NIH, National Cancer Institute, Division of Cancer Treatment and Diagnosis (DCTD) NIH - National Institutes of Health National Cancer Institute DCTD - Division of Cancer Treatment and Diagnosis

Time dependent effect of cold ischemia on the phosphoproteome and protein kinase activity in fresh-frozen colorectal cancer tissue obtained from patients.

Author(s): Buffart TE, van den Oord RAHM, van den Berg A, Hilhorst R, Bastiaensen N, Pruijt HFM, van den Brule A, Nooijen P, Labots M, de Goeij-de Haas RR, Dekker H, Piersma SR, Pham TV, van der Leij T, de Wijn R, Ruijtenbeek R, Jiménez CR, Verheul HMW

Publication: Clin Proteomics, 2021, Vol. 18, Page 8

PubMed ID: 33602116 PubMed Review Paper? No

Purpose of Paper

This paper evaluated the stability of kinase activity and phosphopeptide profiles in snap-frozen colorectal cancer biopsies subjected to cold ischemia times of 0-180 min at room temperature using a peptide microarray assay and LC-MS/MS analysis, respectively.

Conclusion of Paper

Unsupervised cluster analysis of protein tyrosine kinase (PTK) and serine/threonine kinase (STK) signal intensities resulted in specimens clustering by patient, not cold ischemia time (0-180 min), indicating that between-patient variability was larger than the variability introduced by cold ischemia time. The mean kinase activity calculated for each cold ischemia time point did not reveal a trend in the majority (4 of 5) of the patients evaluated. Heat map analysis of data from these patients revealed 1-2 peptides per patient with a change in intensity that was >2-fold across the cold ischemia time course, but there was no overlap in the affected peptides between patients. When data from all five patients were considered, three PTK peptides (Annexin A2, PDGFRB, STAT4) and 1 STK (Phospholemman precursor) peptide demonstrated a >2-fold change in intensity after 180 min of cold ischemia.

Samples from the cold ischemia time course clustered by patient, not cold ischemia time, after unsupervised cluster analysis that was based on the intensity of the phosphopeptides present in control (0 min) cold ischemia specimens. The intensity of 90 phosphopeptides (3.3%), including MAPK1 of the stress response MAPK pathway, changed significantly across the cold ischemia time course relative to the 0 min control (p<0.01). When data from the 3 patients were combined by cold ischemia time point and compared to 0 min controls, a cold ischemia time of 180 min resulted in 52 (0.86%) phosphopeptides, 120 min resulted in 48 phosphopeptides (0.79%), and 60 min resulted in 28 (0.46%) phosphopeptides that displayed a significant difference in intensity (p<0.01). At each time point, the number of significantly affected phosphopeptides that were upregulated or downregulated were approximately equivalent.

Studies

  1. Study Purpose

    This study evaluated the stability of kinase activity in snap-frozen colorectal cancer biopsies subjected to cold ischemia times of 0-180 min at room temperature using a peptide microarray assay. Surgically resected colorectal tissue collected from five patients diagnosed with colorectal cancer (4 adenocarcinomas, 1 mucinous type) was biopsied immediately (0 min), and after 30, 60, 90, 120, 150, and 180 min of cold ischemia time at room temperature.  All tumor specimens were snap-frozen; no further details on snap-freezing, cryosectioning, or the storage temperature or duration of biopsy specimens were provided.  Three cryosections (60 µm thick) were used for protein extraction with Mammalian Protein Extraction Reagent with a protease inhibitor cocktail.  The protein lysate was stored at -80°C and protein concentration was determined using the Bradford Lowry Assay.  Protein tyrosine kinase (PTK) and serine/threonine kinase (STK) activity in protein lysates (5 µg of total protein) was measured by peptide microarrays (PamChip) and a PamStation instrument. Statistical significance was set at p<0.01.

