Direct from polyacrylamide gel infrared laser desorption/ionization

Y. Xu, M.W. Little, D.J. Rousell, J.L. Laboy, K.K. Murray, “Direct from polyacrylamide gel infrared laser desorption/ionization,” Anal. Chem. 76 (2004) 1078–1082. doi:10.1021/ac034879n.


FT-IR-ATR spectra of gel
FT-IR-ATR spectra of dry (black solid line) and wet (dashed line) polyacrylamide gel.

The direct combination of gel electrophoresis and infrared laser desorption/ionization time-of-flight mass spectrometry has been demonstrated. We present results for infrared laser desorption and ionization mass spectrometry of peptides and proteins directly from a polyacrylamide gel without the addition of a matrix. Analyte molecules up to 6 kDa were ionized directly from a vacuum-dried sodium dodecyl sulfate-polyacrylamide gel after electrophoretic separation. Mass spectra were obtained at the wavelength of 2.94 µm, which is consistent with IR absorption by N-H and O-H stretch vibrations of water and other constituents of the gel. A 5-nmol quantity of peptide or protein was loaded per gel slot, although it was possible to obtain mass spectra from a small fraction of the gel spot. This technique shows promise for the direct identification of both parent and fragment masses of proteins contained in polyacrylamide gels.

A nitrocellulose matrix for infrared matrix-assisted laser desorption/ionization of polycyclic aromatic hydrocarbons

S.N. Jackson, S.M. Dutta, K.K. Murray, “A nitrocellulose matrix for infrared matrix-assisted laser desorption/ionization of polycyclic aromatic hydrocarbons,” Rapid Commun. Mass Spectrom.18 (2004) 228–230. doi:10.1002/rcm.1296.


IR-MALDI mass spectra of benzo[a]pyrene with a nitrocellulose matrix
IR-MALDI mass spectra of benzo[a]pyrene with a nitrocellulose matrix at the indicated wavelengths between 2.45 and 3.85 mm. The spectra are an average of five laser shots and are plotted on the same scale.

Infrared matrix-assisted laser desorption/ionization (IR-MALDI) of low molecular weight polycylic aromatic hydrocarbons (PAHs) was performed using a 3 µm mid-infrared laser and nitrocellulose as the matrix.  No other liquid or solid matrix material was added. Positive ion mass spectra obtained with the nitrocellulose matrix contain an intense molecular ion peak with no interference from matrix or alkali cations. PAH mass spectra were obtained at wavelengths between 2.44 to 3.88 mm with the best performance between 2.5 and 3.0 µm and near 3.6 µm. The relative intensities of the analyte signal at the different wavelengths is consistent with absorption of the IR radiation by the nitrocellulose with a possible additional contribution due to the absorption of residual water or solvent.

Characterization of infrared matrix-assisted laser desorption ionization samples by Fourier transform infrared attenuated total reflection spectroscopy

J.L. Laboy, K.K. Murray, “Characterization of infrared matrix-assisted laser desorption ionization samples by Fourier transform infrared attenuated total reflection spectroscopy,” Appl. Spectrosc. 58 (2004) 451–456. doi:10.1366/000370204773580301.


FT-IR ATR spectra
FT-IR ATR spectra of succinic acid in (a) methanol and (b) a 3:1 (v/v) methanol/water mixture. The arrows indicate additional peaks observed in the water-containing sample.

Fourier transform infrared attenuated total reflection (FT-IR ATR) spectroscopy was used to characterize thin films of succinic acid, a matrix compound commonly used with infrared matrix-assisted laser desorption ionization (IR-MALDI) mass spectrometry. IR spectra of succinic acid thin films deposited alone and in combination with the analyte biomolecules insulin and cytochrome c were obtained by FT-IR ATR spectroscopy. Spectra of analyte and matrix alone were similar to those obtained previously from KBr pellets, Nujol mull, or thin-film absorption, although the ATR spectra have significantly lower background interferences. Thin films deposited from mixtures of water and methanol have additional peaks compared to films deposited from a methanol solution. These additional peaks are attributed to carboxylate groups stabilized by residual water molecules. No evidence was found to suggest that residual water absorption contributes to absorption at wavelengths typically used for IR-MALDI. Absorption of energy by analyte vibrational modes with rapid energy transfer to the matrix is suggested as a contributor to desorption and ionization consistent with the FT-IR ATR results.

Characterization of Coarse Particles Formed by Laser Ablation of MALDI Matrixes

S.N. Jackson, S. Mishra, K.K. Murray, Characterization of Coarse Particles Formed by Laser Ablation of MALDI Matrixes, J. Phys. Chem. B.107 (2003) 13106–13110. doi:10.1021/jp030600v.


Aerosynamic Particle Sizer
Schematic diagram of the experimental system for laser ablation particle size measurements.

