On‐target separation of analyte with 3‐aminoquinoline/α‐cyano‐4‐hydroxycinnamic acid liquid matrix for matrix‐assisted laser desorption/ionization mass spectrometry

3‐Aminoquinoline/α‐cyano‐4‐hydroxycinnamic acid (3AQ/CHCA) is a liquid matrix (LM), which was reported by Kumar et al. in 1996 for matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry. It is a viscous liquid and has some advantages of durability of ion generation by a self‐healing surface and quantitative performance. In this study, we found a novel aspect of 3AQ/CHCA as a MALDI matrix, which converges hydrophilic material into the center of the droplet of analyte‐3AQ/CHCA mixture on a MALDI sample target well during the process of evaporation of water derived from analyte solvent. This feature made it possible to separate not only the buffer components, but also the peptides and oligosaccharides from one another within 3AQ/CHCA. The MALDI imaging analyses of the analyte‐3AQ/CHCA droplet indicated that the oligosaccharides and the peptides were distributed in the center and in the whole area around the center of 3AQ/CHCA, respectively. This 'on‐target separation' effect was also applicable to glycoprotein digests such as ribonuclease B. These features of 3AQ/CHCA liquid matrix eliminate the requirement for pretreatment, and reduce sample handling losses thus resulting in the improvement of throughput and sensitivity. Copyright © 2012 John Wiley & Sons, Ltd.     

Aniline/α‐cyano‐4‐hydroxycinnamic acid is a highly versatile ionic liquid for matrix‐assisted laser desorption/ionization mass spectrometry

The performance of a matrix‐assisted laser desorption/ionization (MALDI) ionic liquid matrix (ILM) consisting of α‐cyano‐4‐hydroxycinnamic acid (CHCA) and aniline (ANI) was evaluated to assess whether it could offer possible advantages over conventional matrices. Ultraviolet (UV), Fourier transform infrared (FTIR), nuclear magnetic resonance (NMR) and laser desorption/ionization mass spectrometry (LDI‐MS) experiments were carried out with the aim of confirming the structure of the ANI‐CHCA ILM. Different model analytes such as amino acids, peptides, proteins, lipids, phospholipids, synthetic polymers, and sugars were tested. Mass spectra with similar or improved signal‐to‐noise (S/N) ratio (compared to CHCA) were invariably obtained demonstrating the potential of this ILM as a general purpose matrix. Furthermore, protein identification by peptide mass fingerprinting (PMF) and database search was facilitated compared to CHCA since higher scores and increased sequence coverage were observed. Finally, a complex lipid mixture (i.e. a raw extract of a milk sample) analysed by MALDI‐MS showed improved S/N ratio, a reduced chemical noise and a limited formation of matrix‐clusters. Copyright © 2009 John Wiley & Sons, Ltd.     

A convenient purification and preconcentration of peptides with α‐cyano‐4‐hydroxycinnamic acid matrix crystals in a pipette tip for matrix‐assisted laser desorption/ionization mass spectrometry

Peptide samples derived from enzymatic in‐gel digestion of proteins resolved by gel electrophoresis often contain high amount of salts originating from reaction and separation buffers. Different methods are used for desalting prior to matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry (MS), e.g. reversed‐phase pipette tip purification, on‐target washing, adding co‐matrices, etc. As a suitable matrix for MALDI MS of peptides, α‐cyano‐4‐hydroxycinnamic acid (CHCA) is frequently used. Crystalline CHCA shows the ability to bind peptides on its surface and because it is almost insoluble in acidic water solutions, the on‐target washing of peptide samples can significantly improve MALDI MS signals. Although the common on‐target washing represents a simple, cheap and fast procedure, only a small portion of the available peptide solution is efficiently used for the subsequent MS analysis. The present approach is a combination of the on‐target washing principle carried out in a narrow‐end pipette tip (e.g. GELoader tip) and preconcentration of peptides from acidified solution by passing it through small CHCA crystals captured inside the tip on a glass microfiber frit. The results of MALDI MS analysis using CHCA‐tip peptide preconcentration are comparable with the use of homemade POROS R2 pipette tip microcolumns. Advantages and limitations of this approach are discussed. 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.     

