Cinnamic acid derivatives as matrices for ultraviolet laser desorption mass spectrometry of proteins

The paper reports the discovery of three new matrices for the matrix-assisted laser desorption of proteins. These new matrices (sinapinic, ferulic and caffeic acids) are cinnamic acid derivatives that have several practical advantages over the nicotinic acid matrices previously used. These materials form much less intense photochemically generated adduct peaks in the protein quasimolecular ion signal and the adduct peaks that are present are easier to resolve. These matrices produce intense protonated-molecule ions from all of the proteins (over 50) so far examined. These new matrices are also very stable in a vacuum, allowing for their convenient use in very high vacuum applications (e.g., Fourier transform ion cyclotron resonance mass spectrometry).     

Direct comparison of infrared and ultraviolet wavelength matrix-assisted laser desorption/ionization mass spectrometry of proteins

In an effort to gain an understanding of the processes governing matrix-assisted laser desorption/ionization (MALDI), we made a direct comparison of ultraviolet (UV)- and infrared (IR)-MALDI linear time-of-flight mass spectra of proteins obtained from the same samples and matrices (on the same sample surface), using two different lasers, each having short duration (<10-ns) pulses, i.e., a tunable wavelength Nd:yttrium aluminum garnet (YAG) pumped optical parametric oscillator laser operating at 2.94 µm and a Nd:YAG laser operating at 355 nm. We observed that (1) the IR-MALDI and UV-MALDI spectra of a given protein from the same matrix were strikingly similar; (2) protein ions produced by IR-MALDI experienced less fragmentation than those produced by UV-MALDI; and (3) photochemical adducts produced during UV-MALDI were absent in IR-MALDI. These results lead us to speculate on the mechanisms for the ionization process in UV- and IR-MALDI. Because photons with a wavelength of ～3 µm are unlikely to effect electronic excitation of the matrix at the irradiance used for MALDI, we propose that ionization in IR-MALDI occurs as a natural consequence of the solid-to-gas phase transition induced by the IR irradiation, and involves proton transfer reactions in the intermediate phase between solid and gas. The strikingly similar UV- and IR-MALDI mass spectra leads us to the additional proposal that ionization in UV-MALDI may also be a natural consequence of the phase transition and that electronic excitation may not play a primary role in the ionization process.     

Two-laser infrared and ultraviolet matrix-assisted laser desorption/ionization

Matrix-assisted laser desorption/ionization (MALDI) was performed using two pulsed lasers with wavelengths in the IR and UV regions. A 10.6 micro m pulsed CO(2) laser was used to irradiate a MALDI target, followed after an adjustable delay by a 337 nm pulsed nitrogen laser. The sample consisted of a 2,5-dihydroxybenzoic acid matrix and bovine insulin guest molecule. The pulse energy for both of the lasers was adjusted so that the ion of interest, either the matrix or guest ion, was not produced by either of the lasers alone. The delay time for maximum ion yield occurs at 1 micro s for matrix and guest ions and the signal decayed to zero in approximately 400 micro s. A mechanism is presented for enhanced UV MALDI ion yield following the IR laser pulse based on transient heating.     

基体辅助激光解吸质谱法测定蛋白质分子量

本文叙述用自行研制成功的激光微探针飞行时间质谱仪及采用基体辅助激光解吸的新方法, 对溶菌酶、细胞色素C、肌红蛋白、胰蛋白酶、蛋白酶、白蛋白等多种蛋白质的分子量进行测定, 并对蛋白质混合物进行分析, 得一以满意的结果。此方法测定蛋白质分子量具有速度快(十分钟一个样品), 准确度高(±1%-0.1%), 灵敏度高(10^-^1^2～10^-^1^5mol)等优点, 是传统生物方法难以比拟的。     

A quantitative model of ultraviolet matrix-assisted laser desorption/ionization

A quantitative model of primary ionization in ultraviolet matrix-assisted laser desorption/ionization (UV-MALDI) is presented. It includes not only photochemical processes such as exciton pooling, but also the effects of the desorption event. The interplay of these two is found to be a crucial aspect of the MALDI process. The desorbing plume is modeled as an adiabatic expansion with entrained clusters. The parameters in the model are defined as much as possible via experiment or by analogy with known effects. The model was applied to the matrix 2,5-dihydroxybenzoic acid and found to reproduce the fluence dependence of the fluorescence yield and key features of the picosecond two-pulse ion generation efficiency curves. In addition, the model correctly predicts a fluence rather than irradiance threshold, the magnitude of the threshold, the magnitude of the ion yield, laser wavelength effects, plume temperatures, plume expansion velocities and the spot size effect.     

