Large-scale isotype-specific quantification of Serum amyloid A 1/2 by multiple reaction monitoring in crude sera

Quantification is an essential step in biomarker development. Multiple reaction monitoring (MRM) is a new modified mass spectrometry-based quantification technology that does not require antibody development. Serum amyloid A (SAA) is a positive acute-phase protein identified as a lung cancer biomarker in our previous study. Acute SAA exists in two isoforms with highly similar (92%) amino acid sequences. Until now, studies of SAA have been unable to distinguish between SAA1 and SAA2. To overcome the unavailability of a SAA2-specific antibody, we developed MRM methodology for the verification of SAA1 and SAA2 in clinical crude serum samples from 99 healthy controls and 100 lung adenocarcinoma patients. Differential measurement of SAA1 and SAA2 was made possible for the first time with the developed isotype-specific MRM method. Most healthy control samples had small or no MS/MS peaks of the targeted peptides otherwise, higher peak areas with 10- to 34-fold increase over controls were detected in lung cancer samples. In addition, our SAA1 MRM data demonstrated good agreement with the SAA1 enzyme-linked immunosorbent assay (ELISA) data. Finally, successful quantification of SAA2 in crude serum by MRM, for the first time, shows that SAA2 can be a good biomarker for the detection of lung cancers.

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► MRM analysis has been carried out on large number of crude serum of healthy control and lung cancer patients.
► Two proteins with similar amino acid sequences have been measured separately.
► SAA1 and SAA2 showed similar pattern in their expression.