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Effects of PRSS35 on the extra cellular matrix

ABSTRACT: In the project four sets of label-free proteomics studies were performed to characterize the extra cellular matrix (ECM) proteome of differentially treated cells (see below). All cells were treated with sorbitol to induce the expression of PRSS35. Only in set IV PRSS35 is downregulated. For each set, three biological replicates were performed, which were each fractionated by SDS-PAGE into five fractions. Proteins were in-gel digested with trypsin and analyzed by DDA on an Exploris 480 mass spectrometer:

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Skin, Fibroblast

SUBMITTER: Joern Dengjel

LAB HEAD: Joern Dengjel

PROVIDER: PXD040684 | Pride | 2023-07-25

REPOSITORIES: Pride

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			Action	DRS
	20221007_CR_Col0131A_LC_A1.raw	Raw
	20221007_CR_Col0131A_LC_A2.raw	Raw
	20221007_CR_Col0131A_LC_A3.raw	Raw
	20221007_CR_Col0131A_LC_A4.raw	Raw
	20221007_CR_Col0131A_LC_A5.raw	Raw

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Project description:Background Breast cancer patients who present in the early stage of disease are affected by metastasis to the axillary group of lymph nodes. The first among this group that is affected is called as sentinel lymph node, and metastasis to this lymph node is crucial for the staging of cancer and the quality of surgical intervention. Sentinel Lymph Node Biopsy (SLNB) that is currently used to assess lymph node metastasis is neither sensitive, nor specific, is time-consuming, thereby necessitating the identification of novel biomarkers that can flag sentinel lymph node metastasis. Methods Breast cancer patients were screened, and those with early stage were recruited in the study. Surgical resection of the breast was followed by identification of sentinel lymph nodes by methylene fluorescent technique. Histo-pathology of fresh frozen section biopsy was used as the gold standard to assign the clinical phenotypes of metastatic (SLNM+) and non-metastatic sentinel lymph nodes (SLNM-). Discovery phase of the experiment included isobaric Tags for Relative and Absolute Quantitation (iTRAQ) technique comprising of six comparative experiments coupled with mass spectrometric analysis on Orbitrap Fusion to identify differentially expressed proteins on Proteome Discoverer 2.4. Functional enrichment and pathway analyses of differentially regulated genes was carried out in DAVID functional annotation tool. Validation was done by ELISA and protein concentrations were used to estimate the ROC for computing diagnostic parameters. Results Based on MS/MS spectra there were 2396 unique protein groups and 81 differentially expressed proteins comparing SLNB + and SLNB -. Nineteen proteins up-regulated, and eight proteins that were down regulated in SLNB+ as compared to SLNB-. Bioinformatic analysis showed the implication of extra cellular matrix proteins and ECM-receptor interaction pathways to be implicated in lymph node metastasis. ELISA confirmed the up-regulation of caveolin 1, collagen α-1, desmin, fibrillin-1, and microfibrillar associated glycoprotein 4 in metastatic, as compared to non-metastatic lymph nodes. These proteins are known to be integral in tumorogenesis, cell proliferation, invasion, cell survival and anti-apoptosis. These proteins have 80%-100%, of sensitivity and specificity to differentiate the two clinical phenotypes. Conclusion Identified extra cellular matrix protein biomarkers have requisite diagnostic parameters to be developed as a translational tool to assess the status of sentinel lymph nodes during mastectomy procedure to guide surgical therapy of axillary lymph nodes in early breast oncology.

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Effects of PRSS35 on the extra cellular matrix

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