Microprobe-Capture In-Emitter Elution: An Affinity Capture Technique to Directly Couple a Label-Free Optical Sensing Technology with Mass Spectrometry for Protein Analysis

Abstract

Affinity capture of an analyte by a capture agent is one of the most effective sample preparation approaches in mass spectrometry (MS), especially top-down MS. We describe a new affinity capture technique for protein targets, called microprobe-capture in-emitter elution (MPIE), which can directly couple a label-free optical sensing technology (next-generation biolayer interferometry, BLI) with MS. To implement MPIE, an analyte is first captured on the surface of a microprobe and subsequently eluted from the microprobe inside an electrospray emitter. The capture process is monitored in real-time via BLI. When electrospray is established from the emitter to a mass spectrometer, the analyte is immediately ionized via electrospray ionization (ESI) for MS analysis. By this means, BLI and MS are directly coupled in the form of MPIE-ESI-MS. The performance of MPIE-ESI-MS was demonstrated by the analysis of β-amyloid 1–40 and transferrin using both standard samples and human specimens. In comparison to conventional affinity capture techniques such as bead-based immunoprecipitation, MPIE innovates the affinity capture methodology by introducing real-time process monitoring and providing binding characteristics of analytes, offering more information-rich experiment results. Thus, MPIE is a valuable addition to the top-down MS sample preparation toolbox, and MPIE-ESI-MS can be useful for identification and characterization of targets of interest.

Study of β1-transferrin and β2-transferrin using microprobe-capture in-emitter elution and high-resolution mass spectrometry

“…The performance of MPIE-ESI-MS for Tf analysis was demonstrated and reported elsewhere 27 . MPIE-ESI-MS had a limit of detection for the Tf standard at 0.063 μg/ml, which was translated to no more than 7 fmol Tf molecules captured on a microprobe 27 .…”

“…The performance of MPIE-ESI-MS for Tf analysis was demonstrated and reported elsewhere 27 . MPIE-ESI-MS had a limit of detection for the Tf standard at 0.063 μg/ml, which was translated to no more than 7 fmol Tf molecules captured on a microprobe 27 . The high analytical sensitivity and specificity provided by affinity capture made MPIE-ESI-MS an ideal method to study the Tf molecules.…”

“…Currently MPIE-ESI-MS is not an ideal tool for quantitative analysis due to the variation in capture agent loading and analyte binding on a BLI microprobe, as well as that in MS ionization. In the future, when accurate quantitation is needed, quantitative MPIE-ESI-MS analysis should be achievable by employing internal standards, i.e., preferably stable isotope-labeled analytes 27 .…”

“…An MPIE-ESI-MS experiment consists of two parts: BLI-based affinity capture and in-emitter elution ESI-MS, with the details including instrumentation described elsewhere 27 . The BLI-based affinity capture was implemented in a Gator Plus analyzer (Gator Bio, Palo Alto, CA): a BLI microprobe pre-coated with streptavidin was first dipped into the biotinylated anti-Tf Ab solution for 10 min to load the anti-Tf Ab, then dipped into a sample for 10 min to capture Tf molecules, and rinsed in PBST for 1 min to remove non-specifically bound molecules.…”

“…While HR-MS is an ideal tool to study the Tf glycoforms, the quality of data acquired during HR-MS analysis depends on sample preparation 25 , 26 . In this article, an innovative affinity capture technique for sample preparation, called microprobe-capture in-emitter elution (MPIE), was incorporated with HR-MS to study the Tf glycoforms 27 . MPIE can directly couple a label-free optical sensing technology with MS.…”