norHarmane containing ionic liquid matrices for low molecular weight MALDI‐MS carbohydrate analysis: The perfect couple with α‐cyano‐4‐hydroxycinnamic acid

Cinnamic acid derivatives, particularly α‐cyano‐4‐hydroxycinnamic acid (E‐α‐cyano‐4‐hydroxycinnamic acid or (E)‐2‐cyano‐3‐(4‐hydroxyphenyl)prop‐2‐enoate; CHCA), have been extensively used especially for protein and peptide analysis. Together with the introduction of ionic liquid MALDI matrix (ILM) started the study of applications of IL prepared with CHCA and a counter organic base (ie, aliphatic amines) in which CHCA moiety is the chromophore responsible of UV‐laser absorption. Despite the extensive studies of norharmane (9H‐pyrido[3,4‐b]indole; nHo) applications as matrix and its peculiar basic properties in the ground and electronic excited state, nHo containing ILM was never tested in MALDI‐MS experiments. This pyrido‐indole compound was introduced as MALDI matrix 22 years ago for different applications including low molecular weight (LMW) carbohydrates (neutral, acidic, and basic carbohydrates). These facts encouraged us to use it as a base, for the first time, for ILM preparation. As a rational design of new IL MALDI matrices, E‐α‐cyanocinnamic acid.nHo and E‐cinnamic acid.nHo were prepared and their properties as matrices studied. Their performance was compared with that of (a) the corresponding IL prepared with butylamine as basic component, (b) the corresponding crystalline E‐α‐cyanocinnamic and E‐cinnamic acid, and (c) the classical crystalline matrices (2,5‐dihydroxybenzoic acid, DHB; nHo) used in the analysis of neutral/sulfated carbohydrates. The IL DHB.nHo was tested, too. Herein, we demonstrate the outstanding performance for the IL CHCA.nHo for LMW carbohydrate in positive and negative ion mode (linear and reflectron modes). Sulfated oligosaccharides were detected in negative ion mode, and although the dissociation of sulfate groups was not completely suppressed the relative intensity (RI) of [M ? Na]^? peak was quite high. Additionally, to better understand the quite different performance of each IL tested as matrix, the physical and morphological properties in solid state were studied (optical image; MS image).     

A second‐generation ionic liquid matrix‐assisted laser desorption/ionization matrix for effective mass spectrometric analysis of biodegradable polymers

A second generation ionic liquid matrix (ILM), N,N‐diisopropylethylammonium α‐cyano‐4‐hydroxycinnamate (DEA‐CHCA), was developed for the characterization of polar biodegradable polymers. It is compared with five solid matrices typically used for the characterization of these polymers and one other new ILM. It is shown that use of the ILM, DEA‐CHCA, allows maximum signal with minimum laser intensity which minimizes polymer degradation. In these conditions, the DEA‐CHCA ILM is able to assist in the ionization of analytes in an efficient but soft manner. These qualities produce spectra that allow an accurate and sensitive determination of the number average molecular weights, weight average m.w., and polydispersity index of labile polar polymers. With such polymers, many solid matrices produce spectra showing extensive polymer degradation leading to the underestimation of molecular weights. The distribution of intact analyte peaks obtained with the ILM DEA‐CHCA allows for identification of the fine structure of complex copolymers. ILMs were much less susceptible to effects of extraction delay times on molecular weight determination than were solid matrices. The liquid nature of the matrix is an important reason for the outstanding results obtained for labile analyte polymers. No comparable results could be obtained with any known solid matrices or other ILMs. In many cases, the manufacturers' listed molecular weights and polydispersity measurements for biodegradable polymers are determined by size‐exclusion chromatography and the data obtained by that method may differ considerably from the high‐precision matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) results presented here. Copyright © 2009 John Wiley & Sons, Ltd.     

