Variability of protein and phosphoprotein levels in clinical tissue specimens during the preanalytical phase.
Author(s): Gündisch S, Hauck S, Sarioglu H, Schott C, Viertler C, Kap M, Schuster T, Reischauer B, Rosenberg R, Verhoef C, Mischinger HJ, Riegman P, Zatloukal K, Becker KF
Publication: J Proteome Res, 2012, Vol. 11, Page 5748-62
PubMed ID: 23134551 PubMed Review Paper? No
Purpose of Paper
This paper investigated how warm and cold ischemia times affect protein and phosphoprotein levels quantified by reverse phase protein microarray (RPPA) and mass spectrometry in liver specimens snap-frozen in liquid nitrogen. RPPA results were also compared following different cold ischemia times among case-matched intestine specimens that were snap-frozen or formalin-fixed and paraffin-embedded (FFPE).
Conclusion of Paper
RPPA analysis demonstrated no significant differences between frozen and formalin-fixed intestine specimens. Changes in protein and phosphoprotein profiles as a result of a procedural delay at room temperature were limited to phospho-p44/42 MAPK in intestine beginning after 60 min and caldesmon-1, p44/42 MAPK, Stat 3, and PRAS40 in liver beginning after 30 min. Importantly, changes introduced by a procedural delay were not observed in all intestine or liver specimens, indicating high interpatient variability. LC-MS/MS analysis of liver specimens with comparable warm and cold ischemia times revealed no significant differences for any protein during the first 60 min of cold ischemia and only 1.4% of proteins demonstrated significantly different signal intensities after 180 minutes of cold ischemia, although high interpatient variability was observed. Compared to a pre-surgical biopsy, 74% of the proteins analyzed by RPPA exhibited a significant decline in the resected liver specimens, but only 16% differed significantly between resection specimens subjected to only warm ischemia compared to those experiencing warm and cold ischemia. Of the eight phosphoproteins examined, four exhibited a significant decline in signal intensity in resection specimens compared to the biopsy but the remaining four were unaffected by type or duration of ischemia.
Studies
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Study Purpose
This study investigated the effects of different cold ischemia times among case-matched intestine specimens that were snap-frozen or formalin-fixed and paraffin-embedded (FFPE). A total of 11 nonmalignant colon (8) and small intestine (3) specimens were surgically resected, aliquoted (minimum size of 5 × 5 × 5 mm), and either snap-frozen in liquid nitrogen (LN2) or formalin-fixed in 3.5−3.7% formaldehyde for 24 h immediately or after 30, 60, or 180 min of storage at room temperature in a humidified chamber. Snap-frozen specimens were stored in LN2 and FFPE specimens were stored at 4°C until analysis. Warm ischemia time, defined as the time from vessel ligation until surgical resection, ranged between 3 and 80 min (30 min average). In addition to the experimentally controlled procedural delay (0-180 min in a humidified chamber at room temperature), cold ischemia time also included post-resection time, which the authors defined as the time between surgical resection and arrival in pathology and ranged between 23 and 55 min (37 min average). Protein from cryosections was extracted by homogenization in extraction buffer and sonicated if necessary. Proteins from FFPE tissue were extracted according to the Q proteome FFPE Tissue Kit Handbook. Proteins were analyzed by reverse phase protein array using a BioRad BioOdyssey Calligrapher MiniArrayer.
Summary of Findings:
RPPA analysis of 17 proteins and six phosphoproteins revealed no significant differences between frozen and formalin-fixed specimens after a procedural delay of 0, 30, or 180 minutes (P=0.006, P=0.058, P=0.002; respectively). Statistical results were not provided for the 60-minute time point due to a limited number of specimens. Only phosphop44/42 MAPK levels were affected by a procedural delay with a 2-fold increase in signal intensity from 0 to 60 min and a slight decrease from 60 to 180 min; however, these results were not observed in all specimens, indicating high interpatient variability.