The quantity and size distribution of micrometer-sized particles ejected from thin crystalline films of organic molecules was measured with light scattering particle sizing. Four compounds that are commonly used as matrix materials in matrix-assisted laser desorption ionization (MALDI) were studied:  2,5-dihydroxybenzoic acid (DHB), sinapic acid, 4-nitroaniline, and 2-(4-hydroxyphenylazo)benzoic acid (HABA). Thin films of these matrixes were irradiated at atmospheric pressure with a 4 ns pulsed 337 nm nitrogen laser. Particulate resulting from the ablation was sampled directly into a particle sizing instrument. The mean aerodynamic diameter of the coarse particles formed at a laser fluence of 500 J/m2 was approximately 700 nm for all matrixes. This value does not include particles below 500 nm, which are not accurately measured by the particle sizing instrument. The threshold for detection of particles from the DHB matrix was found to be 300 J/m2 and it was estimated that an average of 1000 particles in the micrometer size range are ejected per laser shot. The fluence threshold and quantity of material ablated are similar to that observed for MALDI ion formation, suggesting that the role of large particle formation in this process is significant.

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

M.W. Little, J.-K. Kim, K.K. Murray, “Two-laser infrared and ultraviolet matrix-assisted laser desorption/ionization,” J. Mass Spectrom.38 (2003) 772–777. doi:10.1002/jms.494.


Matrix‐assisted laser desorption/ionization (MALDI) was performed using two pulsed lasers with wavelengths in the IR and UV regions. A 10.6 µm pulsed CO2 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 µs for matrix and guest ions and the signal decayed to zero in ∼400 µs. A mechanism is presented for enhanced UV MALDI ion yield following the IR laser pulse based on transient heating.

Two-laser IR/UV MALDI
Schematic layout of the two-laser IR/UV MALDI experiments using a linear time-of-flight mass spectrometer (TOF MS). The infrared (IR) and ultraviolet (UV) lasers are directed at the same target spot from opposite sides of the instrument. The computer (PC) controls the delay generator (DG) and digital oscilloscope (DO).
Home-built linear TOF mass spectrometer
Linear time-of-flight mass spectrometer
Linear TOF MS source chamber
Linear time-of-flight mass spectrometer source chamber

A mixed liquid matrix for infrared matrix-assisted laser desorption/ionization of oligonucleotides

S.J. Lawson, K.K. Murray, A mixed liquid matrix for infrared matrix-assisted laser desorption/ionization of oligonucleotides, Rapid Commun. Mass Spectrom.16 (2002) 1248–1250. doi:10.1002/rcm.698.


The use of glycerol as a matrix for oligonucleotide and DNA ionization by infrared matrix-assisted laser desorption/ionization (MALDI) has been the subject of much interest in recent years. Under the proper conditions, double-stranded DNA in excess of 2000 bases in length can be ionized using lasers operating in the 3 µm wavelength region of the mid-IR. However, glycerol can be a difficult matrix to use because the sample can be rapidly depleted and because the quality of the mass spectra obtained is often sensitive to the sample preparation method. The goal of the work described below is to improve the performance of IR MALDI with a glycerol matrix by mixing the glycerol with a solvent that does not absorb the mid-IR laser radiation. In this way, the deposited energy can be varied by changing the energy absorber concentration. The solvent dimethyl sulfoxide (DMSO, (CH3)2SO, also known as methyl sulfoxide) was chosen because it is miscible with water and glycerol and is a useful solvent for oligonucleotides. Since DMSO contains no NH or OH bonds, the IR absorption near 3 µm is low compared to glycerol.8 Mass spectra of oligonucleotides were obtained from frozen mixed matrices consisting of a glycerol energy absorber and DMSO solvent. These mixed matrices are simple to handle when frozen and also have the potential for use with continuous flow IR-MALDI.

Infrared laser desorption/ionization on silicon

S.H. Bhattacharya, T.J. Raiford, K.K. Murray, “Infrared laser desorption/ionization on silicon,” Anal. Chem.74 (2002) 2228–2231. doi:10.1021/ac0112972.


LDI MS of bradykinin at 2.94 µm from (a) silicon and (b) stainless steel targets
Laser desorption/ionization mass spectra of bradykinin at 2.94 µm from (a) silicon and (b) stainless steel targets.

Laser desorption/ionization from a single-crystal silicon surface was performed using a laser operating in the 3-μm region of the mid-infrared. Analyte molecules up to 6 kDa were ionized with no added matrix. As with ultraviolet desorption/ionization from porous silicon (DIOS), IR laser desorption from silicon does not produce matrix ions that can interfere with analysis of low-mass analytes. However, in contrast to UV DIOS, silicon porosity or roughness is not required for ionization using an IR laser. Mass spectra were obtained in the wavelength range between 2.8 and 3.5 μm, which is consistent with energy absorption by a hydrogen-bonded OH group. A mechanism based on desorption of adsorbed solvent molecules is postulated.

