Epsilon Archive for Student Projects

Abstract

Over a decade after its start, Human Protein Atlas (HPA) has been developed as a universal database for human proteome. HPA has used both commercially designed and intra-designed antibodies for protein detection but both types should pass several validation tests like Western blot, immunohistochemistry, protein array and immunofluo-rescence. However, importance of data reliability requires more sensi-tive techniques to confirm specificity of antibodies to avoid any false data in HPA. Immunoprecipitation by help of magnetic beads (IP) coupled with Orbitrap Mass Spectrometry (MS) can be used as an antibody validation method with its incredible sensitivity and high capacity for different biological samples. In this study, it has been tried to test Orbitrap Mass Spectrometry and optimize a protocol for its future application as a validation method in HPA.
Four proteins ATP5B, DDX1, IL18 and KRT7 with high expression (according to RNA data and IHC staining) and high reliability score for their antibodies and four pancreatic markers, GPR44, StradB, TMEM100 and SerpinB10 were selected to be detected in lysates from the cell lines, SIHA, RH-30, BEWO and HACAT respectively (for each cell line one safe and one pancreatic marker). Protein lysates prepared by RIPA buffer were used for Immunoprecipitation and purified samples were run on SDS gel, stained with Coomassie blue and several bands were selected to be cut and prepared for MS run by in-gel digestion method. Peptides were run in Orbitrap Mass Spectrometry for detection.
In total three samples out of seven were detected as correct targets (one ATP5B, and two for KRT7), two samples with high molecular weight were detected as myosin which is a common contamination in proteomic lab and two bands belonging to GPR44 and TMEM100 failed in detection due to incorrect gel cutting.
In total, HPA antibodies were successful in detection of target pro-teins when gel bands were cut at correct molecular weight, but their mono-specificity should be further investigated since KRT7 came below contaminations. For a potential use of orbitrap MS as an anti-body validation test, it is required to use clean facility, to preincubate dynabeads with antibody, to cut the gel at expected molecular weight for target protein and finally to filter out common contaminants in MS analysis to facilitate target protein detection.