Wavelength Dependence of Intact Protein Extraction Using Femtosecond Laser Ablation

Abstract

The extraction and analysis of intact proteins from complex biological samples via femtosecond laser ablation are influenced by wavelength-dependent multiphoton and avalanche ionization processes. To investigate these effects, an intact protein mixture with molecular weights ranging from 9 kDa to 68 kDa as well as individual proteins were sampled using laser wavelengths spanning from the ultraviolet to the near-infrared. Our results suggest that visible and infrared wavelengths enable intact protein extraction, even for proteins with strong absorption in the visible spectrum. Ultraviolet wavelengths also yield intact extraction when long enough to avoid resonant absorption by the aromatic amino acids. Our findings support the hypothesis that minimizing multiphoton ionization helps preserve intact protein signals during femtosecond laser ablation. By isolating the role of laser wavelength, this study provides insight on high intensity laser–biomolecule interactions relevant to analytical techniques such as high-resolution mass spectrometry imaging.

Graphical Abstract

Laser desorption mass spectrometry (LDMS) is a common method of isotope, elemental and molecular analysis in fields like material science,¹ geology,² and bioanalytics.³ Beyond small molecules, LDMS is capable of sampling and ionizing larger biomolecules, including proteins from biological samples, through desorption by impulsive vibrational excitation (DIVE).^4–6 The DIVE process uses pulsed mid-infrared lasers (PIRLs) that target the O–H vibrational modes of water to drive efficient material removal.^7–13 Mass spectrometry imaging (MSI) using PIRLs has provided valuable insights on the functions of metabolites and proteins in complex biological tissues.^14–16 However, PIRLs require sufficient sample hydration to drive the ablation process and are fundamentally limited by diffraction to a minimum laser spot size of ~ 3 μm.

Ultrafast lasers offer a promising alternative mechanism, relying on nonlinear energy deposition via multiphoton, tunneling, and avalanche ionization (AI) rather than resonant vibrational excitation.^3,17,18 This enables femtosecond (fs) laser ablation to operate under a broader range of conditions than PIRLs, including in dry environments. Previous studies have suggested that fs lasers may allow for sub micrometer spatial resolution MSI.¹⁹ However, the specific contributions of protein chromophores, protein size, electron collisional processes, and multiphoton ionization (MPI) to intact protein ejection have not been systematically investigated.

Previous studies have shown intact protein extraction from aqueous solutions and biological tissues using infrared (IR) fs laser ablation coupled with ambient electrospray ionization.^20–22 Other related works using visible fs lasers from ice samples have shown substantial intact signatures for organic molecules, peptides, and small proteins with masses of up to 6 kDa.^23,24 To our knowledge, intact extraction of larger proteins with visible or ultraviolet (UV) ultrafast lasers has not been previously reported. Recent works by the Murray^25,26 and Zhu²⁷ groups have shown successful intact extraction of proteins using nanosecond lasers in the deep UV. These works show the promise of using shorter wavelengths for intact extraction of large biomolecules. However, at the intensities used in ultrafast laser ablation, which are four to five orders of magnitude larger than these nanosecond studies,^25–27 the influence of wavelength on intact protein ejection remains largely unexplored.

As outlined by Linz et al.,¹⁸ laser wavelength determines the extent of MPI and collisional ionization (CI) during fs laser ablation, influencing both ionization efficiency and the potential for damage to the sample’s constituents. For pure water, which is often used to model the ablation dynamics of biological samples like tissue,^17,18,28,29 MPI plays a more significant role at shorter wavelengths in the UV and visible, while CI dominates for longer wavelengths in the IR.¹⁸ When considering biomaterials, proteins and metabolites have a much larger absorptivity across the UV, visible, and near IR spectrum than water (see Figure 1 and Figure S1).^30,31 Consequently, unless a specific absorption peak of water is targeted, MPI will generally occur more readily in proteins and metabolites,³² increasing the likelihood of MPI-induced fragmentation processes in biological samples when compared to exciting vibrational modes of water.

Figure 1.

Optical absorption coefficient of free electrons from the Drude model before the electron avalanche (electron density of 10¹⁰ cm⁻³), at the start of the avalanche process (10¹⁶–10¹⁹ cm⁻³), and in a stable electron avalanche 10²⁰ cm⁻³ estimated using the same assumptions as ref 18, compared to MastR (at 500 μM), HEWL (700 μM), Mb (1000 μM), and water from ref 19.

