Dataset Information

Phosphorylation ratio determination in fresh frozen and formalin-fixed paraffin embedded tissue with targeted mass spectrometry

ABSTRACT: Formalin-fixed paraffin embedded (FFPE) tissues are routinely prepared and collected for diagnostics in pathology departments. Therefore, FFPE tissues are the most accessible research sources in pathology archives. In this study we investigated whether we can apply a targeted and quantitative parallel reaction monitoring (PRM) method for FFPE tissue samples in a sensitive and reproducible way. Normal brain and glioblastoma multiforme (GBM) tissues were used to demonstrate the feasibility of our approach. Two analytical methods (or workflows) were used: PRM measurement of a tryptic digest without phosphopeptide enrichment (Direct-PRM) and after Fe-NTA phosphopeptide enrichment (Fe-NTA-PRM). Applying these two methods, the phosphorylation ratio could be determined for selected four peptide pairs that originate from Neuroblast differentiation-associated protein (AHNAK S5448-p), Calcium/calmodulin-dependent protein kinase type II subunit delta (CAMK2D T337-p), Eukaryotic translation initiation factor 4B (EIF4B S93-p) and Epidermal growth factor receptor (EGFR S1166-p). In normal brain FFPE tissues, using the Fe-NTA-PRM method, we were able to quantify the targeted phosphorylated peptides with a high degree of reproducibility (CV = 14%). Our results indicate that formalin fixation does not impede relative quantification of a phosphosite and its phosphorylation ratio in FFPE tissues. The developed methods open ways to study archival FFPE tissues.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Brain

DISEASE(S): Glioblastoma

SUBMITTER: Lona Zeneyedpour

LAB HEAD: Theo Luider

PROVIDER: PXD017993 | Pride | 2021-04-22

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	190327HFc1_LZ-1819-Spiked-FF-CTRL-1-1-I_001 (F149155).mzid_190327HFc1_LZ-1819-Spiked-FF-CTRL-1-1-I_001_(F149155).pride.mgf.gz	Mzid
	190327HFc1_LZ-1819-Spiked-FF-CTRL-1-1-I_001 (F149155).pride.mztab.gz	Mztab
	190327HFc1_LZ-1819-Spiked-FF-CTRL-1-1-I_001.raw	Raw
	190327HFc1_LZ-1819-Spiked-FF-CTRL-1-1-I_001F149155.mzid.gz	Mzid
	190327HFc1_LZ-1819-Spiked-FF-CTRL-1-1-I_001F149155.mzid_190327HFc1_LZ-1819-Spiked-FF-CTRL-1-1-I_001_F149155.MGF	Mzid

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Publications

Phosphorylation Ratio Determination in Fresh-Frozen and Formalin-Fixed Paraffin-Embedded Tissue with Targeted Mass Spectrometry.

Zeneyedpour Lona L Stingl Christoph C Dekker Lennard J M LJM Mustafa Dana A M DAM Kros Johan M JM Luider Theo M TM

Journal of proteome research 20200908 10

Formalin-fixed paraffin-embedded (FFPE) tissues are routinely prepared and collected for diagnostics in pathology departments. These are, therefore, the most accessible research sources in pathology archives. In this study we investigated whether we can apply a targeted and quantitative parallel reaction monitoring (PRM) method for FFPE tissue samples in a sensitive and reproducible way. The feasibility of this technical approach was demonstrated for normal brain and glioblastoma multiforme tiss ...[more]

PMID: 32811146

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Project description:The heterotransplantation of human tumors to immune-deprived rodents has become an important preclinical tool for evaluating human tumor biology and responsiveness to therapeutic agents, based on the supposition that they reflect their original human origin. However, we demonstrate that such tumors can in fact change genetically, either by becoming tumors of the animal recipient or by becoming a hybrid of the human tumor and the animal hostâs cells. Three human tumors transplanted to the cheek pouch of golden hamsters resulted in permanently transplantable tumors that metastasize in the hamsters. Based on diverse lines of evidence, these transplants from a human glioblastoma multiforme and two Hodgkin lymphomas were malignant hybrids, showing both human and hamster genes in the malignant tumor cells, while retaining the morphology of the original human tumors. We demonstrate that even after being preserved for over 40 years in paraffin blocks, microarray analysis of RNA from these transplants disclosed over 3000 human genes derived from all 23 human chromosome pairs amongst these tumor transplants. A total of 3759 probe sets (ranging from 1040 to 1303 in each transplant) detected human gene transcripts in formalin-fixed, paraffin-embedded sections of the 3 hybrid tumors stored for over 40 years; the probe sets unambiguously map to 3107 unique Human Entrez Gene IDs and are representative of all human chromosomes, although, by karyology, one of the hybrid tumors (GB-749) had a total of 15 human chromosomes in its cells. Among the genes mapped, 39 probe sets, representing transcripts from 33 human genes , were detected in all hybrid tumor samples. Five of these 33 genes encode transcription factors that regulate cell growth and differentiation, five encode cell adhesion and transmigration-associated proteins known to participate in oncogenesis and/or metastasis and invasion, and additional genes encode components of pathways involved with signal transduction, regulation of apoptosis, DNA repair, and multidrug resistance. We posit that in-vivo fusion may disclose genes implicated in tumor progression, as well as gene families coding for the organoid phenotype. Thus, cancer cells can transduce adjacent stromal cells, with the resulting progeny having permanently transcribed genes with malignant and other functions of the donor DNA. Human gene expression profiles were determined by microarray analysis for 3 different Human-Hamster hybrid tumors, two Hodgkin lymphomas (GW-532, GW-584) and a glioblastoma multiforme (GB-749), that were first generated in the hamster cheek pouch after human tumor grafting and then propagated in hamsters and in cell cultures for years. Human gene expression was assessed using total RNA isolated from FFPE samples from GW-532 generations 2 and 34, GW-584 generation 28, and GB-749, in comparison to that for a Hamster control RNA sample isolated from a Hamster melanoma cell line (ATCC CCL-49). Expressed human genes were identified using MAS 5.0 signal expression values and detection P-values in the following manner: 1) Unannotated probe sets, as well as probe sets with no signal value greater than the median signal for AFFX spike-in controls with all Absent Detection Calls, were omitted from further analysis; 2) All remaining signal values for the hamster cell line sample were multiplied by the ratio of the median signal in all FFPE hybrid samples for AFFX spike-in control probe sets called present in all samples divided by the median signal for the same probe sets in the hamster CCL-49 sample; 3) Human transcripts were considered positive in a human-hamster hybrid FFPE sample if (a) a probe set signal exhibited a 2-fold or greater increase in any FFPE hybrid sample compared to the CCL-49 coontrol sample, (b) the fold change was greater than 2 standard deviations for that probe set across the FFPE samples, and (c) was called present (P) or marginal (M) for at least one or more FFPE samples.