High-Throughput Characterization and Optimization of Polyamide Hydrolase Activity Using Open Port Sampling Interface Mass Spectrometry

Abstract

Enzymatic biodegradation of polymers, such as polyamides (PA), has the potential to cost-effectively reduce plastic waste, but enhancements in degradation efficiency are needed. Engineering enzymes through directed evolution is one pathway toward identification of critical domains needed for improving activity. However, screening such enzymatic libraries (100s-to-1000s of samples) is time-consuming. Here we demonstrate the use of robotic autosampler (PAL) and immediate drop on demand technology (I.DOT) liquid handling systems coupled with openport sampling interface-mass spectrometry (OPSI-MS) to screen for PA6 and PA66 hydrolysis by 6-aminohexanoate-oligomer endohydrolase (nylon hydrolase, NylC) in a high-throughput (8−20 s/ sample) manner. The OPSI-MS technique required minimal sample preparation and was amenable to 96-well plate formats for automated processing. Enzymatic hydrolysis of PA characteristically produced soluble linear oligomer products that could be identified by OPSI-MS. Incubation temperatures and times were optimized for PA6 (65 °C, 24 h) and PA66 (75 °C, 24 h) over 108 experiments. In addition, the I.DOT/OPSI-MS quantified production of PA6 linear dimer (8.3 ± 1.6 μg/mL) and PA66 linear monomer (13.5 ± 1.5 μg/mL) by NylC with a lower limit of detection of 0.029 and 0.032 μg/mL, respectively. For PA6 and PA66, linear oligomer production corresponded to 0.096 ± 0.018% and 0.204 ± 0.028% conversion of dry pellet mass, respectively. The developed methodology is expected to be utilized to assess enzymatic hydrolysis of engineered enzyme libraries, comprising hundreds to thousands of individual samples.

Rapid Droplet Sampling Interface for Low-Volume, High-Throughput Mass Spectrometry Analysis

“…A major benefit of a continuous carrier solvent flow is that it can be used to incorporate isotopically labeled internal standards (IS) that eventually mix with the analyte droplet. ,, The IS can be used to correct for matrix effects (e.g., ionization suppression) and to correct for experiment-to-experiment drift in ESI spray conditions, enabling quantitative measurements. To demonstrate quantitation using the RDSI, the carrier solvent was spiked with 50 nM propranolol-D 7 as an IS while analyte droplet propranolol concentrations were measured from 10 nM to 5 μM.…”

“…An OPSI probe was constructed based on previous designs by Van Berkel and Cahill and connected to the same mass spectrometer (see below) as the RDSI. ,, A coaxial tube consisted of an outer stainless steel probe capillary of 1.5 mm (i.d.) (McMaster Carr) and a 0.178 mm i.d.…”

“…Each peak is again baseline-resolved. Integrated peak area of the 300 droplets acquired in Figure 2d resulted in a relative standard deviation (RSD) of 12%, on par with a <10% RSD reported by OPSI-MS. 3,8,9,19 However, as noted below, RSDs between 20 and 30% were common when acquiring with multiple MRMs, which we attribute to an issue in synchronizing the mass spectrometer acquisition speed with droplet ejection (see the Supporting Information).…”

“…In particular, the acoustic and pressure-pulse-induced droplet ejection systems are amenable to high-throughput sample delivery from 96, 384, and 1536 (acoustic) well-plate formats. 1,8,9 Of these techniques, acoustic droplet ejection coupled with OPSI-MS has reported up to 15 Hz sampling throughput with and without multiplexing, respectively, with high signal reproducibility (<10% RSD). 3,17 To achieve such metrics, a carrier solvent flow of 1 mL/min in a modified, high-flow ion source is required to efficiently transport analyte to the mass spectrometer for characterization.…”

“…The highest throughputs achieved to date employ noncontact sampling approaches utilizing droplet ejection systems to dispense single or multiple droplets of sample solution into an atmospheric pressure ionization source. These systems include acoustic, pressure-pulse, and piezoelectric droplet-induced ejection into an open port sampling interface (OPSI), ,− piezoelectric droplet ejection onto a probe-electrospray ionization needle (PESI), , and acoustic mist direct ionization . Droplets in the pL-to-nL range can be reproducibly delivered by such methods, enabling the measure of ultralow sample volumes and reduced consumption of valuable reagents.…”