Quantitative analysis of trace-level proteins for assessing inflammation through clinical sampling

Abstract

Objective To compare the effectiveness between qPCR and trace-level proteins quantitative analysis in detecting the expression of inflammation-associated factors caspase-1 and IL-1β in peripheral blood monocytes from breast cancer patients. Methods Pre- and post-operation peripheral blood samples were collected from 10 breast cancer patients. Monocytes were subsequently isolated and activated by incubation with lipopolysaccharide. The mRNA and protein expression level of caspase-1 and IL-1β in monocytes were examined using qPCR and trace-level proteins quantitative analysis, respectively, and the data were normalized by β-actin. The differences between the two detection methods were compared and evaluated for strengthens and weakness. Results The vast majority of patients showed a notable increase in the number of peripheral blood monocytes after surgical operation. At the mRNA level, compared to preoperative levels, postoperative caspase-1 and IL-1β were both increased in 50% (5/10) and 40% (4/10) of patients, respectively, while the rest showed either a decrease (40% and 50%, respectively) or no significant change (both 10%). In contrast, the results of trace-level proteins quantitative analysis revealed that 60% (6/10) of patients exhibited increased level of postoperative caspase-1 and IL-1β proteins, while the remaining showed a decrease (30% and 20%, respectively) or no significant change (10% and 20%, respectively). Additionally, 50% of patients displayed a significant increase in caspase-1 and IL-1β active cleavage bands by electrophoresis postoperatively. Notably, inconsistent changes in caspase-1 and IL-1β mRNA and protein level after surgery were observed in 30% and 40% of patients respectively. Conclusions The trace-level proteins quantitative analysis is recommended due to its advantages including minimal sample amount requirement, high sensitivity, good repeatability and relatively simple operative procedure. Therefore, it is particularly suitable for assessing the inflammatory status of of patients through clinical sampling of peripheral blood.

Keywords

• inflammation-associated factors|caspase-1|il-1β|trace-level proteins quantitative analysis|real-time quantitative polymerase chain reaction (qpcr)