Quantitation of Peptides on Multiple Surfaces for Matrix-Assisted Laser Desorption/Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry: Comparison of Magnitude versus Absorption Modes

Neuropeptides, particularly in tissues, can be difficult to detect due to their low amounts and similar structures to precursors. With matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), conventional organic acid matrices are used to help ionize analytes but can cause matrix interference in the m/z range of common neuropeptides. The high-resolution of Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) enables distinguishing matrix-derived signals from those of neuropeptides. Another benefit to FTICR MS is the ability to process transients in multiple modes, magnitude-mode and absorption-mode. Using the dried droplet method, leucine enkephalin (LE) samples containing gluten exorphin B5 (B5) internal standard were deposited onto surfaces with a siloxane grid; a stainless-steel target plate (SUS), indium-tin oxide (ITO) slide, and glass slides were used. To improve the crystallization of CHCA, freeze drying was employed. Calibration curves of LE had an R⁵ of 0.88 to 0.93 when processed in magnitude-mode or post-processed in absorption-mode when spotted on SUS, ITO slides, or glass slides. Slopes were nearly equal whether processed in absorption-or magnitude mode and did not impact the limit of detection. However, the nature of substrate impacted calibration curve slopes, with ITO slides providing the steepest slope, followed by non-conductive glass, with stainless steel exhibiting the shallowest slopes and sensitivity. Peak shape was improved in absorption-mode (2.5-2.6x improvement of resolution and 1.9-2.9x improvement of S/N), suggesting that absorption-mode processing should be employed in quantitative FTICR MS applications.