Phosphoproteomic profiling of tumor tissues identifies HSP27 Ser82 phosphorylation as a robust marker of early ischemia.
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.
McKusick-Nathans Institute of Genetic Medicine and Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205 USA.
Muhammad Saddiq
MS
Zahari
McKusick-Nathans Institute of Genetic Medicine and Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205 USA.
Xinyan
X
Wu
Institute of Bioinformatics, International Tech Park, Bangalore, 560066 India.
Sneha M
SM
Pinto
Institute of Bioinformatics, International Tech Park, Bangalore, 560066 India.
Raja Sekhar
RS
Nirujogi
McKusick-Nathans Institute of Genetic Medicine and Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205 USA.
Min-Sik
MS
Kim
Robin Medical, Inc., P.O. Box 2414, Baltimore, MD 21203, USA.
Barry
B
Fetics
Robin Medical, Inc., P.O. Box 2414, Baltimore, MD 21203, USA.
Mathew
M
Philip
Charles River Discovery Research Services, 3300 Gateway Centre Boulevard, Morrisville NC 27560.
Sheri R
SR
Barnes
Charles River Discovery Research Services, 3300 Gateway Centre Boulevard, Morrisville NC 27560.
Beverly
B
Godfrey
Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, USA.|Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, USA.
Edward
E
Gabrielson
Robin Medical, Inc., P.O. Box 2414, Baltimore, MD 21203, USA.
Erez
E
Nevo
McKusick-Nathans Institute of Genetic Medicine and Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205 USA.|Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, USA.|Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, USA.|Adrienne Helis Malvin Medical Research Foundation, New Orleans, LA 70130, USA.|Diana Helis Henry Medical Research Foundation, New Orleans, LA 70130, USA.
Akhilesh
A
Pandey
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D001706:Biopsy; D045744:Cell Line, Tumor; D055551:HSP27 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D007511:Ischemia / Q000378:metabolism* / Q000473:pathology; D016513:Mice, SCID; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D015854:Up-Regulation; D023041:Xenograft Model Antitumor Assays
D015415:Biomarkers; D055551:HSP27 Heat-Shock Proteins; D010750:Phosphoproteins; D010768:Phosphoserine