Analysis of thrombin‐antithrombin complex formation using microchip electrophoresis and mass spectrometry

Abstract

Analysis of thrombin-antithrombin complex formation using microchip electrophoresis and mass spectrometryPreterm birth (PTB) related health problems take over one million lives each year, and currently, no clinical analysis is available to determine if a fetus is at risk for PTB. Here, we describe the preparation of a key PTB risk biomarker, thrombin-antithrombin (TAT), and characterize it using dot blots, MS, and microchip electrophoresis (µCE). The pH for fluorescently labeling TAT was also optimized using spectrofluorometry and spectrophotometry. The LOD of TAT was measured in µCE. Lastly, TAT was combined with six other PTB risk biomarkers and separated in µCE. The ability to make and characterize TAT is an important step toward the development of an integrated microfluidic diagnostic for PTB risk.

3D Printed Microfluidic Devices for Solid-Phase Extraction and On-Chip Fluorescent Labeling of Preterm Birth Risk Biomarkers

“…PTB biomarkers were fluorescently labeled and filtered using previously described procedures , for prelabeled biomarker experiments. The thrombin–antithrombin complex (TAT) was prepared 24 h prior to fluorescent labeling . For on-chip labeling experiments, PTB biomarkers were diluted to the desired concentration in 10 mM BCB.…”

“…The thrombin− antithrombin complex (TAT) was prepared 24 h prior to fluorescent labeling. 32 For on-chip labeling experiments, PTB biomarkers were diluted to the desired concentration in 10 mM BCB.…”

“…Such an assessment may be possible with a panel of nine previously discovered protein and peptide biomarkers, as summarized in Table . Toward development of a PTB risk assessment, some of these protein and peptide biomarkers have been evaluated in microfluidic devices for immunoaffinity extraction, , electrophoretic separations, , and SPE. , However, this entire panel has not previously been evaluated in a microfluidic analysis format.…”

Immunoaffinity monoliths for multiplexed extraction of preterm birth biomarkers from human blood serum in 3D printed microfluidic devices

“…The PTB biomarkers were fluorescently labeled; CRF and TNF were dissolved in 10 mM bicarbonate buffer (BCB, pH 10), and TAT was prepared as in Nielsen et al 25 Alexa Fluor 532-succinimdyl ester was dissolved in DMSO, added to each biomarker and incubated at room temperature overnight. CRF (100 μM), TNF (40 μM), and TAT (26 μM), were labeled at a dye : biomarker molar ratio of 3 : 2, 20 : 1, and 20 : 1, respectively.…”

Structural stability of Human serum albumin is modified in rheumatoid arthritis

“…Such changes in stability would most likely correlate with a change in these proteins’ tertiary structures and would be for a smaller portion of the total HSA (35) (Figure 2B) . We used MS to explore both concentration differences in HSA (and other abundant proteins), as well as HSA tertiary structure changes (by looking at surface reactivity (36, 37)) as potential causes of the characteristic shift in the HDCs. As outlined below, our data support a change in the HSA thermal stability (Figure 2B) .…”