Solid phase extraction as sample pretreatment method for top-down quantitative analysis of low molecular weight proteins from biological samples using liquid chromatography - triple quadrupole mass spectrometry

Targeting and quantifying intact proteins from biological samples is still a very challenging research area. Several crucial steps exist in the analytical workflow, including development of a reliable sample preparation method. Here, we developed and applied for the first time a non-immunoaffinity sample preparation method based on a generally widely available micro-elution solid phase extraction (mu SPE) strategy for the extraction of multiple lower molecular weight intact proteins (<30 kDa) from various biological matrices. Omission of a time-consuming drying and reconstitution step after extraction resulted in a more simple and rapid sample prepa-ration procedure. A model set of eleven intact proteins (molecular weights: 5.5-29 kDa; isoelectric points: 4.5-11.3) were analyzed in multiple biological fluids using reversed-phase liquid chromatography with a triple quadrupole mass spectrometer operated in multiple reaction monitoring mode. Various sample pre-treatment reagents, sorbent types, and washing and elution solvents were experimentally tested and optimized to obtain the mu SPE clean-up condition for a broad mixture of intact proteins having variable physicochemical properties. 1% trifluoroacetic acid and 0.2% Triton 100-X were selected as suitable sample pre-treatment reagents for releasing protein-protein interactions in human serum/plasma and human urine, respectively. Hydrophilic lipophilic balanced mu SPE sorbent was selected as a high performing stationary phase. Addition of 1% tri-fluoroacetic acid to all washing and elution solutions showed the most beneficial effect for the extraction re-covery of the proteins. Under the optimized conditions, reproducible extraction recoveries >65% for all targeted proteins (up to 30 kDa) in human urine and >50% for most of the proteins in serum/plasma were achieved. The selected conditions were applied also for the analysis of clinical serum and urine samples to demonstrate the feasibility of the developed method to target intact proteins directly by more affordable mu SPE sample preparation and triple quadrupole mass spectrometry, which could be beneficial in many application fields.

Automated summary

In this study, a non-immunoaffinity sample preparation method based on micro-elution solid phase extraction was developed and applied for the extraction of multiple lower molecular weight intact proteins from various biological matrices. By omitting the drying and reconstitution step, a more simple and rapid sample preparation procedure was achieved. The optimized conditions showed high extraction recoveries for targeted proteins in human urine and serum/plasma.