Mass analysis of biological macromolecules at atmospheric pressure using nonresonant femtosecond laser vaporization and electrospray ionization

A nonresonant femtosecond laser pulse, with an intensity of 10(13) Wcm(-2), vaporizes proteins and biomolecules intact, regardless of molecular structure, size or electronic structure for subsequent electrospray ionization and transfer into a mass spectrometer. Rapid, direct analysis from dried sample, aqueous solution and cellular material is demonstrated at atmospheric pressure using laser electrospray mass spectrometry (LEMS). Measurements are presented for lysozyme (14.3 kDa), hemoglobin from human blood, ovalbumin (45 kDa) from hen egg white and phospholipids from hen egg yolk. Mass analysis of biological material is performed without dilution, extraction or sample preparation, other than placing the biological material onto the sample plate.     

An automated noncontact deposition interface for liquid chromatography matrix-assisted laser desorption/ionization mass spectrometry

A new multichannel deposition system was developed for off-line liquid chromatography/matrix-assisted laser desorption/ionization mass spectrometry (LC/MALDI-MS). This system employs a pulsed electric field to transfer the eluents from multiple parallel columns directly onto MALDI targets without the column outlets touching the target surface. The deposition device performs well with a wide variety of solvents that have different viscosities, vapor pressures, polarities, and ionic strengths. Surface-modified targets were used to facilitate concentration and precise positioning of samples, allowing for efficient automation of high-throughput MALDI analysis. The operational properties of this system allow the user to prepare samples using MALDI matrixes whose properties range from hydrophilic to hydrophobic. The latter, exemplified by alpha-cyano-4-hydroxycinnamic acid, were typically processed with a multistep deposition method consisting of precoating of individual spots on the target plate, sample deposition, and sample recrystallization steps. Using this method, 50 amol of angiotensin II was detected reproducibly with high signal-to-noise ratio after LC separation. Experimental results show that there is no significant decrease in chromatographic resolution using this device. To assess the behavior of the apparatus for complex mixtures, 5 microg of a tryptic digest of the cytosolic proteins of yeast was analyzed by LC/MALDI-MS and more than 13,500 unique analytes were detected in a single LC/MS analysis.     

Use of nitrocellulose membranes for protein characterization by matrix-assisted laser desorption/ionization mass spectrometry

We present an improved method for MALDI-MS analysis of proteins that have been electroblotted onto a nitrocellulose (NC) membrane. With this approach, electroblotted proteins can be analyzed directly for intact molecular weight determination or after on-membrane digestion by dissolution of the nitrocellulose in MALDI matrix solution containing 70% acetonitrile and 30% methanol. This solution helps maintain solubility of proteins and peptides while dissolving the NC membrane, which is dissolved by 100% acetone in other protocols. On-membrane tryptic digestion using this method requires half the time of in-gel digestion and results in fewer missed cleavages and better protein coverage. For the membrane proteins studied, bovine uroplakins II and III, the protein coverage was almost twice that provided by conventional in-gel digestion, and the transmembrane domains of both uroplakins were detected only after on-membrane digestion. We also demonstrated the compatibility with MALDI-MS of a new dye, MemCode, which is specifically designed for staining NC membrane-immobilized proteins and is faster and more sensitive than Ponceau-S. Our improved on-membrane digestion protocol greatly improves the study of soluble and, particularly strikingly, integral membrane proteins by mass spectrometry.     

Quantitative analysis of cyanobacterial toxins by matrix-assisted laser desorption ionization mass spectrometry

Microcystins (MCs) are a growing problem in drinking water supplies worldwide. Common analytical techniques used to determine MC concentrations have several shortcomings, including extensive sample handling and lengthy analysis times. A simple, rapid method for quantitation of MCs by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is presented. Four potential internal standards were tested, including an 15N-labeled MC. For MC-LR in mixed standard solutions, a linear range of 0.11-5.0 microM (R2 = 0.98) was achieved, with a method detection limit (MDL) of 0.015 microM. Matrix effects due to extracted cell components decreased the MC-LR linear range slightly to 0.19-5.0 microM (R2 = 0.99), with MDL = 0.058 microM. Extensive analysis of possible internal standards indicates that nodularin was preferred over [15N]10-microcystin-YR or angiotensin I. The ionization efficiency and analyte-analyte suppression for four MCs of varying polarity are presented; the three polar congeners exhibited good ionization efficiency and acceptable levels of analyte-analyte suppression. These results indicate that MALDI-TOF MS represents a viable alternative for the quantitative measurement of MCs in field samples.     