Homogeneous bottleneck model of matrix-assisted ultraviolet laser desorption of large molecules

The factors affecting the yield of high-mass molecules by matrix-assisted ultraviolet laser volatilization are examined in a simple model. The key material factors appear to be a low heat of sublimation, subcritical concentration of the guest molecules and a high irradiance input in a short time compared to the sublimation induction period. The model is homogeneous in that the energy density is taken to be uniform within the ‘hot region’ of the matrix. The two competing effects are the rates of energy transfer from the matrix to the guest molecules and the desorption by sublimation. It is the bottleneck for energy transfer to the embedded guest molecules that makes their energy content lag behind that of the matrix. This is particularly the case for an initially cold sample. When a sufficiently high rate of sublimation can be achieved (e.g., using a high-power laser), the guest molecules (or adduct ions) will desorb internally cold and will thus not fragment. Numerical simulations of the sublimation kinetics using realistic laser and material parameters support the conclusions and delineate the ranges of the critical factors.     

Liquid supports for ultraviolet atmospheric pressure matrix-assisted laser desorption/ionization

Atmospheric pressure (AP) liquid matrices for ultraviolet (UV) matrix-assisted laser desorption/ionization (MALDI) are presented. Doping a known organic chromophore, alpha-cyano-4-hydroxycinnamic acid (CHCA), into liquid media yielded a homogenous sample system with simplified sample preparation, increased sample lifetime, and added utility for APMALDI ion sources. Compared with vacuum situations, AP matrices are not as limited by vapor pressure, so liquid matrix formulations can focus on desorption and ionization versus vacuum stability and source contamination. The parameters studied include chromophore concentration, liquid support variations, and quantitation capability. Chromophore concentration adjustments provided insight into the necessary absorbance for UV-APMALDI and demonstrated the importance of laser penetration depth. Liquid support variations allowed adjustments of sample lifetime and analyte solvents. Extended sample lifetime is beneficial for instrument tuning and source optimization; however, increased liquid viscosity lowers signal intensity. The shot-to-shot reproducibility, as examined with individual ion packets, suggests that the liquid matrix can alleviate some inconsistencies seen with solid MALDI, suggesting a possibility for better quantitation. The measurements for laser penetration depth, solution viscosity, and solvent additives could add to the information on MALDI mechanisms. The liquid matrix offers advantages that complement current MALDI methods.     

Singlet-singlet annihilation in ultraviolet matrix-assisted laser desorption/ionization studied by fluorescence spectroscopy

Laser-induced fluorescence spectroscopy was carried out on microcrystalline samples of three typical matrices under conditions of matrix-assisted laser desorption/ionization (MALDI). The emitted fluorescence intensity was determined as a function of incident laser fluence and a sublinear increase of the fluorescence intensity with laser fluence was found. A very good fit was obtained when the experimental fluorescence vs. fluence data were compared with a numerical model assuming that under typical MALDI fluence conditions a large fraction of molecules in the excited singlet state undergoes singlet-singlet annihilation. Throughout the fluence range relevant for MALDI, however, the experimental data could not be fit well to a model assuming resonant two-photon absorption as the process depopulating the singlet state. In a separate set of experiments, the singlet lifetimes of several typical crystalline MALDI matrices were determined and found to be considerably shorter than previously reported. While both singlet-singlet annihilation and resonant two-photon absorption have been discussed in the literature as candidates for pathways to primary matrix ion generation in MALDI, the data presented here suggest that singlet-singlet annihilation is the dominant mechanism for depopulating the singlet state in a matrix crystal excited at typical MALDI fluences.     

Acid hydrolysis of proteins in matrix assisted laser desorption ionization matrices

Sample preparation is crucial to the success of experiments in biological mass spectrometry. In proteomics, digestion of the proteins into peptides is a key step for “bottom-up” approaches. Often, the use of enzymes requires physiological conditions, producing peptides that must be extracted or further purified before mass analysis. Chemical cleavage reagents offer more flexibility and can be more compatible with downstream mass analysis. Expanding on prior work using acid hydrolysis, proteolysis with matrix-assisted laser desorption ionization (MALDI) matrices is presented. This sample preparation can be performed rapidly with a minimum of reagents and sample handling, but it must first be evaluated in terms of digestion efficiency, missed cleavages, and side reactions before implementation for in-gel digestion and in-solution digestion using minimal volumes of protein. Time courses of acid hydrolysis are shown for protein standards, illustrating the sensitivity of this type of sample preparation, minimization of side reactions, and performance for proteins in mixtures. To illustrate the potential for sensitive detection of a specific protein, MALDI matrix hydrolysis is used to digest a protein immunoprecipitated from cell lysate.     

Factors that influence the observed fast fragmentation of peptides in matrix-assisted laser desorption

Fragmentation processes that occur very early during matrix-assisted laser desorption ionization (MALDI) of peptides are examined by utilization of delayed pulsed ion extraction with a linear time-of-flight mass spectrometer. The oxidized B chain of bovine insulin (MW=3495. 95 u), which produces a wide range of fragment ions, is utilized as a probe to examine the effects of several experimental parameters on this process. Experimental evidence suggests that this MALDI process is not prompt fragmentation and involves metastable ion decay that is quite different from that which is observed with postsource decay experiments. This conclusion is based upon the significant differences observed in the fragmentation products produced by the two techniques. This metastable ion decay process also appears to be over within the minimum pulse delay period (320 ns) that is possible with the current pulsed ion extraction hardware. These two observations suggest that either different activation processes are involved in the two techniques or that the much different time frame of the methods influences the observed ion decay pathways. This fast MALDI metastable ion fragmentation also is shown to be influenced by both the MALDI matrix and the laser fluence.     

