A portrait of tissue phosphoprotein stability in the clinical tissue procurement process.
Author(s): Espina V, Edmiston KH, Heiby M, Pierobon M, Sciro M, Merritt B, Banks S, Deng J, VanMeter AJ, Geho DH, Pastore L, Sennesh J, Petricoin EF, Liotta LA
Publication: Mol Cell Proteomics, 2008, Vol. 7, Page 1998-2018
PubMed ID: 18667411 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
This study investigated the stability of phosphoprotein levels after a cold ischemia time (post-excision delay at room temperature) of up to 120 min. Eleven tissue specimens from breast, colon, lung, ovary, uterus, skin were analyzed. Phosphoprotein levels were compared to controls that were processed as soon as possible; notably, although controls are termed "time zero" actual cold ischemia time was specimen-specific.
Summary of Findings:
Every tissue type examined displayed robust alterations in the post-translational levels of 9 or more phosphoproteins (+/- 20% change compared to specimen-specific "time zero" controls) within the first 60 min of cold ischemia, although tissue-specific effects were observed. Phosphoproteins significantly affected by cold ischemia times of 60 min or less included those functioning in apoptosis, hypoxia, proliferation/survival, stress/inflammation, and as transcription factors. More specifically, significant fluctuations in phosphoprotein levels were observed in uterine specimens after 10 min of cold ischemia compared to case-matched controls frozen within 4 minutes of excision, and in lung specimens after 15 min of cold ischemia compared to case-matched controls frozen within 10 minutes of excision. Although time-dependent alterations in phosphoprotein levels were observed among both normal and cancerous lung tissue, the direction and magnitude of response were protein-specific and influenced by disease state.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
- Neoplastic - Melanoma
- Neoplastic - Carcinoma
- Neoplastic - Benign
Platform:
Analyte Technology Platform Protein Reverse phase protein microarray Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Biospecimen location Breast
Colon
Lung
Ovary
Uterus
Skin
Preaquisition Diagnosis/ patient condition Normal
Neoplastic
Biospecimen Acquisition Cold ischemia time < 60 min
60-120 min
> 120 min
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Study Purpose
The purpose of this study was to determine if the temperature of post excision storage (4 degrees C, room temperature) differentially affects phosphoprotein levels. A single uterine specimen was analyzed after cold ischemia times of 30 min (baseline), 45 min, 60 min, 90 min, and 120 min.
Summary of Findings:
Significant and duration-dependent deviations from the baseline were observed among case-matched uterine specimens held at 4 degrees C or room temperature. Although maximal differences in phosphoprotein levels were similar among specimens stored at different temperatures, the onset of effect was modestly delayed for several phosphoproteins in specimens stored at 4 degrees C compared to room temperature prior to freezing.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Protein Reverse phase protein microarray Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage temperature Room temperature
4 degrees C
Biospecimen Acquisition Cold ischemia time 30 min
45 min
60 min
90 min
120 min
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Study Purpose
The purpose of this study was to determine if specimen incubation with phosphatase and kinase inhibitors (alone or in combination) could attenuate ischemia-induced effects on phosphoprotein levels. Breast and melanoma specimens in the presence or absence of inhibitors were assessed.
Summary of Findings:
Representative phosphoproteins that increased progressively with ischemia duration were further elevated in breast specimens incubated with phosphatase inhibitors during cold ischemia; conversely, incubation with kinase inhibitors attenuated ischemia-induced increases in phosphoprotein levels. Examination of a larger subset of phosphoproteins in melanoma specimens buttressed findings in breast, with 85% of the phosphoproteins examined elevated above vehicle controls after incubation with phosphatase inhibitors during 30 min of ischemia, and 20% depressed after incubation with kinase inhibitors. Incubation of breast specimens with phosphatase and kinase inhibitors in combination attenuated ischemia-induced alterations in 15% of the 32 phosphoproteins analyzed, with specimens subjected to 5 or 90 minutes of cold ischemia time. Specimen incubation with phosphatase and kinase inhibitors in a methanol or ethanol based solution adequately preserved cellular morphology.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Melanoma
- Neoplastic - Benign
Platform:
Analyte Technology Platform Protein Reverse phase protein microarray Morphology H-and-E microscopy Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Cold ischemia time 5 min
10 min
30 min
60 min
90 min
120 min
Biospecimen Acquisition Biospecimen location Breast
Skin
Biospecimen Preservation Kinase inhibitor Genistein
None
Staurosporine
Biospecimen Preservation Enzyme inhibitor Beta-glycerophosphate
None
Sodium orthovanadate