Guanidino labeling derivatization strategy for global characterization of peptide mixtures by liquid chromatography matrix-assisted laser desorption/ionization mass spectrometry

Guanidination performed with isotopic isoforms of O-methylisourea was used in combination with reversed-phase liquid chromatography (LC) matrix-assisted laser desorption/ionization to characterize, both qualitatively and quantitatively, protein mixtures. Synthesis of (13)C- and (15)N(2)-labeled O-methylisourea sulfate produces a molecule that is 3 Da heavier than the light isotopic variant. Protein mixtures containing identical components in different concentration are pooled together following parallel derivatization. Relative quantification of protein mixtures is achieved by mass spectrometry. A difference of 3 Da allows negligible interference between the two isotopic clusters for quantification of peptides up to 1400 Da. Under these conditions, the chromatographic resolution achieved allows separation of different pairs of derivatized peptides without altering the retention time of structurally identical isotopic isoforms. Concomitant isolation of both chemically modified precursors is followed by tandem mass analysis. Activation of the ions via collisions with an inert gas produces isotopically derivatized fragment ions, which appear as doublets in the product ion spectrum. Since the modification occurs on the C-terminal lysine, ions incorporating the guanidino moiety on the C-terminus can be distinguished from those containing the original unmodified peptide N-terminus. Knowledge of the location of the proton can be beneficial to data interpretation and peptide sequencing.     

Two-step matrix application technique to improve ionization efficiency for matrix-assisted laser desorption/ionization in imaging mass spectrometry

A novel matrix application protocol for direct tissue mass spectrometry is presented. Matrix-assisted laser desorption/ionization is a popular ionization procedure for direct tissue analysis and imaging mass spectrometry. Usually, matrixes are applied by dispensing droplets through either pipettes or automated dispensing machines, or by airbrushing. These techniques are very simple, but it was difficult to obtain uniform matrix crystals on the tissue surface, and nonuniform crystals degrade the spectrum qualities. Here we report a new matrix application protocol, which is a combination of spraying and dispensing droplets, and we have succeeded in overcoming these problems in conventional matrix applications on tissue surfaces. We call our new technique the "spray-droplet method". In this technique, tiny matrix crystals formed by spraying act as seeds for crystal growth. Our technique leads to matrix spots that are filled homogeneously with minute crystals. Such matrix crystals dramatically improve peak intensity and signal-to-noise ratio. In an example on a rat brain section, the number of detectable peaks was increased and signal intensity of m/z 5440 in our method was approximately 30.6 times higher than that in conventional methods. We used this spray-droplet method with a chemical ink-jet technology for matrix deposition to succeed in MALDI imaging of signals, which were undetectable from the conventional matrix applications.     

Use of bioactive glass slides for matrix-assisted laser desorption/ionization analysis: application to microorganisms

Glass slides are widely used in high-throughput analysis and are available commercially with surfaces activated, etched, and channeled. Thin glass microscope slides are shown here to be suitable sample supports for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. As a demonstration, lectins immobilized on glass slides with activated surfaces are used to concentrate and purify agglutinated Bacillis spores. It is expected that such slides will provide a rapid, inexpensive way to evaluate and implement new strategies involving MALDI MS readout.     

Electrowetting-based microfluidics for analysis of peptides and proteins by matrix-assisted laser desorption/ionization mass spectrometry

A new technique for preparing samples for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is reported. The technique relies on electrowetting-on-dielectric (EWOD) to move droplets containing proteins or peptides and matrix to specific locations on an array of electrodes for analysis. Standard MALDI-MS reagents, analytes, concentrations, and recipes are demonstrated to be compatible with the technique. Mass spectra are comparable to those collected by conventional methods. Nonspecific adsorption of analytes to device surfaces is demonstrated to be negligible. The results suggest that EWOD may be a useful tool for automating sample preparation for high-throughput proteomics and other applications of MALDI-MS.     

