Peptide Mass Spectra from Micrometer-Thick Ice Films Produced with Femtosecond Pulses

We present a cryogenic mass spectrometry protocol with the capability to detect peptides in the attomole dilution range from ice films. Our approach employs femtosecond laser pulses and implements neither substrate modification nor proton donor agents in the aqueous solution, known to facilitate analyte detection in mass spectrometry. In a systematic study, we investigated the impact of temperature, substrate composition, and irradiation wavelength (513 and 1026 nm) on the bradykinin signal onset. Our findings show that substrate choice and irradiation wavelength have a minor impact on signal intensity once the preparation protocol is optimized. However, if the temperature is increased from -140 to 0 degrees C, which is accompanied by ice film thinning, a somehow complex picture of analyte desorption and ionization is recognizable, which has not been described in the literature yet. Under cryogenic conditions (-140 degrees C), obtaining a signal is only possible from isolated sweet spots across the film. If the thin ice film is between -100 and -70 degrees C of temperature, these sweet spots appear more frequently. Ice sublimation triggered by temperatures above -70 degrees C leads to an intense and robust signal onset that could be maintained for several hours. In addition to the above findings, we notice that a vibrant fragmentation pattern produced is strikingly similar with both wavelengths. Our findings suggest that while following an optimized protocol, femtosecond mass spectrometry has excellent potential to analyze small organic molecules and peptides with a mass range of up to 2.5 kDa in aqueous solution without any matrix, as employed in matrix-assisted laser desorption/ionization (MALDI) or any substrate surface modification, found in surface-assisted laser desorption/ionization (SALDI).

Automated summary

We developed a cryogenic mass spectrometry method using femtosecond laser pulses to detect peptides in ice films at the attomole dilution range. Our study found that temperature, substrate composition, and irradiation wavelength had minimal impact on signal intensity once the preparation protocol was optimized. We observed a complex behavior of analyte desorption and ionization with thinning ice films at increased temperatures. Cryogenic conditions enabled signal detection only from isolated spots, while higher temperatures led to more frequent detection and intense signal onset. Additionally, we found that the fragmentation pattern produced by both irradiation wavelengths was similar. Our findings highlight the potential of femtosecond mass spectrometry for analyzing small organic molecules and peptides in aqueous solution without the need for matrix or substrate surface modification.