On‐chip solid‐phase extraction pre‐concentration/focusing substrates coupled to atmospheric pressure matrix‐assisted laser desorption/ionization ion trap mass spectrometry for high sensitivity biomolecule analysis

Atmospheric pressure matrix‐assisted laser desorption/ionization (AP‐MALDI) has proven a convenient and rapid method for ion production in the mass spectrometric (MS) analysis of biomolecules. AP‐MALDI and electrospray ionization (ESI) sources are easily interchangeable in most mass spectrometers. However, AP‐MALDI suffers from less‐than‐optimal sensitivity due to ion losses during transport from the atmosphere into the vacuum of the mass spectrometer. Here, we study the signal‐to‐noise ratio (S/N) gains observed when an on‐chip dynamic pre‐concentration/focusing approach is coupled to AP‐MALDI for the MS analysis of neuropeptides and protein digests. It was found that, in comparison with conventional AP‐MALDI targets, focusing targets showed (1) a sensitivity enhancement of approximately two orders of magnitude with S/N gains of 200–900 for hydrophobic substrates, and 150–400 for weak cation‐exchange (WCX) substrates; (2) improved detection limits as low as 5 fmol/µL for standard peptides; (3) significantly reduced matrix background; and (4) higher inter‐day reproducibility. The improved sensitivity allowed successful tandem mass spectrometric (MS/MS) sequencing of dilute solutions of a derivatized tryptic digest of a protein standard, and enabled the first reported AP‐MALDI MS detection of neuropeptides from Aedes aegypti mosquito heads. 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.     

Rapid and specific high‐performance liquid chromatography for the in vitro quantification of d‐Lys6–GnRH in a microemulsion‐type formulation in the presence of peptide oxidation products

A high‐performance liquid chromatography (HPLC) method for assay of d ‐Lys⁶–GnRH contained in a microemulsion‐type formulation is described. The peptide is extracted from the microemulsion matrix and quantified using a two‐step gradient method. Separation from microemulsion compounds and potential peptide oxidation products was achieved on a Jupiter C₁₈ column at 40°C, using a gradient of 10–35% CH₃CN for peptide elution. The correlation of peak intensity measured at 220 nm and peptide concentration was linear over the range 2.5–60 µg/mL with a correlation coefficient of 0.9997 and a y‐intercept not significantly different from zero (p > 0.05). Intraday and interday variability of the assay was less than 5% for multiple injections of samples containing 7.5, 30 and 60 µg/mL. The lower limit of quantitation was calculated to be 0.38 µg/mL, and the lower limit of detection was 0.13 µg/mL. The assay was applied to samples that were stressed under physiological conditions (37°C, pH 7.4) over 4 days. Three degradation peaks were well resolved from the parent peptide, demonstrating the selectivity of the assay. Off‐line MALDI TOF mass spectrometry was applied to identify these degradation species as oxidation products of the peptide. Copyright © 2009 John Wiley & Sons, Ltd.     

Controlling matrix suppression for matrix‐assisted laser desorption/ionization analysis of small molecules

The need for high‐throughput methodologies providing both qualitative and quantitative information has grown substantially in the pharmaceutical laboratory in recent years. Currently, tandem mass spectrometry (MS/MS) using quadrupole technology offers analysis in the minutes time scale. The use of matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) offers the advantage of speed and automation and enables analysis in the seconds time scale with accurate mass capabilities that are not typically found in quadrupole MS/MS. However, one of the limitations of MALDI for the analysis of small molecules is the abundance of interfering matrix peaks in the low molecular weight region of the mass spectrum. Described herein is an evaluation of a pre‐prepared MALDI target plate that has been coated with a thin layer of α‐cyano‐4‐hydroxycinnamic acid (CHCA) and nitrocellulose. This modified plate has been shown to suppress or eliminate CHCA matrix signals without any significant loss of analyte sensitivity when compared with analysis of the same sample using an unmodified target plate. Copyright © 2004 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.     

