The identification of angiogenesis-related proteins is important for the development of new antiangiogenic therapies, and such proteins are potential new biomarkers for gliomas. The aim of this study was to identify proteins that are exclusively present in glioma neovasculature and not in the vasculature of normal brain. We combined advanced proteomics techniques to compare the expression profiles of microdissected blood vessels from glioma with blood vessels of normal control brain samples. We measured the enzymatic generated peptide profiles from these microdissected samples by MALDI-FTMS. Subsequently, the samples were fractionated by nano-LC prior to MALDI-TOF/TOF. This combined approach enabled us to identify four proteins that appeared to be exclusively expressed in the glioma blood vessels. Two of these proteins, fibronectin and colligin 2, were validated on tissue sections using specific antibodies. We found that both proteins are present in active angiogenesis in glioma, other neoplasms, and reactive conditions in which neoangiogenesis takes place. This work proves that gel-free mass spectrometric techniques can be used on relatively

The discovery of the IDH1 R132H (IDH1 mut) mutation in low‐grade glioma and the associated change in function of the IDH1 enzyme has increased the interest in glioma metabolism. In an earlier study, we found that changes in expression of genes involved in the aerobic glycolysis and the TCA cycle are associated with IDH1 mut. Here, we apply proteomics to FFPE samples of diffuse gliomas with or without IDH1 mutations, to map changes in protein levels associated with this mutation. We observed significant changes in the enzyme abundance associated with aerobic glycolysis, glutamate metabolism, and the TCA cycle in IDH1 mut gliomas. Specifically, the enzymes involved in the metabolism of glutamate, lactate, and enzymes involved in the conversion of α‐ketoglutarate were increased in IDH1 mut gliomas. In addition, the bicarbonate transporter (SLC4A4) was increased in IDH1 mut gliomas, supporting the idea that a mechanism preventing intracellular acidification is active. We also found that enzymes that convert proline, valine, leucine, and isoleucine into glutamate were increased in IDH1 mut glioma. We conclude that in IDH1 mut glioma metabolism is rewired (increased input of lactate and glutamate) to preserve TCA‐cycle activity in IDH1 mut gliomas.

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Background: A Java™ application is presented, which compares large numbers (n > 100) of raw FTICR mass spectra from patients and controls. Two peptide profile matrices can be produced simultaneously, one with occurrences of peptide masses in samples and another with the intensity of common peak masses in all the measured samples, using the peak-and background intensities of the raw data. In latter way, more significantly differentially expressed peptides are found between groups than just using the presence or absence in samples of common peak masses. The software application is tested by searching angiogenesis related proteins in glioma by comparing laser capture micro dissected-and enzymatic by trypsin digested tissue sections.

Laser microdissection is an effective technique to harvest pure cell populations from complex tissue sections. In addition to using the microdissected cells in several DNA and RNA studies, it has been shown that the small number of cells obtained by this technique can also be used for proteomics analysis. Combining laser capture microdissection and different types of mass spectrometers opened ways to find and identify proteins that are specific for various cell types, tissues, and their morbid alterations. Although the combination of microdissection followed by the currently available techniques of proteomics has not yet reached the stage of genome wide representation of all proteins present in a tissue, it is a feasible way to find significant differentially expressed proteins in target tissues. Recent developments in mass spectrometric detection followed by proper statistics and bioinformatics enable to analyze the proteome of not more than 100-200 cells. Obviously, validation of result is essential. The present review describes and discusses the various methods developed to target cell populations of interest by laser microdissection, followed by analysis of their proteome.