Assessment of ApoC1, LuzP6, C12orf75 and OCC-1 in cystic glioblastoma using MALDI-TOF mass spectrometry, immunohistochemistry and qRT-PCR

Abstract

Mass spectrometric analysis of glioblastoma cyst fluids has disclosed a protein peak with m/z 6424-6433. Among the proteins, potentially generating this peak are ApoC1 and LuzP6. To further elucidate protein expression of glioblastoma cells, we analyzed MALDI-TOF results of cyst fluid, performed immunohistochemistry and mRNA analysis. MALDI-TOF protein extraction from 24 glioblastoma cyst fluids was performed with a weak cation exchange. 50 glioblastoma samples were stained with two custom-made antibodies against LuzP6 and commercial antibodies against ApoC1, C12orf75 and OCC-1 and analyzed. For mRNA detection, 16 tissue samples were stored in RNAlater, extracted using the miRNeasy kit and reversely transcribed. For 12 patients, synopsis of results from all three examinations was possible. MALDI-TOF confirmed the peak at 6433 Da in 75% of samples. Immunohistochemically, LuzP6 was detected in 92% (LuzP6_1-29) and 96% (LuzP6_30-58) of samples and ApoC1 in 66%. Mean mRNA levels were highest for ApoC1, followed by LuzP6. No correlation between mRNA expression, immunohistochemical staining and intensity of the MALDI-TOF peaks was found. An unequivocal identification of one protein as the source for the 6433 peak is not possible, but our results point to ApoC1 and LuzP6 as the underlying proteins.

Keywords: ApoC1; C12orf75; Cystic glioblastoma; LuzP6; MALDI–TOF; Malignant glioma; OCC-1.

Fig. 1

Strong positive LuzP6_30-58 immunohistochemical staining of cystic glioblastomas. a Light brown cytoplasmatic staining of astrocyte-shaped cells around a vessel. b Dark brown nuclear staining (both original magnification 200×)

Fig. 2

Significant correlation between relative peak intensities in the 19 samples containing peaks at 6433 and 6632 Da. Both protein bands can result from ApoC1

Fig. 3

Mean values of the semiquantitative immunoreactivity score for the five antibodies, separately shown for cystic and non-cystic glioblastomas. Bars show standard deviation

Fig. 4

Mean RNA levels of the four proteins in 16 glioblastoma samples. Bars give the standard deviation

Fig. 5

Results of RNA detection in the 16 samples and the OcL control

Fig. 6

Correlation of RNA levels and immunostaining scores for Occ1 and LuzP6 in 12 concomitant samples. Nb: the staining scores for LuzP6_1-29 and LuzP6_30-58 were averaged. Due to the low values of Occ1 mRNA, not all 12 markers are visible in the plot