Biospecimens
Preservative Types
- Formalin
- Frozen
Diagnoses:
- Neoplastic - Benign
Platform:
Analyte Technology Platform Protein Reverse phase protein microarray Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Cold ischemia time 0 min
30 min
60 min
180 min
Reverse phase protein microarray Specific Targeted peptide/protein Phospho-PTEN
PTEN
Phospho-Akt (Ser473)
Akt
Phospho-GSK-3β
GSK-3β
Phospho-p44/42 MAPK (Thr202/Tyr204)
p44/42 MAPK (Erk1/2)
Phospho-p38 MAPK (Thr180/Tyr182)
p38 MAPK
Phospho-Stat3
Stat3
Phospho-NF-κB p65
NF-κB p65
FAK
c-Fos
Cleaved Caspase-3
E-Cadherin
Egr1
HIF-1α
Hsp70
β-Actin
GAPDH
Biospecimen Preservation Type of fixation/preservation Snap frozen
Formaldehyde
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Study Purpose
This study investigated the effects of warm and cold ischemia time on protein levels in liver specimens from patients diagnosed with metastatic colon cancer. Eleven biopsies were obtained before vessel ligation and immediately snap-frozen and stored in LN2. Seventeen specimens were surgically resected, aliquoted (minimum size of 5 × 5 × 5 mm), and snap-frozen in LN2 immediately or after 30−360 min at room temperature in a humidified chamber. Cold ischemia time also included post-resection time, which the authors defined as the time between surgical resection and arrival in pathology, was limited to 4 min or less. Warm ischemia time, defined as the time from vessel ligation until surgical resection, ranged between 15 and 195 min (88 min average). Proteins from cryosections were extracted by homogenization in extraction buffer and sonicated if necessary. Specimens were prefractionated by one-dimensional SDS-PAGE under reducing conditions, peptides were eluted from the gel by two repetitive extractions with 60% acetonitrile/0.1% trifluoric acetic acid, and protein levels were investigated by RPPA, Western blot analysis, and LC−MS/MS.
Summary of Findings:
Of the 30 proteins investigated by RPPA, only p44/42 MAPK (Erk-1/2) demonstrated a statistically significant increase after procedural delays of 30, 60, 180, and 360 minutes (P<0.05, P<0.05, P<0.01 and P<0.05; respectively). Caldesmon-1 and Stat3 showed a significant increase in signal intensity after 30, 180, and 360 minutes (P<0.05, P<0.01, and P<0.05 for both; respectively) and PRAS40 increased after 180 and 360 minutes (P<0.01, both). Importantly, interpatient variability was observed among signal intensities following a procedural delay. When four specimens with comparable warm and cold ischemia times were analyzed by LC-MS/MS, only 18 (1.4%) of 1254 proteins identified displayed a significantly different signal intensity (9 decreased and 9 increased) after a procedural delay of 180 minutes when compared to the reference specimen and no significant differences were observed for any protein during the first 60 min of a delay. Detailed analysis of CK18 in these four specimens revealed an increase of signal intensity due to a procedural delay (1.4, 2.3, and 4.3-fold increase after 30, 180, and 360 min; respectively), althougha high degree of interpatient variability (5-fold increase for two patients and only a 2.4-fold increase for the other two) was observed and confirmed by Western Blot. Analysis of CK18 expression in all 17 specimens revealed a nonsignificant increase in signal intensity after 360 min. RPPA analysis of liver biopsies obtained prior to blood flow restriction revealed high interpatient variability for each protein examined, with a 6.9-fold mean maximum change for all analyzed proteins (n = 31) which decreased to 2.8-fold when normalized to GAPDH (data not provided). The authors stated that 23 (74%) of the 31 proteins analyzed by RPPA displayed a significantly lower signal intensity in the resection specimens which experienced warm ischemia times (average 126 min) compared to the respective biopsy, but only 5 (16%) proteins differed significantly between resection specimens that experienced warm ischemia and those that were exposed to both warm and cold ischemia (which included a post-resection time of 4 min and a procedural delay of 30 min). Of the eight phosphoproteins examined, approximately 60% displayed significantly lower signal intensities in resection specimens exposed to warm and cold ischemia than biopsy specimens; specifically, affected proteins included phospho-PTEN and phospho-Akt (P<0.05 each) and phospho-GSK-3β and phospho-PRAS40 (P<0.01 each). In contrast, levels of phospho-ERK1/2, phospho-p38 MAPK, phosphor-NFκB, and phospho-Stat3 were unaffected by type or duration of ischemia.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Normal Adjacent
Platform:
Analyte Technology Platform Protein LC-MS or LC-MS/MS Protein Western blot Protein Reverse phase protein microarray Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Warm ischemia time 15 - 195 min (88 min average)
Reverse phase protein microarray Specific Targeted peptide/protein PI3 Kinase p85
Phospho-PTEN
PTEN
Phospho-Akt (Ser473)
Akt
Phospho-GSK-3β
GSK-3β
Phospho-PRAS40
PRAS40
Phospho-p44/42 MAPK (Thr202/Tyr204)
p44/42 MAPK (Erk1/2)
Phospho-p38 MAPK (Thr180/Tyr182)
p38 MAPK
Phospho-Stat3
Stat3
Phospho-NF-κB p65
NF-κB p65
FAK
c-Fos
Cleaved Caspase-3
Desmin
Caldesmon-1
E-Cadherin
Vimentin
Egr1
HIF-1α
Hsp70
α-Tubulin
β-Actin
GAPDH
Biospecimen Acquisition Cold ischemia time 0 min
30 min
60 min
120 min
180 min
360 min
Western blot Specific Targeted peptide/protein CK18
GAPDH