An improved sample preparation method for the sensitive detection of peptides by MALDI‐MS

We describe here an optimization study of the sample preparation conditions for sensitive detection of peptides by matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS). Among many factors in the conditions, we varied the percent acetonitrile in the peptide solution, the percent acetonitrile in the matrix solution and the α‐cyano‐4‐hydroxycinnamic acid (CHCA) concentration in the matrix solution. CHCA was chosen because it is the most frequently used matrix for analyzing peptides. The well‐established dried‐droplet method was employed for sample deposition. The examined range of the concentration of CHCA was from 0.01 to 10 mg/ml, and the MeCN content of the solvent for matrix/analyte was 10% to 50%. The indicator for the detection sensitivity was the S/N ratio of the peaks of peptides used. Highly increased sensitivity (100‐ to 1000‐fold) was observed for the optimal CHCA concentration of 0.1 mg/ml in 20% MeCN/0.1% aq. trifluoroacetic acid (TFA), as compared with the conventional concentration (10 mg/ml) in 50% MeCN/0.1% aq. TFA. For example, the limit of detection of human ACTH 18–39 was 10 amol/well for the optimal condition but 10 fmol/well for the conventional condition. The optimal condition (0.1 mg/ml CHCA in 20% MeCN/0.1% aq. TFA) was verified with five model peptides and provided significant improvement in sensitivity (by two to three orders of magnitude) compared with the conventional conditions. Optimizing the CHCA concentration and solvent composition significantly improved the detection sensitivity in the analysis of peptides by MALDI‐MS. Copyright © 2013 John Wiley & Sons, Ltd.     

Quantitative reproducibility of mass spectra in matrix‐assisted laser desorption ionization and unraveling of the mechanism for gas‐phase peptide ion formation

In a previous study on matrix‐assisted laser desorption ionization (MALDI) of peptides using α‐cyano‐4‐hydroxycinnamic acid (CHCA) as a matrix, we found that the patterns of single‐shot spectra obtained under different experimental conditions became similar upon temperature selection. In this paper, we report that absolute ion abundances are also similar in temperature‐selected MALDI spectra, even when laser fluence is varied. The result that has been obtained using CHCA and 2,5‐dihydroxybenzoic acid as matrices is in disagreement with the hypothesis of laser‐induced ionization of matrix as the mechanism for primary ion formation in MALDI. We also report that the total number of ions in such a spectrum is unaffected by the identity, concentration and number of analytes, i.e. it is the same as that in the spectrum of pure matrix. We propose that the generation of gas‐phase ions in MALDI can be explained in terms of two thermal reactions, i.e. the autoprotolysis of matrix molecules and the matrix‐to‐analyte proton transfer, both of which are in quasi‐equilibrium in the early matrix plume. Copyright © 2013 John Wiley & Sons, Ltd.     

Evaluation of matrix‐assisted laser desorption/ionization (MALDI) preparation techniques for surface characterization of intact Fusarium spores by MALDI linear time‐of‐flight mass spectrometry

