Water-based Cryogenic Mass Spectrometry with Ultra-short Laser Pulses

Abstract

The advent of soft ionization techniques such as MALDI and ESI has facilitated the study of non-volatile and fragile molecules with high masses in the absence of fragmentation. Since then, mass spectrometry (MS) has become a vital tool in biochemical and medical research, and these methods have paved the way for new research fields such as proteomics. A central yet unanswered scientific question is identifying and investigating every type of protein and its location within a single cell. Recent technologies have targeted clusters of cells to analyze and investigate diseases such as cancer. However, an in-depth understanding of cancer can only be reached when all protein functions and the evolution of malfunctions in a single cell are investigated and understood. With more than a billion different protein species, this is a challenging task and the requirements for scientific instrumentation are demanding. Although MALDI and ESI are the most frequently employed analytical methods due to their robustness and maintainability, they also pose severe challenges, especially during sample preparation. The present work investigates new soft ionization techniques, which operate close to in vivo conditions while requiring minimal sample preparation - a foundational step in single-cell proteomics. First, a novel water-based sample preparation protocols is developed. Subsequently, two laser systems are employed to desorb and ionize the biomolecules in a resonant or non-resonant regime, which defines how laser pulses interact with water. The first method is coined resonant infrared laser-based matrix-assisted laser desorption and ionization (resonant IR-MALDI), and the second non-resonant femtosecond laser desorption and ionization (non-resonant fs IR-LDI). Resonant IR-MALDI is especially useful as this technique targets water as an intermediate energy carrier to extract biomolecules from their native environment. Rigorous samples preparation and other pre-experimental steps are obsolete as water can be found in all living organisms. The present results show that such an approach can successfully produce mass spectra of peptides and proteins embedded in frozen aqueous solutions. Several sample preparation techniques were developed, which greatly simplify the established preparation protocols for specimens investigated under cryogenic conditions. Most importantly, an sample preparation protocol is reproducible and can be conducted within a few minutes. With these achievements, the IR laser-based MS is more streamlined and accessible to a broader scientific community. In addition to highly reproducible sample preparation, superior sensitivity have been achieved using various ablation techniques. We have discovered that oversampling, rastering the specimen area with distances smaller than the laser diameter, greatly enhances the quality of the results, particularly when the step size is limited to a few micrometers. Ultimately, we can obtain a sensitivity of 200 fmol per laser shot, which is suitable for most biological and biochemical specimens, but with greatly reduced effort in sample preparation and access to in vivo conditions. In follow-up experiments glycerol was used as an additive to push the limit of detection leading ultimately to a sensitivity of 1 fmol per laser shot. In the second project, non-resonant fs IR-LDI is used in the second project to detect peptides around 1 kDa by irradiating specimens deposited on different substrates. An important finding is that no surface modification is necessary to obtain high-quality analyte signals. A systematic study with temperature-dependent measurements reveals that a low water-analyte ratio is beneficial for the signal onset. Furthermore, an exceptional sensitivity of 25 amol and a mass limit of 2.5 kDa have been achieved. Finally, a vibrant fragmentation pattern for the investigated analytes have been observed, but those fragments show only a fraction of the intensity of the intact species. In this work, the developed method have shown that water targets can be detected without rigorous sample preparation or surface modification and that 2D imaging is possible for different specimens.