Optimized collision energy based multiple reaction monitoring facilitated validation of immune cell activated proteins in 3D cancer – Immune cell co-culture model

Multiple reaction monitoring (MRM), is a highly sensitive targeted mass spectrometry technique that is used to validate discovery phase mass spectrometry data. Through this simple technique, relative protein expressions can be acquired without using commercially available antibodies. Our previous work showed Gipie silencing of UM-HACC-2A (adenoid cystic carcinoma) cells resulted in upregulation of a plethora of immune proteins in immune cells in 3D ACC - immune co-culture model. MRM enabled determining relative protein expression of significantly upregulated immune proteins due to silencing of Gipie. Granzyme A, CD48, granzyme B, HLA class I, antigen B, HLA class I, antigen A, galectin 1, vimentin, endoplasmic reticulum chaperone BiP, and dipeptidyl peptidase 1 differential expressions were validated. Whereas coactosin-like protein expressions were found non-significant among control and Gipie silenced ACC. This manuscript presents the validation data of previous discovery phase mass spectrometry and also accentuate the use of MRM in translational or preclinical studies in the field of biomedical sciences.