Unambiguous identification of mycotoxin‐producing fungal species as Fusarium is of great relevance to agriculture and the food‐producing industry as well as in medicine. Protein profiles of intact fungal spores, such as Penicillium, Aspergillus and Trichoderma, derived from matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) were shown to provide a rapid and straightforward method for species identification and characterization. In this study, we applied this approach to five different Fusarium spp. strains which are known to affect the growth of different grain plants. To obtain a suitable MALDI matrix system and sample preparation method, thin‐layer, dried‐droplet and sandwich methods and several MALDI matrices, namely CHCA, DHB, FA, SA and THAP dissolved in various solvent mixtures (organic solvents such as ACN, MeOH, EtOH and iPrOH and for the aqueous phase water and 0.1% TFA), were evaluated in terms of mass spectrometric pattern and signal intensities. The most significant peptide/protein profiles were obtained with 10 mg ferulic acid (FA) in 1 mL ACN/0.1% TFA (7:3, v/v) used as matrix system. Mixing the spores with the matrix solution directly on the MALDI target (dried‐droplet technique) resulted in an evenly distributed spores/matrix crystal layer, yielding highly reproducible peptide/protein profiles from the spore surfaces. Numerous abundant ions throughout the investigated m/z range (m/z 1500–15 000) could be detected. Differences in the obtained mass spectral patterns allowed the differentiation of spores of various Fusarium species. Copyright © 2009 John Wiley & Sons, Ltd.     

Novel approach to enhance sensitivity in surface‐assisted laser desorption/ionization mass spectrometry imaging using deposited organic‐inorganic hybrid matrices

Sample pretreatment is key to obtaining good data in matrix‐assisted laser desorption/ionization mass spectrometry imaging (MALDI‐MSI). Although sublimation is one of the best methods for obtaining homogenously fine organic matrix crystals, its sensitivity can be low due to the lack of a solvent extraction effect. We investigated the effect of incorporating a thin film of metal formed by zirconium (Zr) sputtering into the sublimation process for MALDI matrix deposition for improving the detection sensitivity in mouse liver tissue sections treated with olanzapine. The matrix‐enhanced surface‐assisted laser desorption/ionization (ME‐SALDI) method, where a matrix was formed by sputtering Zr to form a thin nanoparticle layer before depositing MALDI organic matrix comprising α‐cyano‐4‐hydroxycinnamic acid (CHCA) by sublimation, resulted in a significant improvement in sensitivity, with the ion intensity of olanzapine being about 1800 times that observed using the MALDI method, comprising CHCA sublimation alone. When Zr sputtering was performed after CHCA deposition, however, no such enhancement in sensitivity was observed. The enhanced sensitivity due to Zr sputtering was also observed when the CHCA solution was applied by spraying, being about twice as high as that observed by CHCA spraying alone. In addition, the detection sensitivity of these various pretreatment methods was similar for endogenous glutathione. Given that sample preparation using the ME‐SALDI‐MSI method, which combines Zr sputtering with the sublimation method for depositing an organic matrix, does not involve a solvent, delocalization problems such as migration of analytes observed after matrix spraying and washing with aqueous solutions as sample pretreatment are not expected. Therefore, ME‐Zr‐SALDI‐MSI is a novel sample pretreatment method that can improve the sensitivity of analytes while maintaining high spatial resolution in MALDI‐MSI.     

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.     

Negative ion production from peptides and proteins by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Negative ion production from peptides and proteins was investigated by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry. Although most research on peptide and protein identification with ionization by MALDI has involved the detection of positive ions, for some acidic peptides protonated molecules are not easily formed because the side chains of acidic residues are more likely to lose a proton and form a deprotonated species. After investigating more than 30 peptides and proteins in both positive and negative ion modes, [M–H]⁻ ions were detected in the negative ion mode for all peptides and proteins although the matrix used was 2,5‐dihydroxybenzoic acid (DHB), which is a good proton donor and favors the positive ion mode production of [M+H]⁺ ions. Even for highly basic peptides without an acidic site, such as myosin kinase inhibiting peptide and substance P, good negative ion signals were observed. Conversely, gastrin I (1‐14), a peptide without a highly basic site, will form positive ions. In addition, spectra obtained in the negative ion mode are usually cleaner due to absence of alkali metal adducts. This can be useful during precursor ion isolation for MS/MS studies. Copyright © 2008 John Wiley & Sons, Ltd.     