Quantitative analysis of synthetic polymers using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Quantitative analyses of synthetic polymers were accomplished using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS). Many factors have hindered the development of quantitative measurement of polymers via MALDI TOF MS, e.g., laser power, matrix, cation salt, and cocrystallization. By probing the optimal conditions, two sets of polymers were studied. Fair repeatability of the samples ensures acceptable results. In set 1, two poly(ethylene glycols) with different end groups showed equal desorption/ionization efficiencies. Two synthetic polymers in set 2 with different chemical properties resulted in different MALDI responses. Good linearity was achieved by plotting the relationship between the sample concentration ratio and the total signal intensity ratio in both sets.     

Fluorinated matrix approach for the characterization of hydrophobic perfluoropolyethers by matrix-assisted laser desorption/ionization time-of-flight MS

Characterization of fluorinated polymers in MALDI is often unsuccessful because commonly used matrixes, such as 2,5-dihydroxybenzoic acid, Indole acrylic acid, alpha-cyano-4-hydroxycinnamic acid, etc., do not desorb/ionize fluorinated polymers efficiently. This could be in part attributed to the unfavorable interaction between the matrix molecules and fluorinated oligomers due to differences in their hydrophobicities. Moreover, the relative cation affinity between the matrix molecules and the fluorinated oligomers may not favor the gas-phase cationization process of the fluorinated oligomers. To overcome these limitations, fluorinated derivates of benzoic acid (pentafluorobenzoic acid) and cinnamic acid (Pentafluoro cinnamic acid) were employed for the desorption/ionization of perfluoropolyethers. Presence of fluorine atoms in the matrix might improve the interaction between the matrix and perfluoroether during the crystallization or ionization step. With a pentafluorobenzoic acid matrix, intact silver cationized oligomers were desorbed, whereas with a pentafluorocinnamic acid matrix, loss of end group was observed. This loss could be rationalized by the dissociation of the silver cationized oligomers via an ion-dipole mechanism. This work shows the possibility of characterizing yet another important class of fluorinated polymer by MALDI-TOFMS.     

Ionic liquids as matrixes for matrix-assisted laser desorption/ionization mass spectrometry

Room-temperature ionic liquids are useful as solvents for organic synthesis, electrochemical studies, and separations. We wished to examine whether their high solubalizing power, negligible vapor pressure, and broad liquid temperature range are advantageous if they are used as matrixes for UV-MALDI. Several different ionic matrixes were synthesized and tested, using peptides, proteins, and poly(ethylene glycol) (PEG-2000). All ionic liquids tested have excellent solubilizing properties and vacuum stability compared to other commonly used liquid and solid matrixes. However, they varied widely in their ability to produce analyte gas-phase ions. Certain ionic matrixes, however, produce homogeneous solutions of greater vacuum stability, higher ion peak intensity, and equivalent or lower detection limits than currently used solid matrixes. Clearly, ionic liquids and their more amorphous solid analogues merit further investigation as MALDI matrixes.     

Functional microfabricated sample targets for matrix-assisted laser desorption/ionization mass spectrometry analysis of ribonucleic acids

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful analytical tool for the structural characterization of proteins and nucleic acids. However, many proteomics or genomics methodologies that employ MALDI-MS require external sample manipulation, which limits the overall throughput of analysis. We have focused on fabricating functional MALDI sample plates that would permit the on-probe characterization of nucleic acids. Here, we present results arising from the fabrication of functional sample plates composed of poly(methyl methacrylate) (PMMA). The PMMA sample plates were fabricated by a CNC milling technique. The key structural feature of our microfabricated samples plates is the presence of individual cylindrical posts (360 microm x 360 microm), which serve as individual sample targets within the overall PMMA-based MALDI sample plate. Functionality is added to these microposts via the covalent attachment of enzymes. As an example of the applicability of these microfabricated sample plates, enzymatic digestion of ribonucleic acids was performed on probe (i.e., on the micropost) with subsequent analysis by MALDI-MS. Advantages to such an approach include a reduction in sample handling (and concomitant sample losses) and a reduction in the amount of sample required for analysis due to the small surface area of the microposts.     