Nanomaterial-based surface-assisted laser desorption/ionization mass spectrometry of peptides and proteins

We have investigated six nanomaterials for their applicability as surfaces for the analyses of peptides and proteins using surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS). Gold nanoparticles (NPs) were useful nanomateriais for small analytes (e.g., glutathione); Pt nanosponges and Fe₃O₄ NPs were efficient nanomaterials for proteins, with an upper detectable mass limit of ca. 25 kDa. Nanomateriais have several advantages over organic matrices, including lower limits of detection for small analytes and lower batch-to-batch variations (fewer problems associated with “sweet spois”), when used in laser desorption/ionization mass spectrometry.     

Laser-induced desorption of proteins

Laser-induced desorption mass spectrometry has been applied to a number of proteins in the mass range 5000-150,000 u. The beam from an excimer-laser-pumped dye-laser at 266 nm has been focused to a spot of about 50 microns in diameter with irradiances in the 10(7) W/cm2 region. A linear time-of-flight mass spectrometer has been used for mass spectrometric measurements, where positive and negative secondary ions of large proteins have been studied. The effect of different experimental parameters on the protein ion-signal intensities are discussed.     

Matrix-assisted laser desorption/ionization mass spectrometry of nucleic acids with wavelengths in the ultraviolet and infrared.

A number of different matrices have been tested and compared for ultraviolet and infrared (UV and IR) matrix-assisted laser desorption/ionization (MALDI) of oligodeoxyribonucleotides and mixtures thereof, as well as ribonucleic acids (tRNA from yeast and rRNA from E. coli). A new technique for removing alkali cations from nucleic acid samples during sample preparation on the sample support is demonstrated. The amount of oligonucleotide sample consumed during a typical measurement in IR-MALDI-MS was determined.     

Electrospray-assisted laser desorption/ionization and tandem mass spectrometry of peptides and proteins

We have constructed an electrospray-assisted laser desorption/ionization (ELDI) source which utilizes a nitrogen laser pulse to desorb intact molecules from matrix-containing sample solution droplets, followed by electrospray ionization (ESI) post-ionization. The ELDI source is coupled to a quadrupole ion trap mass spectrometer and allows sampling under ambient conditions. Preliminary data showed that ELDI produces ESI-like multiply charged peptides and proteins up to 29 kDa carbonic anhydrase and 66 kDa bovine albumin from single-protein solutions, as well as from complex digest mixtures. The generated multiply charged polypeptides enable efficient tandem mass spectrometric (MS/MS)-based peptide sequencing. ELDI-MS/MS of protein digests and small intact proteins was performed both by collisionally activated dissociation (CAD) and by nozzle-skimmer dissociation (NSD). ELDI-MS/MS may be a useful tool for protein sequencing analysis and top-down proteomics study, and may complement matrix-assisted laser desorption/ionization (MALDI)-based measurements.     

An ultraviolet/infrared matrix-assisted laser desorption ionization sample stage integrating scanning knife-edge and slit devices for laser beam analysis

A sample stage is described that allows the on-line analysis of laser intensity profiles and spot sizes directly in the ion source of a matrix-assisted laser desorption ionization (MALDI) mass spectrometer. The detector uses either a scanning knife-edge or a narrow slit in combination with diffusing disks for scattering of photons and a pyroelectric sensor for recording the light pulses. The setup was integrated into the sample holder of a oMALDI2(TM) ion source (AB Sciex) and allows parallel analysis of UV- and IR-laser beams at typical UV-/IR-MALDI laser fluences. The concept could be especially useful for a precise control of the laser spot size in MALDI imaging applications.     

Suppression of matrix ions in ultraviolet laser desorption: Scanning electron microscopy and raman spectroscopy of the solid samples

Suppression of low-mass ion peaks in matrix-assisted ultraviolet laser desorption has been found to occur at low matrix-to-analyte molar ratios when using nicotinic acid as matrix, independent of the angle of illumination. Microscopic Raman scattering spectroscopy has been employed to investigate the matrix–analyte solid-state composition. The matrix-to-analyte molar ratios employed in preparing the solutions are reliable guides to the relative amounts of matrix and analyte molecules in the solid crystals, given the method of sample preparation employed involving drying under a stream of nitrogen. A qualitative model based on the proton supply-and-demand argument is tentatively proposed to explain the suppression phenomenon.     

New type of matrix for matrix-assisted laser desorption mass spectrometry of polysaccharides and proteins

3-Aminoquinoline is introduced as matrix for matrix-assisted laser desorption mass spectrometry of polysaccharides and proteins. The first MALD mass spectrum of the polysaccharide inulin is reported. 3-Aminoquinoline seems to be more effective than the commonly used acidic matrix 2,5-dihydroxybenzoic acid, assisting in the desorption and ionisation of inulin. A higher sensitivity, sharper peaks and a lower background were observed. 3-Aminoquinoline is also quite effective when measuring the protein myoglobin.