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.


The synthesis of TA-COOEt.
1.03 g of TA, 0.31 g of DMAP, 4.24 mL of ethanol and 40 mL of CH 2 Cl 2 were placed in a flask and stirred. The reaction mixture was cooled to 0 °C, and a solution of 1.54 g DCC in 10 mL CH 2 Cl 2 was added dropwise. The reaction mixture was stirred at 0 °C for 1 h and then stirred at room temperature for 24 h. The precipitate was filtered and evaporated to give a yellow oil. The mixture was purified by flash chromatography and evaporated to give a yellow oil.

The synthesis of TA-NHNH 2 .
1.0 g of TA-COOEt, 1.4 mL of NH 2 NH 2 ·H 2 O，and 18 mL of methanol were mixed and stirred at 40 °C for 12 h. The mixture was diluted with ethyl acetate and the organic layer was washed with brine, dried with MgSO 4 , then filtered and concentrated. The mixture was purified by flash chromatography and evaporated to give a yellow solid.

The synthesis of TAPA.
132 mg of TA-NHNH 2 and 97.7 mg of APA were dissolved in 30 mL of methanol. 50 μL of TFA was added and the mixture was stirred at room temperature for 24 h in the dark. The mixture was concentrated and purified by flash chromatography and evaporated to give a yellow solid. The mass spectrum of TAPA was shown in Fig. S1.

LC-MS/MS conditions
The chromatographic separation was achieved on a Hedera ODS-2 column (5 µm, 150 × 2.1 mm, Hanbon Science and Technology, China), protected by a security guard C 18 column (5 µm, 4 × 2.0 mm, Phenomenex, Torrance, CA, USA). The mobile phase was composed of methanol (mobile phase A) and 0.1% formic acid solution (mobile phase B). The gradient elution produce was started at 60% A for 2 min, changing linearly to 100% A within 0.5 min, then maintained for 2 min and finally brought back to the initial condition during 0.5 min and re-equilibrated for 4.0 min. The injection volume was 5 µL. The autosampler was maintained at 8 °C.
The mass spectrometer was operated in the positive ESI mode. Quantification was performed using multiple reaction monitoring of the transitions of m/z 123.1 -78.0 for APA and m/z 239.0 -122.0 for the IS nitrofurantoin. Declustering Potential (DP) set for APA and IS was 60 V and 80 V, respectively. The Collision Energy (CE) was set at 32 V and 28 V for APA and IS, respectively. The collision gas and curtain gas were maintained at 12 and 30 psi, respectively. The ion source gas 1 and ion source gas 2 were both 30 psi. The ionspray voltage and temperature were kept at 4000 V and 450 °C. The system control and data analysis were carried out by Analyst (AB Sciex, version 1.5.2).

Optimization of experimental conditions
In order to obtain the best performance, some experimental conditions such as pH, the concentration of NaCl and the incubation time were optimized.
To investigate the effect of pH on the connection between aptamers and thrombin, a series of 25 mM Tris-HCl buffer (142 mM NaCl, 5 mM MgCl 2 , 15 mM KCl) with different pH (6.0, 6.5, 7.0, 7.4, 8.2, 9.0) was tested. As shown in Fig. S4a, there was no obvious change of mass intensities with the change of pH, so we chose the pH of physiological environment 7.4 as the final pH condition.
Aptamers need the assistance of metal ions to form G-quadruplex for the recognition of thrombin. In this experiment, different concentrations of NaCl (50, 100, 142, 200, 250 mM) in 25 mM Tris-HCl buffer was investigated. As shown in Incubation time is also important factor for the reaction. As shown in Fig. S4c, it can be seen that when aptamers reacted with thrombin for 2 h, the intensity reached a plateau and kept stable. Therefor, 2 h is consider as the optimum incubation time.