Internal energy of ions generated by matrix-assisted laser desorption/ionization

To provide an objective measure of the correlation between the internal energy content of ions generated by matrix-assisted laser desorption/ionization (MALDI) and the matrix properties, a series of well-characterized benzyl-substituted benzylpyridinium salts were used as thermometer molecules (TMs). To determine the internal energy variations of analyte ions, the survival yields of TM molecular ions were measured in three different matrixes, alpha-cyano-4-hydroxycinnamic acid (CHCA), 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid, SA), and 2,5-dihydroxybenzoic acid (DHB). Statistical analysis of extensive survival yield data indicated that there were discernible differences among the studied matrixes. The experimental survival yields of the TM ions were used to calculate the unimolecular decomposition rate coefficient. Corresponding theoretical reaction rate coefficients were calculated based on the Rice-Ramsperger-Kassel-Marcus (RRKM) theory for different internal energies of the TMs. The internal energies of the ions were obtained by projecting the experimental rate coefficient values onto the theoretical curves obtained by the RRKM calculations. Molecular ions of the analytes showed decreasing survival yields and consequently increasing internal energies in the three matrixes in the following order: CHCA, SA, and DHB with "cold", "intermediate", and "hot" characteristics, respectively. Qualitatively, this could be interpreted as a significant departure from earlier observations suggesting an opposite trend. The classification as hot and cold matrixes should be further qualified by accounting for the influence of laser pulse energy and the nature of the analyte. Higher laser pulse energy led to an elevated level of energy transferred to the analyte, which in turn resulted in a diminished survival yield of the analyte molecular ion. It is quite possible that the assignment of hot and cold reverses as the analyte or the laser energy changes. These findings can help predict the outcome of postsource decay experiments and clarify the concept of hot and cold matrixes in MALDI mass spectrometry.     

Attomole peptide analysis by high-pressure matrix-assisted laser desorption/ionization Fourier transform mass spectrometry

A new high-pressure matrix-assisted laser desorption/ionization (HP-MALDI) source for FTMS has recently been described (O'Connor et al. J. Am. Soc. Mass Spectrom., in press). Improvements to the source design, including the incorporation of a new high-pressure gas channel plate, resulted in ions devoid of metastable fragmentation and also in increased sensitivity compared to the HP-MALDI prototype source design. The focus of this contribution is the evaluation of the current HP-MALDI FTMS configuration. The use of nonconductive sample surfaces, such as Parafilm and Teflon, was explored, and spectra from 30 amol of peptide applied to these surfaces were routinely obtained. In addition, the current limit of detection for this configuration is demonstrated to be 300 zmol for the phosphopeptide RRREEE(pS)EEEAA using multishot accumulation of the ions from 15 laser shots in the hexapole and 1 scan. In addition, the performance of the new HP-MALDI FTMS configuration and its potential application for high-throughput proteomics analyses are discussed.     

A technique for obtaining matrix-assisted laser desorption/ionization time-of-flight mass spectra of poorly soluble and insoluble aromatic polyamides

Wet grinding methods for obtaining matrix-assisted laser desorption/ionization time-of-flight mass spectra of poorly soluble and insoluble low molecular mass oligomers (<4600 Da) of Nomex and Kevlar are described. Optimum conditions for sample preparation are given along with a detailed analysis of the spectra obtained. Two matrix materials were employed in this analysis, 1,8-dihydroxyanthrone (dithranol) and 3-aminoquinoline with potassium trifluoroacetate used as the cationizing agent. The spectra obtained in this study are sensitive to the matrix, molar mixing ratios of matrix/polymer/cationizing agent, and the sample preparation method.     

Application of matrix-assisted laser desorption/ionization mass spectrometric imaging for photolithographic structuring

The aim of this contribution is the application of matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) in the area of photolithographic structuring. As proof of concept, this method was used to image an UV exposed negative photoresist layer, which is generally used to manufacture printed circuit boards (PCB) for electronic components. The negative photoresist layer consisting of the main component novolac, benzophenone as the active component, and the solvent tetrahydrofuran was mixed with the matrix dithranol and the salt additive LiTFA and spin-coated onto an ITO-conductive glass slide. To imprint an image on the created surface, a transparency with a printed wiring diagram was placed on top of it and irradiated by UV light for 15 min. The inspection of the efficient imprinting of the microstructure onto the photoresist layer was performed by MALDI-MSI. This unique application represents a further step toward the surface analysis of polymer films by this emerging life science imaging technique.     

