Effect of difluoroacetic acid and biological matrices on the development of a liquid chromatography-triple quadrupole mass spectrometry method for determination of intact growth factor proteins.

In biological systems, variable protein expression is a crucial marker for numerous diseases, including cancer. The vast majority of liquid chromatography-triple quadrupole mass spectrometry-based quantitative protein assays use bottom-up methodologies, where proteins are subjected to proteolytic cleavage prior to analysis. Here, the effect of difluoroacetic acid and biological matrices on the developement of a multiple reaction monitoring based top-down reversed-phase liquid chromatography-triple quadrupole mass spectrometry method for analysis of cancer-related intact proteins was evaluated. Seven growth factors (5.5-26.5 kDa; isoelectric points: 4.6-9.9) were analyzed on a wide-pore C4 column. The optimized method was performed at 30°C, using a 0.2 mL/min flow rate, a 10 %B/min gradient slope, and 0.05% v/v difluoroacetic acid as a mobile phase modifier. The increase of mass spectrometry sensitivity due to the difluoroacetic acid (estimated limits of detection in biological matrices 1-500 ng/mL) significantly varied for proteins with lower and higher charge state distributions. Matrix effects, as well as the specificity of the method were assessed for variable biological samples and pretreatment methods. This work demonstrates method development to improve the ability to target intact proteins directly by more affordable triple quadrupole mass spectrometry instrumentation, which could be beneficial in many application fields.