Effects of substrate surfaces in matrix‐assisted laser desorption/ionization mass spectrometry

For matrix‐assisted laser desorption/ionization (MALDI) mass spectra, undesirable ion contamination can occur due to the direct laser excitation of substrate materials (i.e., laser desorption/ionization (LDI)) if the samples do not completely cover the substrate surfaces. In this study, comparison is made of LDI processes on substrates of indium and silver, which easily emit their own ions upon laser irradiation, and conventional materials, stainless steel and gold. A simultaneous decrease of ion intensities with the number of laser pulses is observed as a common feature. By the application of an indium substrate to the MALDI mass spectrometry of alkali salts and alkylammonium salts mixed with matrices, 2,5‐dihydroxybenzoic acid (DHB) or N‐(4‐methoxybenzylidene)‐4‐butylaniline (MBBA), the mixing of LDI processes can be detected by the presence of indium ions in the mass spectra. This method has also been found to be useful for investigating the intrinsic properties of the MALDI matrices: DHB samples show an increase in the abundance of fragment ions of matrix molecules and cesium ions with the number of laser pulses irradiating the same sample spot; MBBA samples reveal a decrease in the level of background noise with an increase in the thickness of the sample layer. Copyright © 2009 John Wiley & Sons, Ltd.     

Continuous flow infrared matrix‐assisted laser desorption electrospray ionization mass spectrometry

Continuous flow infrared matrix‐assisted laser desorption electrospray ionization (CF IR MALDESI) mass spectrometry was demonstrated for the on‐line analysis of liquid samples. Samples in aqueous solution were flowed through a 50 µm i.d. fused‐silica capillary at a flow rate of 1–6 µL/min. As analyte aqueous solution flowed through the capillary, a liquid sample bead formed at the capillary tip. A pulsed infrared optical parametric oscillator (OPO) laser with wavelength of 2.94 µm and a 20 Hz repetition rate was focused onto the capillary tip for sample desorption and ablation. The plume of ejected sample was entrained in an electrospray to form ions by MALDESI. The resulting ions were sampled into an ion trap mass spectrometer for analysis. Using CF IR MALDESI, several chemical and biochemical reactions were monitored on‐line: the chelation of 1,10‐phenanthroline with iron(II), insulin denaturation with 1,4‐dithiothreitol, and tryptic digestion of cytochrome c. Copyright © 2010 John Wiley & Sons, Ltd.     

Online deposition of nano‐aerosol for matrix‐assisted laser desorption/ionization mass spectrometry

An online nano‐aerosol sample deposition method for matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry is described in which matrix and analyte particles between 50 and 500 nm are aerodynamically focused onto a tight spot, ca. 200 µm in diameter, on the target plate under vacuum. MALDI analysis of the target is performed without additional sample preparation. The method is evaluated with insulin as the analyte and alpha‐cyano‐4‐hydroxycinnamic acid (CHCA) as the matrix. Two preparation modes are compared with conventional dried‐droplet deposition: mixture deposition where a single layer is deposited consisting of particles that contain both matrix and analyte, and layered deposition where an underlayer of matrix particles and an overlayer of analyte particles are deposited separately. Desalting is performed by adding ammonium sulfate to the solution used to generate the matrix aerosol. With mixture deposition, the optimum matrix‐to‐analyte mole ratio is about 500:1 compared with 5000:1 for the conventional dried‐droplet method. With layered deposition, the thicknesses of the matrix and analyte layers are more important determinants of the analyte signal intensity than the matrix‐to‐analyte mole ratio. Analyte signal intensities are independent of matrix layer thickness above 200 nm, and the optimum analyte signal is obtained with an analyte layer thickness of about 100 nm. Copyright © 2009 John Wiley & Sons, Ltd.     

Inline pneumatically assisted atmospheric pressure matrix‐assisted laser desorption/ionization ion trap mass spectrometry

