An LC-MS/MS method for protein detection based on a mass barcode and dual-target recognition strategy

A mass barcode mediated signal amplification strategy was developed and applied to the determination of protein. A new compound, N'-((2-aminopyridin-3-yl)methylene)-5-(1,2-dithiolan-3-yl)pentanehydrazide (TAPA), was synthesized from the linker and the signal barcode, and used as the bonding barcode. For the realization of signal transduction, TAPAs and the target catcher aptamers, were both modified on gold nanoparticles (AuNPs) to establish the relationship between TAPAs and the target. Owing to the fact that the amount of TAPAs was much greater than the target, the signal of the target was not only transduced to the signal of the mass barcodes, but also amplified greatly. Thrombin, an important biomarker for coagulation abnormality diseases, was selected as a model analyte. Two kinds of thrombin recognition aptamers, aptamer 29 (apt29) and aptamer 15 (apt15), were modified onto the magnetic beads (MBs) and AuNPs, respectively. The modified AuNPs were further functionalized with lots of TAPA and formed apt15-AuNPs-TAPA. MBs-apt29 and apt15-AuNPs-TAPA could both recognize the target thrombin and form the sandwich complex (MBs-apt29/thrombin/apt15-AuNPs-TAPA). After the complex was separated by an extra magnetic field, NaClO oxidant solution was added to release the signal barcodes, 2-Amino-3-pyridinecarboxaldehyde (APA), which were then collected after centrifuging and analyzed by LC-MS/MS. Under optimized conditions, the mass response intensity was proportional to thrombin concentration in the range of 0.05-10 nM, with a 0.007 nM detection limit. This method was applied to the determination of thrombin in spiked serum samples, and the average recoveries ranged from 89.6% to 110.4%, which confirmed the applicability of this method.