During the fs laser ablation, AI is substantially a CI process driven by photoabsorption of liberated electrons. Following the Drude model, the one-photon absorption cross-section for free electrons is given by:¹⁸

$${\sigma }_{1\mathit{\text{pt}}}=\frac{t_{\mathit{\text{col}}}{e}^2}{({\omega }^2{t}_{\mathit{\text{col}}}^2+1)c{n}_0{\epsilon }_0{m}_c}$$

where t_col is the Drude collision time, e is the charge of an electron, ω is the angular frequency of the incident laser, n₀ is the index of refraction of the medium, m_c is the mass of the liberated electron, c is the speed of light and σ_1pt is the single photon absorption cross section. As the electron density (n(t)) evolves throughout the ablation process, the roles of MPI and energy absorption by free electrons, generated by MPI and tunneling ionization, can be compared using the absorptivity, defined by the absorption coefficient (α(t)=σ_1ptn(t)). Figure 1 shows a wavelength-dependent comparison of the single-photon absorption coefficient of free electrons during the ablation process - at an electron density of 10¹⁰ cm⁻³, at the onset of the avalanche process (10¹⁶–10¹⁹ cm⁻³), and during a stable electron avalanche (10²⁰ cm⁻³) - alongside the absorption coefficients for the proteins: hen egg white lysozyme (HEWL), myoglobin (Mb), and Mastigocladopsis repens rhodopsin (MastR). We propose that the significant absorption coefficient of free electrons at densities between 10¹⁶ and 10¹⁹ cm⁻³ allows the AI process to dominate the absorption process, limiting protein fragmentation from MPI. Previously, a similar electron density was found to correspond to the threshold required for AI to drive the absorption process when modeling the ablation of water with 250 fs laser pulses.¹⁸

Although photoinduced fragmentation can be mitigated by selecting the appropriate wavelength, the risk of dissociation from CI during the ablation process must be addressed. To preserve protein integrity, the energy of electrons in the low-density plasma, produced during ablation, must remain below the electron collisional dissociation threshold of the analyte molecules. Fortunately, under near ablation threshold conditions (between 10¹² and 10¹³ W/cm², depending on wavelength), the average energy of electrons is below the collisional dissociation threshold of most proteins (~10 eV^33,34).^18,28 Therefore, electron collisional dissociation is unlikely to significantly contribute to fragmentation and reduction in intact protein signal.

The contributions of protein chromophores were explored with three model proteins which span a range of structural complexity and photoactivity. These proteins were not chosen to mimic tissue environments, but rather to enable the controlled study of energy transfer and ablation product formation across proteins with distinct absorption profiles. Establishing these principles under well-defined conditions is a prerequisite to extending laser ablation methods to more complex biological systems.

HEWL serves as a valuable reference point as it only absorbs near 280 nm, has a well-characterized structure and a long history of use in conventional mass spectrometry (MS).^35,36 Mb contains a heme chromophore and exhibits a well-characterized optical response and ionization behavior with a strong absorption peak at 400 nm and a weak absorption peak near 500 nm^37,38 (see Figure S1). As a membrane protein, MastR is of particular interest due to its structural similarity to light activated proteins like bacteriorhodopsin.³⁹ MastR belongs to the microbial rhodopsin family and acts as a light-activated chloride pump containing a retinal chromophore with an absorption peak at 535 nm (near 515 nm).

To examine the influence of molecular weight on protein extraction, a protein mixture was used, with approximate masses of 9 kDa, 12 kDa, 21 kDa, 29 kDa, 50 kDa, and 68 kDa (details in Experimental Methods Section of the Supporting Information).

Two different Orbitrap MS data acquisition methods were used to guarantee the efficient detection and processing of protein signals across a broad mass range for each wavelength used: ensemble Fourier-transform-based (for HEWL and Mb), and individual ion based^40–42 (for MastR and the protein mixture). See the experimental methods section of the supplement for more details. Only data sets generated using the same analysis techniques are compared.