Atmospheric pressure (AP) matrix‐assisted laser desorption/ionization (MALDI) is known to suffer from poor ion transfer efficiencies as compared to conventional vacuum MALDI (vMALDI). To mitigate these issues, a new AP‐MALDI ion source utilizing a coaxial gas flow was developed. Nitrogen, helium, and sulfur hexafluoride were tested for their abilities as ion carriers for a standard peptide and small drug molecules. Nitrogen showed the best ion transport efficiency, with sensitivity gains of up to 1900% and 20% for a peptide standard when the target plate voltage was either continuous or pulsed, respectively. The addition of carrier gas not only entrained the ions efficiently but also deflected background species and declustered analyte–matrix adducts, resulting in higher absolute analyte signal intensities and greater signal‐to‐noise (S/N) ratios. With the increased sensitivity of pneumatically assisted (PA) AP‐MALDI, the limits of detection of angiotensin I were 20 or 3 fmols for continuous or pulsed target plate voltage, respectively. For analyzing low‐mass analytes, it was found that very low gas flow rates (0.3–0.6 l min^?1) were preferable owing to increased fragmentation at higher gas flows. The analyte lability, type of gas, and nature of the extraction field between the target plate and mass spectrometer inlet were observed to be the most important factors affecting the performance of the in‐line PA‐AP‐MALDI ion source. Copyright © 2010 John Wiley & Sons, Ltd.     

Study of the cyanoacrylate fuming mechanism by matrix‐assisted laser desorption/ionization mass spectrometry

Despite cyanoacrylate fuming being widely used in the forensic science field, its mechanism is not well understood. In this study, matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry is used to study latent fingerprints that have been cyanoacrylate fumed in an attempt to gain insight into the fuming mechanism. In the negative mode mass spectrometry data, four compounds related to the polymerization of cyanoacrylate are identified and their structures are determined from accurate mass and MS/MS. A mechanism is proposed for the formation of these compounds that are regarded as intermediates in the polymerization reaction. In addition, based on the fuming of standard endogenous compounds, we suggest that fatty acids and amino acids are the major catalytic nucleophiles that initiate the polymerization reactions.     

A binary matrix for improved detection of phosphopeptides in matrix‐assisted laser desorption/ionization mass spectrometry

Application of matrix‐assisted laser‐desorption/ionization mass spectrometry (MALDI MS) to analysis and characterization of phosphopeptides in peptide mixtures may have a limitation, because of the lower ionizing efficiency of phosphopeptides than nonphosphorylated peptides in MALDI MS. In this work, a binary matrix that consists of two conventional matrices of 3‐hydroxypicolinic acid (3‐HPA) and α‐cyano‐4‐hydroxycinnamic acid (CCA) was tested for phosphopeptide analysis. 3‐HPA and CCA were found to be hot matrices, and 3‐HPA not as good as CCA and 2,5‐dihydroxybenzoic acid (DHB) for peptide analysis. However, the presence of 3‐HPA in the CCA solution with a volume ratio of 1:1 could significantly enhance ion signals for phosphopeptides in both positive‐ion and negative‐ion detection modes compared with the use of pure CCA or DHB, the most common phosphopeptide matrices. Higher signal intensities of phosphopeptides could be obtained with lower laser power using the binary matrix. Neutral loss of the phosphate group (?80 Da) and phosphoric acid (?98 Da) from the phosphorylated‐residue‐containing peptide ions with the binary matrix was decreased compared with CCA alone. In addition, since the crystal shape prepared with the binary matrix was more homogeneous than that prepared with DHB, searching for ‘sweet’ spots can be avoided. The sensitivity to detect singly or doubly phosphorylated peptides in peptide mixtures was higher than that obtained with pure CCA and as good as that obtained using DHB. We also used the binary matrix to detect the in‐solution tryptic digest of the crude casein extracted from commercially available low fat milk sample, and found six phosphopeptides to match the digestion products of casein, based on mass‐to‐charge values and LIFT TOF‐TOF spectra. Copyright © 2009 John Wiley & Sons, Ltd.     

Electrospray ionization and atmospheric pressure matrix‐assisted laser desorption/ionization mass spectrometry of antioxidants applied in lubricants

The aim of this study was to investigate the utility of ion trap mass spectrometry (ITMS) in combination with the two desorption/ionization methods, electrospray (ESI) and atmospheric pressure matrix‐assisted laser desorption/ionization (AP‐MALDI), for the detection of antioxidants which are applied in lubricants. These experiments should form the base for future investigations of antioxidants in tribologically formed thin layers on the surface of frictional systems. Seventeen different antioxidants were selected out of the group of hindered phenolic and aromatic aminic compounds. Practically all antioxidants could be characterized by positive ion ESI‐ and AP‐MALDI‐ITMS, forming various types/species of molecular ions (e.g. [M]^{+ .} , [M+H]⁺, [M+Na]⁺ or [M–2H+H]⁺). A few compounds could be analyzed by negative ion ESI‐MS, too, but none by negative ion AP‐MALDI‐MS. The influence of target materials in AP‐MALDI‐MS (gold‐ and titanium nitride (TiN)‐covered stainless steel, micro‐diamond‐covered hard metal, hand‐polished and sand‐blasted stainless steel targets) with respect to the molecular ion intensity and type of molecular ion of two selected antioxidants was evaluated. The surface properties are of particular interest because in friction tests different materials with different surface characteristics are used. However, the MS results indicate that optimal target surfaces have to be found for individual antioxidants in AP‐MALDI‐MS but in general smooth surfaces were superior to rough surfaces. Finally the gold‐covered stainless steel MALDI target provided the best mass spectra and was selected for all the antioxidants investigated. Copyright © 2009 John Wiley & Sons, Ltd.     

