J Korean Med Sci.  2014 Jul;29(7):934-940. 10.3346/jkms.2014.29.7.934.

Imaging Mass Spectrometry in Papillary Thyroid Carcinoma for the Identification and Validation of Biomarker Proteins

Affiliations
  • 1Department of Pathology, Konkuk University School of Medicine, Seoul, Korea. wskim@kuh.ac.kr
  • 2Department of Molecular Biotechnology, WCU Program, Konkuk University, Seoul, Korea.
  • 3SMART Institute of Advanced Biomedical Science, Konkuk University, Seoul, Korea.
  • 4The Research Institute of Medical Science, Konkuk University School of Medicine, Seoul, Korea.
  • 5Department of Surgery, Konkuk University School of Medicine, Seoul, Korea.

Abstract

Direct tissue imaging mass spectrometry (IMS) by matrix-assisted laser desorption ionization and time-of-flight (MALDI-TOF) mass spectrometry has become increasingly important in biology and medicine, because this technology can detect the relative abundance and spatial distribution of interesting proteins in tissues. Five thyroid cancer samples, along with normal tissue, were sliced and transferred onto conductive glass slides. After laser scanning by MALDI-TOF equipped with a smart beam laser, images were created for individual masses and proteins were classified at 200-microm spatial resolution. Based on the spatial distribution, region-specific proteins on a tumor lesion could be identified by protein extraction from tumor tissue and analysis using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Using all the spectral data at each spot, various intensities of a specific peak were detected in the tumor and normal regions of the thyroid. Differences in the molecular weights of expressed proteins between tumor and normal regions were analyzed using unsupervised and supervised clustering. To verify the presence of discovered proteins through IMS, we identified ribosomal protein P2, which is specific for cancer. We have demonstrated the feasibility of IMS as a useful tool for the analysis of tissue sections, and identified the tumor-specific protein ribosomal protein P2.

Keyword

Pathology; Proteins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Thyroid Gland; Neoplasms

MeSH Terms

Aged
Amino Acid Sequence
Biological Markers/*analysis
Carcinoma/*diagnosis/metabolism/pathology
Chromatography, High Pressure Liquid
Cluster Analysis
Female
Humans
Image Processing, Computer-Assisted
Male
Middle Aged
Molecular Sequence Data
Molecular Weight
Phosphoproteins/analysis/metabolism
Proteome/analysis
Proteomics
Reproducibility of Results
Ribosomal Proteins/analysis/metabolism
*Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
*Tandem Mass Spectrometry
Thyroid Gland/metabolism/pathology
Thyroid Neoplasms/*diagnosis/metabolism/pathology
Biological Markers
Phosphoproteins
Proteome
Ribosomal Proteins

Figure

  • Fig. 1 Gross and microscopic findings of the thyroid. (A) The right thyroid showed a papillary carcinoma (black arrow) and nodular hyperplasia (black dash line arrow). Immunohistochemistry was positive in CK19 (B) and Galectin-3 (C).

  • Fig. 2 Selected molecular ion images from a papillary thyroid carcinoma section. The thyroid slide stained hematoxylin and eosin revealed papillary carcinoma. Imaging mass spectrometry based on Principal Component Analysis, molecular ions corresponding to 11,661 m/z and 9,282 m/z.

  • Fig. 3 Protein identification (include mass spectrum for protein in HPLC fractions). Identification of proteins of differential expression was carried out by digesting of separated proteins with trypsin and sequencing by LC-MS/MS. MS/MS spectra showing a peptide sequences. YVASYLLAALGGNSSPSAK of fraction 50 (pooled fraction 4): Xc value is 2.420, ΔCn value is 0.261 (A), NIEDVIAQGIGK of fraction 50 (pooled fraction 4): Xc value is 4.347, ΔCn value is 0.394 (B), LASVPAGGAVAVSAAPGSAAPAAGSAPAAAEEK of fraction 50 (pooled fraction 4): Xc value is 4.308 (C), ΔCn value is 0.608 and tolerances of m/z of the peptide were ± 2 and ± 1 for full mass and MS/MS fragmentation, respectively.

  • Fig. 4 In imaging mass spectrometry, the distribution of detecting protein (11,661 m/z) displayed high concentration and corresponded with concentration of ribosomal protein p2 (11,657 m/z) in tumor lesion (Δmass: 4 m/z).


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