Figure 2 shows the mass spectra of extracted HEWL using fs laser ablation pulses at wavelengths between 1030 and 257 nm. These spectra are compared to a control sample drop cast on a glass slide, which was introduced into the mass spectrometer using nanospray desorption electrospray ionization (nano-DESI).⁴³ Intact HEWL peaks are visible when ablated with wavelengths of 1030, 515, and 343 nm. However, the intact HEWL signal intensity is inversely correlated with the increased photon energy of shorter wavelengths. At 1030 nm, MPI initiates free-electron generation, which in turn seeds an electron avalanche that is responsible for the majority of the energy absorption (see Figure 1 to compare absorption after seed electron generation).^18,28 Based on rate equation modeling for pure water, for a 1030 nm, 250 fs pulse at 10¹³ W/cm², there would be approximately 100 times more ionization events from the avalanche process than from strong field ionization (see ref 18 for details). For a visible and UV pulse of the same duration and intensity, approximately 10 times more ionization occurs from AI than MPI at 515 nm and about 3 times at 343 nm, indicating an increased significance of MPI.¹⁸ These results suggest that as the wavelength decreases, the protein absorbs more energy via MPI leading to increased fragmentation. When ablated with 257 nm, there are no intact protein peaks present, suggesting fragmentation of the protein. However, specific fragmentation peaks could not be assigned to a high degree of certainty. HEWL has no chromophore absorbing in the visible range and only absorbs strongly at 280 nm due to the aromatic amino acids. This suggests that during laser ablation at 257 nm the photon energy is deposited into the protein via MPI, likely leading to extensive protein fragmentation and preventing identification from the mass spectra.

Figure 2.

Mass spectra of HEWL following fs laser ablation and subsequent analysis by nano-DESI using a catch-and-release method. Charge states corresponding to the intact protein are labeled in each spectrum. Compared to the unablated drop-cast nano-DESI control, the ablation spectra exhibit increased charge state broadening and a decreased signal-to-noise ratio at shorter laser wavelengths. This trend reflects a wavelength-dependent reduction in intact protein signal. Corresponding signal intensities are presented in Supplementary Figure S2.

Analysis of Mb (Figure 3) shows a similar loss of intact protein signal as wavelength decreases (see supplementary Figure S3). In Mb, the heme group absorbs around 500 nm, making 515 nm a near-resonant wavelength. The intact protein, including the heme group, remains detectable down to 343 nm. However, the change in peak intensity of 515 to 1030 nm is much more significant in Mb with an 87% decrease in signal, than HEWL (37% decrease). This difference may reflect an increased role of MPI in the ablation process at 515 nm for Mb compared to HEWL, resulting in increased protein fragmentation and a lower intact protein signal. At 257 nm, the spectrum lacks any intact protein peak, isotopic peaks, or charge state series, suggesting fragmentation.

Figure 3.

Mass spectra of Mb post ultrafast laser ablation sampling using a catch and release method with nano-DESI. The charge states of the intact protein are indicated in each spectrum. The signal intensities are shown in Figure S3.

MastR (data shown in supplement Figure S4 and with mirror plot comparisons in Figure S5), behaves similarly to Mb despite a significantly different linear absorption spectrum. Specifically, MastR’s deconvoluted mass spectra at 1030 nm and with a PIRL are nearly identical, confirming that fs laser ablation at 1030 nm supports intact protein extraction. Likewise, the loss of intact protein signatures at 257 nm is consistent with the other proteins studied. However, unlike Mb, MastR has approximately 7.5 times lower linear absorption at 343 nm than 515 nm. At the high intensities used, this would suggest that resonant enhanced multiphoton absorption would be more significant at 515 nm than 343 nm (following ref 44), yet as wavelength decreases we still observe a decrease in MastR’s intact protein signal. This contradiction indicates a more complicated process than pure MPI. We hypothesize that at 515 nm, AI will contribute an order of magnitude more ions than MPI,¹⁸ resulting in the observed intact protein signal. In contrast, when water is ablated at 343 nm, MPI accounts for approximately one-third of the ionization from AI, indicating a significantly greater contribution of MPI to the ablation process.¹⁸ Due to the higher absorptivity of MastR relative to water at 343 nm, the role of MPI will be greater in the protein mixture, with a reduced contribution from AI compared to that observed at 515 nm. We suggest that the decrease in AI compared to 515 nm allows for direct absorption of energy by the protein, leading to more fragmentation and a decrease of the observed intact protein signal. To validate this proposed mechanism, high intensity transient absorption spectroscopy experiments and rate equation modeling that account for the contributions of chromophores to the MPI process are required. Additionally, future studies should compare the on-resonance and near resonance cases by using a fs laser tuned exactly to an absorption peak of a protein between 400 and 600 nm to drive ablation directly into a mass spectrometer for direct top-down proteomic analysis.

