title: Nanoproteomic analysis of ischemia-dependent changes in signaling protein phosphorylation in colorectal normal and cancer tissue.
abstract: BACKGROUND
  Clinical diagnostic research relies upon the collection of tissue samples, and for those samples to be representative of the in vivo situation. Tissue collection procedures, including post-operative ischemia, can impact the molecular profile of the tissue at the genetic and proteomic level. Understanding the influence of factors such as ischemia on tissue samples is imperative in order to develop both markers of tissue quality and ultimately accurate diagnostic tests.
  
  
  METHODS
  Using NanoPro1000 technology, a rapid and highly sensitive immunoassay platform, the phosphorylation status of clinically relevant cancer-related biomarkers in response to ischemia was quantified in tissue samples from 20 patients with primary colorectal cancer. Tumor tissue and adjacent normal tissue samples were collected and subjected to cold ischemia prior to nanoproteomic analysis of AKT, ERK1/2, MEK1/2, and c-MET. Ischemia-induced relative changes in overall phosphorylation and phosphorylation of individual isoforms were calculated and statistical significance determined. Any differences in baseline levels of phosphorylation between tumor tissue and normal tissue were also analyzed.
  
  
  RESULTS
  Changes in overall phosphorylation of the selected proteins in response to ischemia revealed minor variations in both normal and tumor tissue; however, significant changes were identified in the phosphorylation of individual isoforms. In normal tissue post-operative ischemia, phosphorylation was increased in two AKT isoforms, two ERK1/2 isoforms, and one MEK1/2 isoform and decreased in one MEK1/2 isoform and one c-MET isoform. Following ischemia in tumor tissue, one AKT isoform showed decreased phosphorylation and there was an overall increase in unphosphorylated ERK1/2, whereas an increase in the phosphorylation of two MEK1/2 isoforms was observed. There were no changes in c-MET phosphorylation in tumor tissue.
  
  
  CONCLUSIONS
  This study provides insight into the influence of post-operative ischemia on tissue sample biology, which may inform the future development of markers of tissue quality and assist in the development of diagnostic tests.
authors.affiliation: Indivumed GmbH, Falkenried 88, 20251, Hamburg, Germany. unger@indivumed.com.
authors.forename: Florian T
authors.initials: FT
authors.lastname: Unger
authors.affiliation: Indivumed GmbH, Falkenried 88, 20251, Hamburg, Germany. lange@indivumed.com.
authors.forename: Nicole
authors.initials: N
authors.lastname: Lange
authors.affiliation: Indivumed GmbH, Falkenried 88, 20251, Hamburg, Germany. krueger@indivumed.com.
authors.forename: Jana
authors.initials: J
authors.lastname: Krüger
authors.affiliation: Arizona State University, Phoenix, AZ, USA. Carolyn.Compton@asu.edu.
authors.forename: Carolyn
authors.initials: C
authors.lastname: Compton
authors.affiliation: Biorepositories and Biospecimen Research Branch,National Cancer Institute, National Institutes of Health, Bethesda, MD, USA. moorehe@mail.nih.gov.
authors.forename: Helen
authors.initials: H
authors.lastname: Moore
authors.affiliation: Biorepositories and Biospecimen Research Branch,National Cancer Institute, National Institutes of Health, Bethesda, MD, USA. lokesh.agrawal@nih.gov.
authors.forename: Lokesh
authors.initials: L
authors.lastname: Agrawal
authors.affiliation: Indivumed GmbH, Falkenried 88, 20251, Hamburg, Germany. juhl@indivumed.com.
authors.forename: Hartmut
authors.initials: H
authors.lastname: Juhl
authors.affiliation: Indivumed GmbH, Falkenried 88, 20251, Hamburg, Germany. david@indivumed.com.
authors.forename: Kerstin A
authors.initials: KA
authors.lastname: David
mesh_terms: D001681:Biological Assay; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D007511:Ischemia / Q000378:metabolism*; D008163:Luminescent Measurements; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D036103:Nanotechnology / Q000379:methods*; D010766:Phosphorylation; D020033:Protein Isoforms / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D020033:Protein Isoforms; D066246:ErbB Receptors; D019859:Proto-Oncogene Proteins c-met; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D020929:Mitogen-Activated Protein Kinase Kinases