On-line single droplet deposition for MALDI mass spectrometry

X. Zhang, D.A. Narcisse, K.K. Murray, On-line single droplet deposition for MALDI mass spectrometry, J. Am. Soc. Mass Spectrom.15 (2004) 1471–1477. doi:10.1016/j.jasms.2004.06.016.


A single droplet generator was coupled with a rotating ball inlet matrix-assisted laser desorption/ionization (MALDI) time of flight (TOF) mass spectrometer. Single droplets with 100 picoliter volume were ejected by a piezoelectric-actuated droplet generator and deposited onto a matrix-coated rotating stainless steel ball at atmospheric pressure. The single droplet deposit was transported to the vacuum side of the instrument where ionization was accomplished using a UV pulsed laser. Using this on-line interface, it was possible to obtain protonated molecule signal from as little as 10 fmol analyte.

Diagram of the single droplet deposition device: A, 19 mm diameter stainless steel ball; B, drive shaft; C, gasket; D, ISO 100 flange; E, ground grid; F, droplet generation electronics; G, syringe pump for matrix solution; H, syringe pump for solvent; I, capillary; J, translation stage for capillary; K, droplet generator; L, translation stage for droplet generator; M, cleaning system (Teflon holder and felt); N, peek plastic tube for waste; O, syringe pump for analyte solution.

On-line laser desorption/ionization mass spectrometry of matrix-coated aerosols

S.N. Jackson, S. Mishra, K.K. Murray, On-line laser desorption/ionization mass spectrometry of matrix-coated aerosols, Rapid Commun. Mass Spectrom.18 (2004) 2041–2045. doi:10.1002/rcm.1590.


Aerosol MALDI
Aerosol MALDI instrument at LSU, 2003

Matrix‐assisted laser desorption/ionization (MALDI) was used for the on‐line analysis of single particles. An aerosol was generated at atmospheric pressure and particles were introduced into a time‐of‐flight (TOF) mass spectrometer through a single‐stage differentially pumped capillary inlet. Prior to entering the mass spectrometer, a matrix was added to the particles using a heated saturator and condenser. A liquid matrix, 3‐nitrobenzyl alcohol (NBA), and a solid matrix, picolinic acid (PA), were used. Particles were ablated with a 351 nm excimer laser and the resulting ions were mass‐separated in a two‐stage reflectron TOF mass spectrometer. Aerosol particles containing the biomolecules erythromycin and gramicidin S were analyzed with and without the matrix addition step. The addition of NBA to the particles resulted in mass spectra that contained an intact molecular ion mass peak. In contrast, PA‐coated particles did not yield molecular ion peaks from matrix‐coated particles.

Aerosol MALDI instrument
Aerosol MALDI mass spectrometer schematic (top view)

Online CE-MALDI-TOF MS using a rotating ball interface

H. Musyimi, D.A. Narcisse, X. Zhang, W. Stryjewski, S.A. Soper, K.K. Murray, Online CE-MALDI-TOF MS using a rotating ball interface, Anal. Chem.76 (2004) 5968–5973. doi:10.1021/ac0489723.


A schematic representation of the rotating ball inlet interface for CE-MS indicating: ball A, driveshaft B, teflon gasket C, ISO-100 flange D, extraction lens E, matrix capillary F, buffer capillary G, anode and running buffer H, electrophoresis capillary I, cleaning solvent capillary J, felt pad and holder K and solvent drain L.

We report on the construction and performance of a rotating ball interface for online coupling of capillary electrophoresis (CE) to matrix-assisted laser desorption ionization (MALDI) mass spectrometry with a time-of-flight (TOF) mass analyzer. The interface is based on a rotating stainless steel ball that transports samples from atmospheric pressure to the high vacuum of the mass spectrometer for desorption and ionization. The sample is deposited directly from a 50-μm-i.d. separation capillary onto the 19-mm ball that is rotating at 0.03 to 0.3 rpm. The sample is mixed online with matrix flowing from a separate 50-μm-i.d. capillary. The sample deposit dries before it is rotated past a polymer gasket and into the laser ionization region. Cleaning of the interface is accomplished using solvent-saturated felt, which cleans the ball surface after it rotates out of the ionization chamber. On-line CE−MALDI is demonstrated, and the performance is evaluated with the analysis of a mixture of three peptides:  [Lsy8] vasopressin, substance P, and neurotensin. The rotating ball interface to MALDI-TOF MS demonstrated mass detection limit in the high femtomole range. The interface has negligible memory effect and shows no significant electrophoretic peak broadening when operated under optimized conditions.

A schematic diagram showing the top view of the chip to ball direct contact deposition: (A) sample reservoir, (B) waste reservoir, (C) buffer reservoir, (D) fluidic channel exit tip, (E) (E) ball. Panels on the right show two scanning electron micros- copy (SEM) images of (1) the brass mold insert and (2) an embossed microchip tip.
Rotating ball inlet detail
Rotating ball inlet with capillary electrophoresis microfluidic chip.

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.