Microdeposition device interfacing capillary electrochromatography and microcolumn liquid chromatography with matrix-assisted laser desorption/ionization mass spectrometry

A sample deposition device has been constructed and optimized for interfacing CEC and capillary LC columns to MALDI mass spectrometry. For CEC analysis, the device is composed of an inlet buffer reservoir and an outlet buffer reservoir connected to a matrix reservoir through a connection sleeve. The matrix reservoir is connected to a deposition capillary via another connection sleeve. CEC eluent is transported to the matrix reservoir via a capillary that is connected to the deposition capillary by the connection sleeve inside the matrix reservoir. This connection sleeve also acts as a mixing chamber, allowing the CEC eluent to be mixed with matrix prior to deposition. Complex glycan mixtures can be separated by CEC using hydrophilic-phase monolithic columns, with capillary eluent being deposited on a standard MALDI plate along with a suitable matrix solution. Thousands of discrete, highly homogeneous dots can be generated for a subsequent mass spectrometric analysis. With minor modifications, this device is also applicable to capillary LC of peptides using gradient elution. In this configuration, the outlet of the LC column is connected to a deposition capillary inside a matrix reservoir through a connection sleeve that allows mixing of the LC effluent with an appropriate matrix. The device has been evaluated with the tryptic digests of proteins.     

Automation of data collection for matrix-assisted laser desorption/ionization mass spectrometry using a correlative analysis algorithm

Automation of data collection in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry using a correlative analysis algorithm is demonstrated. This algorithm was employed to compensate for mass spectral jittering in MALDI data collection (e.g., peak shifts along the m/z axis, signal intensity deviations, etc.). Several important parameters for performing correlative analysis, such as the minimum correlation coefficient to be used and number of mass spectra to acquire prior to correlation, have been investigated and optimized. In addition, the correlation algorithm improved mass resolution of low- and high-molecular-weight compounds by as much as a factor of 4. Signal reproducibility in MALDI quantitative analysis also is improved when correlation is employed for data collection. This data collection algorithm can be used in conjunction with other instrumental optimization programs to allow for fully automated MALDI analysis, which is required for the routine applications carried out in many analytical laboratories.     

Top-down sequencing of O-glycoproteins by in-source decay matrix-assisted laser desorption ionization mass spectrometry for glycosylation site analysis

The sites of mucin-type O-glycosylation are largely unpredictable, making structural analysis by mass spectrometry (MS) indispensible. On the peptide level, a site localization and characterization of O-linked glycans in situ using tandem MS with electron-transfer dissociation or matrix-assisted laser desorption ionization (MALDI) MS with postsource decay have been reported. The top-down sequencing on the protein level by MALDI-MS is based on the in-source decay (ISD) of intact glycoproteins induced by hydrogen radical transfer from the matrix. It allows a ladder sequencing from both termini with assignment of O-glycosylation sites based on intense c-, y-, and z-type ions. The feasibility of ISD-MALDI-MS in the localization of O-glycosylation sites was demonstrated with synthetic O-glycopeptides, the tandem repeat domain of recombinant MUC1, and the natural bovine glycoproteins asialofetuin and desialylated κ-casein. Ladder sequencing of the 17-18.5 kD MUC1 hexarepeat domains revealed (1) cell-specific glycosylation site patterns on comparison of probes expressed in human HEK-293 or Drosophila S2 cells, and (2) a site-specific microheterogeneity at the Thr/Ser sites with variations of the glycan compositions from zero to four monosaccharides. Novel O-glycosylation sites in the C-terminal domains of fetuin (T334) and κ-caseinoglycopeptide (S154 and T156) were assigned, the former representing a sequence conflict with the published T154.     

End-group analysis of blue light-emitting polymers using matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry

An analytical method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been applied to provide information on the structure of a copolymer, e.g., repeat unit and end group. Seven conjugated polymers, which have been demonstrated as the active component in blue light-emitting diodes, were synthesized through Suzuki polycondensation reaction in the presence of Pd(PPh3)4 catalyst. Their molecular weights were obtained using gel permeation chromatography analysis. MALDI-TOF MS was used to investigate the structure information in detail. The proposed end-group structures were confirmed by the identity between the observed and the simulated isotopic distribution of each polymer. The results demonstrate that these synthetic polymers possess various end groups and even contain macrocycles. The catalyst Pd(PPh3)4 was found to introduce phenyl end groups via aryl-aryl exchange between the catalytic palladium intermediate and the triphenylphosphine ligand. All these results are based on the analysis of the mass spectrum data, which suggests that MALDI-TOF MS is an extraordinarily strong tool in synthetic polymer structure analysis.