The mass spectra for HEWL, Mb, and MastR provide empirical support for longer wavelengths favoring intact protein extraction. These results are consistent with the hypothesis that, when electron avalanche processes dominate energy absorption, direct MPI of proteins is suppressed, reducing fragmentation. By looking at a protein mixture, we analyzed the dependency of protein size for intact extraction (see Figure 4). Our results show that the fs pulses have a lower relative intensity peak for the intact protein around 68 kDa when compared to nano-DESI and PIRL sampling. We suggest that the proportional increase in the inelastic electron collisional absorption cross section with protein mass leads to a reduction in intact signal for larger proteins due to more CI events within the electron cloud. To validate this hypothesis, detailed studies of plasma energy during laser ablation would be required. Overall, our observations are consistent with the hypothesis that by selecting wavelengths which avoid absorption in the aromatic amino acids, it is possible to minimize the likelihood of fragmentation for proteins. However, for larger proteins the intact peak intensity drops, suggesting protein fragmentation.

Figure 4.

Protein ladder with masses from 9 kDa to 68 kDa sampled using UV, visible and IR fs lasers, PIRL and nano-DESI sampling of a drop cast sample.

In this work, we explore the influence of laser wavelength on intact protein extraction during laser desorption. We minimized matrix effects by using aqueous protein samples to isolate the fundamental desorption processes in pure and mixed analyte solutions. These insights provide a starting point for applying visible and UV ultrafast lasers to more complex biological systems, where protein concentrations can range from below 100 pg/μL to over 10 μg/μL.^45,46 Given the concentration independence of the extraction mechanism, as demonstrated by the application of fs laser sampling in several publications,^20–24,47 our results support the broad applicability of fs extraction techniques in proteomics.

One promising application of our findings is subcellular MSI using fs lasers.¹⁹ However, achieving sufficient proteoform depth from micron and submicron voxels requires improvements in ion transfer efficiency and MS sensitivity to detect the limited number of proteins present. In our experiment, protein standards were prepared in undersaturated aqueous solutions, likely favoring the ejection of individual molecules. From tissue, proteins may be ejected as individual molecules, clusters, or larger particulates, each with distinct ionization and photochemical behaviors. Although prior work suggests that particulate size can vary with optical penetration depth,³⁰ this effect is minimal across the wavelengths examined and is further constrained by plasma shielding, which limits optical penetration at longer wavelengths.^18,30,48,49 Because our system favors the ejection of individual molecules and exhibits minimal variability in optical penetration depth across wavelengths, we expect particulate size to play a minor role in our findings. This factor, however, may be more significant in tissue imaging, where matrix effects can promote the formation and ejection of larger particulates.

Despite the inherent challenges to high-resolution protein sampling, our study represents an initial step toward understanding the physical and photochemical processes that enable intact protein extraction using ultrafast laser ablation. We show evidence that fs lasers with IR, visible and UV wavelengths long enough to avoid resonant absorption in the aromatic amino acids, near 280 nm, effectively extract intact proteins. This result is notable as shorter wavelengths allow for smaller ablation spot sizes than PIRL techniques (see Table S1 in the Supporting Information for the specific laser parameters used in our study). Although prior work using nanosecond lasers in the deep UV demonstrated intact protein extraction,^25–27 the intensities used for ultrafast ablation are four to five orders of magnitude higher, leading to nonlinear processes dominating.^44,50 As such, we cannot extend our findings to comment on the behavior of deep UV lasers. However, significant differences in absorption behavior are expected between ultrafast and longer pulsed lasers. Ultrafast laser ablation can be confined to depths on the order of 100 nm.^51,52 In contrast, ablation with longer pulses and lower peak intensities produce crater profiles defined by the absorption depth and highly nonlinear plasma screening, making reproducible profiles more difficult to achieve than with fs lasers.^29,51

Our observations support the hypothesis that avoiding resonance with aromatic amino acids allows for intact extraction. Our results indicate a wavelength range from ~ 300 nm to 1 μm that offers a favorable trade-off between reduced fragmentation and the ability to ablate material at submicron resolution.³ Notably, proteins with chromophores showed strong intact signals when irradiated at wavelengths near their visible absorption peaks, suggesting that AI and electron shielding play a role in protecting molecular integrity. Further studies using lasers tuned to specific protein absorption maxima could help elucidate these protective mechanisms. By understanding the wavelength-dependent response of different proteins under fs laser ablation, this study provides foundational insight into how photon energy and nonlinear ionization dynamics influence intact biomolecule extraction. As ultrafast laser technology becomes more widely available,^53,54 our findings offer important insight toward applying visible and UV wavelengths for proteomic studies in complex tissue environments and subcellular MS workflows.

Supplementary Material

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jpclett.5c01440.

Detailed outline of the protein preparation and laser parameters used; fully annotated mass spectra for each of the proteins examined and annotated supporting data (PDF)

Transparent Peer Review report available (PDF)

Data Availability Statement

RAW MS files are available from the MassIVE repository under the identifier MSV000097234.