detection
Clin. Chem., April 1, 2003; 49(4): 533 - 534.
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