Combining Fibrinogen-Conjugated Gold Nanoparticles with a Cellulose Membrane for the Mass Spectrometry-Based Detection of Fibrinolytic-Related Proteins

摘要

In this paper, we describe a pulsed-laser desorption/ionization mass spectrometry (LDI-MS) approach for the detection of plasmin with subnanomolar sensitivity through the analysis of gold (Au) clusters desorbed from fibrinogen-modified gold nanoparticles (Fib-Au NPs) on a mixed cellulose ester membrane (MCEM). The mechanism of action of this probe is based on the plasmin-mediated cleavage of the Fib-Au NPs and the reduced interaction between Fib-Au NPs and MCEM. The Fib-Au NPs were deposited onto the MCEM to form a highly efficient background-free surface-assisted LDI substrate. Under pulsed laser irradiation (355 nm), the cleaved Fib-Au NPs decreased the adsorbed on MCEM. As a result, the intensities of the signals of the Au clusters decreased in the mass spectra. This approach provided a highly amplified target-labeling indicator for the analysis of plasmin. Under optimized conditions, this probe was highly sensitive (limit of detection: ca. 0.1 nM) and selective (by at least 1000-fold over other enzymes and proteins) toward plasmin; it also improved the reproducibility (<5%) of ion production by presenting a more-homogeneous substrate surface relative to surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) analysis, thereby enabling LDI-MS to be used for the accurate and precise quantification of plasminogen in human serum in the presence of urokinase (an activator that converts plasminogen to plasmin). Relative to conventional assays, this new probe for plasmin offers the advantages of high sensitivity and selectivity and high throughput, with great potential for practical studies of fibrinolytic-related proteins.