Quantification of hepcidin using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Hepcidin is known to be a key systemic iron‐regulatory hormone which has been demonstrated to be associated with a number of iron disorders. Hepcidin concentrations are increased in inflammation and suppressed in hemochromatosis. In view of the role of hepcidin in disease, its potential as a diagnostic tool in a clinical setting is evident. This study describes the development of a matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) assay for the quantitative determination of hepcidin concentrations in clinical samples. A stable isotope labeled hepcidin was prepared as an internal standard and a standard quantity was added to urine samples. Extraction was performed with weak cation‐exchange magnetic nanoparticles. The basic peptides were eluted from the magnetic nanoparticles using a matrix solution directly onto a target plate and analyzed by MALDI‐TOF MS to determine the concentration of hepcidin. The assay was validated in charcoal stripped urine, and good recovery (70–80%) was obtained, as were limit of quantitation data (5 nmol/L), accuracy (RE <10%), precision (CV <21%), within ‐day repeatability (CV <13%) and between‐day repeatability (CV <21%). Urine hepcidin levels were 10 nmol/mmol creatinine in healthy controls, with reduced levels in hereditary hemochromatosis (P < 0.000005) and elevated levels in inflammation (P < 0.0007). In summary a validated method has been developed for the determination of hepcidin concentrations in clinical samples. Copyright © 2009 John Wiley & Sons, Ltd.     

Imaging mass spectrometry for examining localization of polymeric composition in matrix‐assisted laser desorption/ionization samples

The localization of polymeric composition in samples prepared for matrix‐assisted laser desorption/ionization (MALDI) analysis has been investigated by imaging mass spectrometry. Various matrices and solvents were used for sample spot preparation of a polybutyleneglycol (PBG 1000). It was shown that in visibly homogeneous spots, prepared using the ‘dried droplet’ method, separation between matrix and polymer takes place. Moreover, using α‐cyano‐4‐hydroxycinnamic acid (CCA) as matrix and methanol as solvent molecular mass separation of the polymer homologues in the spots was detectable. In contrast to manually spotted samples, dry spray deposition results in homogeneous layers showing no separation effects. Copyright © 2009 John Wiley & Sons, Ltd.     

Exploring the frontiers of synthetic eumelanin polymers by high‐resolution matrix‐assisted laser/desorption ionization mass spectrometry

New trends in material science and nanotechnologies have spurred growing interest in eumelanins black insoluble biopolymers derived by tyrosinase‐catalysed oxidation of tyrosine via 5,6‐dihydroxyindole (DHI) and its 2‐carboxylic acid (DHICA). Efficient antioxidant and photoprotective actions, associated with peculiar optoelectronic properties, are recognised as prominent functions of eumelanin macromolecules within the human and mammalian pigmentary system, making them unique candidates for the realisation of innovative bio‐inspired functional soft materials, with structure‐based physical–chemical properties. An unprecedented breakthrough into the mechanism of synthetic eumelanin buildup has derived from a detailed investigation of the oxidative polymerization of DHI and its N‐methyl derivative (NMDHI) by linear and reflectron matrix‐assisted laser/desorption ionization mass spectrometry. Regular collections of oligomers of increasing masses, spanning the entire m/z ranges up to 5000 Da (>30‐mer) and 8000 Da (> 50‐mer) for the two building blocks, respectively, were disclosed. It is the first time that the in vitro polymerisation of dihydroxyindoles to form synthetic eumelanins is explored up to its high mass limits, giving at the same time information on the polymerisation mode, whether it follows a stepwise pattern (being this the conclusion in our case) or a staking sequencing of small‐sized entities. It also highlighted the influence of the N‐methyl substituent on the polymerization process; this opens the way to the production of N‐functionalized, synthetic eumelanin‐inspired soft materials, for possible future technological applications. Copyright © 2012 John Wiley & Sons, Ltd.