A rotating ball inlet for on-line MALDI mass spectrometry

H. Orsnes, T. Graf, H. Degn, K.K. Murray, “A rotating ball inlet for on-line MALDI mass spectrometry,” Anal. Chem. 72 (2000) 251–254. doi:10.1021/ac9905773.


Diagram of the vacuum version of the online ROBIN-MALDI probe. A, 10 mm in diameter stainless steel ball; B, drive shaft; C, gasket; D, adjustment screw; E, repeller; F, extraction grid; G, ground grid; H, capillary. The ball is rotated through the shaft, which is connected to a gear motor positioned outside the vacuum chamber.
Rotating ball inlet

The rotating ball inlet (ROBIN) is presented in a new design for on-line matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). This method uses a capillary to deliver a matrix and analyte solution to the surface of a rotating ball upon which MALDI is carried out. The ball is in contact with a polymer gasket surrounding the capillary. Sample adhering to the surface of the ball is dragged past the gasket into the vacuum of the mass spectrometer where it is irradiated by a pulsed UV laser, and the resulting ions are mass-separated in a linear time-of-flight mass spectrometer. The mechanical sample introduction prevents clogging of the vacuum interface by matrix crystals or frozen solvent. Preliminary results from now injection analysis (FIA) suggest that the new interface does not introduce a significant peak-tailing or memory effect. The system is capable of 20-30 h of continuous operation with a now rate of 2 mu L/min before cleaning of the ball is needed. With the prototype inlet, concentration detection limits are at the low micromolar level.

337 nm Matrix‐assisted laser desorption/ionization of single aerosol particles

L. He, K.K. Murray, 337 nm Matrix‐assisted laser desorption/ionization of single aerosol particles, J. Mass Spectrom. 34 (1999) 909–914. doi:10.1002/(SICI)1096-9888(199909)34:9<909::AID-JMS849>3.0.CO;2-A.


Single particle aerosol MALDI
Single particle aerosol MALDI mass spectrometer

Matrix-assisted laser desorption/ionization (MALDI) mass spectra were obtained from single particles injected directly into a time-of-flight mass spectrometer. Aerosol particles were generated at atmospheric pressure using a piezoelectric single-particle generator or a pneumatic nebulizer and introduced into the mass spectrometer through a series of narrow-bore tubes. Particles were detected by light scattering that was used to trigger a 337 nm pulsed nitrogen laser and the ions produced by laser desorption were mass separated in a two-stage reflectron time-of-flight mass spectrometer. MALDI mass spectra of single particles containing bradykinin, angiotensin II, gramicidin S, vitamin B12 or gramicidin D were obtained at mass resolutions greater than 400 FWHM. For the piezoelectric particle generator, the efficiency of particle delivery was estimated to be approximately 0.02%, and 50 pmol of sample were consumed for each mass spectrum. For the pneumatic nebulizer, mass spectra could be obtained from single particles contg. less than 100 amol of analyte, although the sample consumption for a typical mass spectrum was over 400 pmol.

Aerosol MALDI instrument
Aerosol MALDI instrument at Emory University

Infrared matrix-assisted laser desorption/ionization using OH, NH and CH vibrational absorption

J.D. Sheffer, K.K. Murray, Infrared matrix-assisted laser desorption/ionization using OH, NH and CH vibrational absorption, Rapid Commun. Mass Spectrom. 12 (1998) 1685-1690; doi:10.1002/(SICI)1097-0231(19981130)12:22<1685::AID-RCM389>3.0.CO;2-S.


MALDI of bovine insulin with succinic acid matrix
MALDI mass spectra of bovine insulin with succinic acid matrix at 2.90 and 3.50 µm.

The matrices succinic acid (butanedioc acid), caffeic acid (3,4‐dihydroxycinnamic acid), and 4‐nitroaniline were used to obtain matrix‐assisted laser desorption/ionization (MALDI) time‐of‐flight mass spectra using a Nd:YAG‐pumped optical parametric oscillator (OPO). The matrices were used to ionize the protein bovine insulin at wavelengths between 2.6 and 4.0 μm. Protonated insulin was observed between 2.81 and 3.55 μm with succinic acid, between 2.73 and 3.5 μm with caffeic acid, and in two distinct bands with 4‐nitroaniline: one from 2.85 to 3.1 μm and another between 3.4 and 3.5 μm. The best signal and mass resolution were found at approximately 2.9 μm for all matrices. The minimum laser fluence required for MALDI (threshold fluence) was compared to the IR absorption spectra of the solid matrix. We found a good correlation between IR absorption and inverse threshold fluence for the NH and CH stretch absorption bands of 4‐nitroaniline. The threshold fluence was lower than expected between 2.81 and 3.3 μm for succinic acid and between 2.73 and 2.85 μm for caffeic acid. The low threshold fluence and good MALDI performance in the 2.9 μm region can be explained by free OH group or residual water absorption.