Atmospheric pressure infrared (10.6 µm) laser desorption electrospray ionization (IR‐LDESI) coupled to a LTQ Fourier transform ion cyclotron resonance mass spectrometer

We present the implementation of a CO₂ laser emitting infrared laser irradiation at 10.6 µm onto the versatile atmospheric pressure ionization platform. Infrared laser desorption electrospray ionization (IR‐LDESI) is demonstrated from liquid‐state samples at atmospheric pressure with and without ESI postionization. Multiply charged proteins ranging in molecular mass from 8.6 to 17 kDa were detected from liquid‐state samples without the addition of matrix. Copyright © 2009 John Wiley & Sons, Ltd.     

Estimation of peptide N–Cα bond cleavage efficiency during MALDI‐ISD using a cyclic peptide

Matrix‐assisted laser desorption/ionization in‐source decay (MALDI‐ISD) induces N–C_α bond cleavage via hydrogen transfer from the matrix to the peptide backbone, which produces a c′/z? fragment pair. Subsequently, the z? generates z′ and [z + matrix] fragments via further radical reactions because of the low stability of the z?. In the present study, we investigated MALDI‐ISD of a cyclic peptide. The N–C_α bond cleavage in the cyclic peptide by MALDI‐ISD produced the hydrogen‐abundant peptide radical [M + 2H]⁺? with a radical site on the α‐carbon atom, which then reacted with the matrix to give [M + 3H]⁺ and [M + H + matrix]⁺. For 1,5‐diaminonaphthalene (1,5‐DAN) adducts with z fragments, post‐source decay of [M + H + 1,5‐DAN]⁺ generated from the cyclic peptide showed predominant loss of an amino acid with 1,5‐DAN. Additionally, MALDI‐ISD with Fourier transform‐ion cyclotron resonance mass spectrometry allowed for the detection of both [M + 3H]⁺ and [M + H]⁺ with two ¹³C atoms. These results strongly suggested that [M + 3H]⁺ and [M + H + 1,5‐DAN]⁺ were formed by N–C_α bond cleavage with further radical reactions. As a consequence, the cleavage efficiency of the N–C_α bond during MALDI‐ISD could be estimated by the ratio of the intensity of [M + H]⁺ and [M + 3H]⁺ in the Fourier transform‐ion cyclotron resonance spectrum. Because the reduction efficiency of a matrix for the cyclic peptide cyclo(Arg‐Gly‐Asp‐D‐Phe‐Val) was correlated to its tendency to cleave the N–C_α bond in linear peptides, the present method could allow the evaluation of the efficiency of N–C_α bond cleavage for MALDI matrix development. Copyright © 2016 John Wiley & Sons, Ltd.     

Characterization of [peptide+(Ag)n]+ complexes using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Silver ion complexes of peptides [M + (Ag)_n]⁺, M = angiotensin I or substance P where n = 1–8 and 17–23 for angiotensin I and n = 1–5 for substance P, are identified and characterized using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS). The Ag⁺ coordination number exceeds the number of available amino acid residues in angiotensin I whereas the number of observed complexes in substance P is less than the number of amino acid residues in it. The larger coordination number of angiotensin I with Ag⁺ indicates the simultaneous binding of several Ag⁺ ions to the amino acid residue present in it. The lower number of observed complexes in substance P suggests the binding of two or more residues to one Ag⁺ ion. The presence of trifluoroacetic acid in the peptide samples reduces the Ag⁺ coordination ability in both the peptides which indicates that the basic residues in it are already protonated and do not participate in the Ag⁺‐binding process. The Ag⁺ ion also forms a complex with the α‐cyano‐4‐hydroxycinnamic acid (CHCA) matrix and is observed in the MALDI mass spectra and the formation of [CHCA + Ag]⁺, [CHCA + AgNO₃]⁺ and [(CHCA)₂ + Ag]⁺ ions is due to the high binding affinity of Ag⁺ to the CN group of CHCA. Copyright © 2010 John Wiley & Sons, Ltd.