Phosphoproteomic profiling of tumor tissues identifies HSP27 Ser82 phosphorylation as a robust marker of early ischemia

Abstract

Delays between tissue collection and tissue fixation result in ischemia and ischemia-associated changes in protein phosphorylation levels, which can misguide the examination of signaling pathway status. To identify a biomarker that serves as a reliable indicator of ischemic changes that tumor tissues undergo, we subjected harvested xenograft tumors to room temperature for 0, 2, 10 and 30 minutes before freezing in liquid nitrogen. Multiplex TMT-labeling was conducted to achieve precise quantitation, followed by TiO2 phosphopeptide enrichment and high resolution mass spectrometry profiling. LC-MS/MS analyses revealed phosphorylation level changes of a number of phosphosites in the ischemic samples. The phosphorylation of one of these sites, S82 of the heat shock protein 27 kDa (HSP27), was especially abundant and consistently upregulated in tissues with delays in freezing as short as 2 minutes. In order to eliminate effects of ischemia, we employed a novel cryogenic biopsy device which begins freezing tissues in situ before they are excised. Using this device, we showed that the upregulation of phosphorylation of S82 on HSP27 was abrogated. We thus demonstrate that our cryogenic biopsy device can eliminate ischemia-induced phosphoproteome alterations, and measurements of S82 on HSP27 can be used as a robust marker of ischemia in tissues.

Figure 1. Phosphoproteomic profiling of xenograft tumors during ischemia.

A schematic workflow of the strategy used to profile the phoshoproteomic changes resulting from ischemia. Whole xenograft tumors from the breast cancer cell line HCC1395 were harvested from mice and exposed to room temperature for 0, 2, 10 and 30 minutes before snap freezing in liquid nitrogen. After protein extraction and digestion with trypsin, each sample was labeled with different versions of TMT reagents followed by pooling, desalting, and enrichment using TiO₂ beads. The enriched phosphopeptides were then analyzed on LTQ-Orbitrap Elite mass spectrometer without further fractionation. Figure drawn by M.S.Z.

Figure 2. Upregulation of S82 phosphorylation of HSP27 during ischemia.

(A) A representative MS/MS spectrum for HSP27 (QLsSGVSEIR) and the relative intensities of the TMT reporter ions for the different time points are shown on upper left. (B) Western blot analysis of the ischemia samples with the indicated antibodies. β-actin is used as loading control.

Figure 3. Development of novel cryogenic biopsy device.

(A) A picture of the cryogenic biopsy device made using a conventional TSK biopsy gun with a tube feeding cryogen gas and a modified cryoablation needle (inset). (B) A picture of the simple cryogenic system containing manual pressure regulator to set the input pressure to the needle, a safety valve, and a solenoid for computer controlled operation

Figure 4. Prevention of hyperphosphorylation of HSP27 S82 by the novel cryogenic biopsy device.

(A) A distribution of ratios of all phosphopeptides identified in the mass spectrometry analysis of NCI-N87 gastric xenograft tumors biopsied using conventional/cryogenic device. (B) A representative MS/MS spectrum for HSP27 (QLsSGVSEIR) and the relative intensities of the TMT reporter ions for the different biopsy devices are shown on upper left (C,D) Western blot analysis of the NCI-N87 gastric xenograft tumor and U87MG glioma xenograft tumor biopsied with the cryogenic and conventional devices using the indicated antibodies. β-actin is used as loading control.