Rapid and selective screening for sulfhydryl analytes in plasma and urine using surface-enhanced transmission mode desorption electrospray ionization mass spectrometry

Abstract

Nylon mesh substrates were derivatized to include VICAT(SH), a biotinylated reagent that contains both a photolabile linking group and a thiol specific capture agent. The enhanced mesh substrates were then used to capture sulfhydryl analytes directly from urine and plasma samples via covalent reaction between the reactive thiols of the analytes and the iodoacetaminyl unit of VICAT(SH). Photocleavage of the labile linker was followed by direct analysis of the mesh surface via transmission mode desorption electrospray ionization (TM-DESI). This chemoselective capture method promoted enrichment of sulfhydryl analytes and reduced matrix interferences, thereby resulting in increased analytical performance of surface enhanced TM-DESI-MS when compared to standard DESI-MS. The present work describes the manufacture of the derivatized mesh substrates and the quality control assessments made during the manufacturing process, the optimization of the chemoselective capture method, and results of experiments pertinent to biological applications. Integration of the chemoselective capture materials with ambient ionization and tandem mass spectrometry results in a powerful combination of speed and selectivity for targeted analyte screening.

Figure 1

Sulfhydryl analyte structures.

Figure 2

Schematic view of surface-enhanced transmission mode desorption electrospray ionization employing VICAT_SH as a thiol capture reagent attached to a neutravidin-coated mesh.

Figure 3

The distribution of VICAT_SH across the derivatized mesh was monitored by TM-DESI-MS. The 1 cm square mesh was affixed to the backing plate and scanned at a rate of 250 μm/sec, resulting in exposure of the mesh to the DESI spray for approximately 0.66 min. Photocleavage of unreacted VICAT_SH followed by desorption electrospray ionization and spontaneous loss of CO₂ produces the stable ion of m/z 257, which corresponds to protonated 4-iodoacetamidyl-butylamine (IABA). Collisional induced dissociation (MS³) results in the formation of the product ion of m/z 198. (XIC shown)

Figure 4

Surface-enhanced TM-DESI analysis of a 1 mL plasma sample containing mercaptopurine (50 μM). The extracted ion chronogram of the collisionally induced dissociation product ion of m/z 193 is shown on the left, while MS² and MS³ spectra for the captured analyte are shown on the right.

Figure 5

Surface-enhanced TM-DESI analysis of a 40 mL urine sample containing penicillamine (20 μM). The extracted ion chronogram of the collisionally induced dissociation product ion of m/z 217 is shown on the left, while MS² and MS³ spectra for the captured sulfhydryl analyte are shown on the right. In this case, two mesh materials were submerged in the urine sample and analyzed as duplicates.

Figure 6

Analysis of VICAT_SH-modified mesh substrates submerged in urine samples. Mesh 1 was rinsed with H₂O prior to TM-DESI-MS analysis while Mesh 2 was air dried without any additional rinsing. The extracted ion chronogram for the ion of m/z 198 (blue) illustrates that the response for VICAT_SH is dramatically reduced when the sample was not rinsed. The extracted ion chronogram for the ion of m/z 86 (gold) corresponds to the presence of creatinine, an abundant component of urine whose presence causes ion suppression of lower abundance analytes. Together the two chronograms illustrate the impact of effective matrix removal following analyte capture.

Figure 7

Overlaid extracted ion chronograms for surface-enhanced TM-DESI analysis of mixtures of captopril and mercaptopurine in urine. The molar ratio of analytes is given as captopril:mercaptopurine. In each case the chronogram corresponds to the primary MS³ ion listed in Table 1.

Scheme 1

Work flow for the manufacture of surface-enhanced materials and the capture and analysis of sulfhydryl compounds using surface enhanced TM-DESI-MS.

Scheme 2

Schematic overview of the photocleavage reaction for a sulfhydryl-containing analyte captured by VICAT_SH. The biotinylated linker molecule remains attached to the mesh surface while the mass tagged analyte is released. The initial photocleavage product contains a carbamic acid that undergoes spontaneous decarboxylation prior to ionization to give the final photocleavage product (shown in protonated form). Capture and release results in a mass shift of 129 Da of the analyte.