    Summary of Findings:

    Restricting analysis to ATP-dependent signals that were present in ≥20% of arrays resulted in 93 PTK and 117 STK peptides suitable for further study.  Technical replicates had a coefficient of variation (CV) of 19% for TK signals and 14% for STK signals; sample preparation replicates (lysed and extracted separately) had a CV of 19% and 15% for PTK and STK signals, respectively.  Unsupervised cluster analysis of PTK and STK signal intensities resulted in specimens clustering by patient, not cold ischemia time, indicating that between-patient variability was larger than the variability introduced by cold ischemia time. The mean kinase activity calculated for each cold ischemia time point did not reveal a trend in four of the five patients; signal intensity of PTK peptides increased and STK peptides decreased during the cold ischemia time course in the remaining patient (Patient 1). Heat map analysis of the four patients in whom no trend was observed revealed 1-2 peptides per patient with a change in intensity that was >2-fold across the cold ischemia time course, but there was no overlap in the affected peptides between patients. The remaining patient (Patient 1) displayed a significant difference in phosphorylation signal in 56 (60.2%) and 79 (67.5%) of PTK and STK peptides, respectively; 35.5% (33 peptides) and 53.0% (62 peptides) of these changes in PTK and STK peptides, respectively, were >2-fold at the end of the time course. When data from all five patients were considered, three PTK peptides (Annexin A2, PDGFRB, STAT4) and 1 STK (Phospholemman precursor) peptide demonstrated a >2-fold change in intensity after 180 min of cold ischemia.

    Biospecimens
    Preservative Types
    • Frozen
    Diagnoses:
    • Neoplastic - Carcinoma
    Platform:
    AnalyteTechnology Platform
    Protein Enzyme assay
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Biospecimen Acquisition Cold ischemia time 0 min
    30 min
    60 min
    90 min
    120 min
    150 min
    180 min
  2. Study Purpose

    This paper evaluated the stability of phosphopeptide profiles in snap-frozen colorectal cancer biopsies subjected to cold ischemia times of 0-180 min at room temperature by LC-MS/MS analysis. Surgically resected colorectal tissue collected from three patients diagnosed with colorectal cancer (adenocarcinoma) was biopsied immediately (0 min) and after 60, 120, and 180 min of cold ischemia time at room temperature.  All tumor specimens were snap-frozen; no further details on snap-freezing, cryosectioning, or storage temperature or duration were provided.  Protein from sixty 10 µm-thick cryosections of each biopsy specimen as lysed in HEPES buffer containing orthovanadate, pyrophosphate, and β-glycerophosphate.  Protein concentration was quantified using the bicinchoninic acid (BCA) assay.  Protein lysate samples were peptide-enriched with titanium dioxide beads.  Peptides were separated by nanoLC-MS/MS. Phosphopeptide intensities were normalized to the median intensity of all phosphopeptides identified.  The threshold for biological relevance was set to a change in intensity that was >2-fold. Statistical significance was set at p<0.01.

    Summary of Findings:

    LC-MS/MS global phosphoproteome analysis resulted in the identification of 10,488 phosphopeptides in total and 2,715 phosphopeptides that were present in all control samples for the cold ischemia time course (0 min cold ischemia). Samples from the cold ischemia time course clustered by patient, not cold ischemia time, after unsupervised cluster analysis that was based on the intensity of the phosphopeptides present in control (0 min) cold ischemia specimens.The intensity of 90 phosphopeptides (3.3%) changed significantly with cold ischemia relative to the 0 min control (p<0.01), one of which was MAPK1 of the stress response MAPK pathway. When data from the 3 patients were combined by cold ischemia time point and compared to 0 min controls, a cold ischemia time of 180 min resulted in the greatest number of phosphopeptides that displayed a significant difference in intensity (p<0.01)(52 phosphopeptides, 0.86%), followed by a cold ischemia time of 120 min (48 phosphopeptides, 0.79%), and finally 60 min (28 phosphopeptides, 0.46%). At each time point, the number of significantly affected phosphopeptides that were upregulated or downregulated were approximately equivalent: 180 min: 31 upregulated, 21 downregulated, 120 min: 23 upregulated, 25 downregulated, 60 min: 18 upregulated, 15 downregulated.

    Biospecimens
    Preservative Types
    • Frozen
    Diagnoses:
    • Neoplastic - Carcinoma
    Platform:
    AnalyteTechnology Platform
    Protein LC-MS or LC-MS/MS
    Peptide LC-MS or LC-MS/MS
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Biospecimen Acquisition Cold ischemia time 0 min
    60 min
    80 min
    120 min

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