Direct Analysis of Intact Biological Macromolecules by Low-Energy, Fiber-Based Femtosecond Laser Vaporization at 1042 nm Wavelength with Nanospray Postionization Mass Spectrometry

Abstract

A fiber-based laser with a pulse duration of 435 fs and a wavelength of 1042 nm was used to vaporize biological macromolecules intact from the condensed phase into the gas phase for nanospray postionization and mass analysis. Laser vaporization of dried standard protein samples from a glass substrate by 10 Hz bursts of 20 pulses having 10 μs pulse separation and <50 μJ pulse energy resulted in signal comparable to a metal substrate. The protein signal observed from an aqueous droplet on a glass substrate was negligible compared to either a droplet on metal or a thin film on glass. The mass spectra generated from dried and aqueous protein samples by the low-energy, fiber laser were similar to the results from high-energy (500 μJ), 45-fs, 800-nm Ti:sapphire-based femtosecond laser electrospray mass spectrometry (LEMS) experiments, suggesting that the fiber-based femtosecond laser desorption mechanism involves a nonresonant, multiphoton process, rather than thermal- or photoacoustic-induced desorption. Direct analysis of whole blood performed without any pretreatment resulted in features corresponding to hemoglobin subunit-heme complex ions. The observation of intact molecular ions with low charge states from protein, and the tentatively assigned hemoglobin α subunit-heme complex from blood suggests that fiber-based femtosecond laser vaporization is a "soft" desorption source at a laser intensity of 2.39 × 10(12) W/cm(2). The low-energy, turnkey fiber laser demonstrates the potential of a more robust and affordable laser for femtosecond laser vaporization to deliver biological macromolecules into the gas phase for mass analysis.

Ambient Molecular Analysis of Biological Tissue Using Low-Energy, Femtosecond Laser Vaporization and Nanospray Postionization Mass Spectrometry

“…The analysis of leaf samples using Ti:Sapphire-LEMS also revealed higher ion yields at 505 and 1120 μJ, whereas negligible anthocyanin and sugar molecular features were observed at 160 μJ. This suggests that sufficiently high laser pulse energy is required to detect signal in agreement with previous Ti:Sapphire-LEMS and F-LEMS measurements of biological macromolecules [35]. The successful analysis of plant samples using fiber laser vaporization suggests that the low-energy laser vaporization is suitable for the direct analysis of complex biological systems.…”

“…The laser vaporization source was described in detail previously for the Ti:Sapphire-LEMS [29] and has been recently modified to couple the fiber laser to the electrospray inlet of a quadrupole time of flight (QTOF) mass spectrometer (micrOTOF-Q II; Bruker Daltonik GmbH, Bremen, Germany) [35,36]. Briefly, the fiber laser delivers 100 kHz, 435 fs, 1042 nm pulses with maximum pulse energy of 50 μJ.…”

“…However, the higher cost and lower robustness of the Ti:Sapphire amplified femtosecond laser in comparison with the more common nanosecond UV and IR lasers limit the wide application of Ti:Sapphire laser as a desorption source. In recent studies, we successfully implemented a low-energy, femtosecond fiber laser for vaporization and a nanospray for postionization in the analysis of proteins, whole blood [35], and substituted benzylpyridinium salts [36]. The studies show that the low-energy fiber laser resulted in comparable internal energy distributions to nanospray [36] and retained intact biological macromolecules after laser radiation [35].…”

“…In recent studies, we successfully implemented a low-energy, femtosecond fiber laser for vaporization and a nanospray for postionization in the analysis of proteins, whole blood [35], and substituted benzylpyridinium salts [36]. The studies show that the low-energy fiber laser resulted in comparable internal energy distributions to nanospray [36] and retained intact biological macromolecules after laser radiation [35]. The application of a femtosecond fiber laser rather than a solid state amplifier laser to more complex biological samples is thus of interest for the broad and potential applicability of LEMS as a robust, portable, and affordable analytical tool to non-specialists.…”

Nonresonant, femtosecond laser vaporization and electrospray post-ionization mass spectrometry as a tool for biological tissue imaging

“…have been successfully vaporized and analyzed without matrix deposition. LEMS represents a universal tool to analyze a variety of samples intact, for samples including peptides [25], lipids [37], proteins [38][39][40], explosives [41,42], narcotics [26], smokeless powders [43], pharmaceuticals [26], and tissues [44,45].…”