Matrix pre-coated MALDI MS targets for small molecule imaging in tissues

A new sample preparation method for MALDI tissue imaging has been developed for the analysis of low molecular weight compounds that employs matrix pre-coated MALDI targets. Tissue sections need only to be transferred onto the pre-coated target before analysis for fast and easy sample preparation. Pre-coated targets have a homogenous matrix coating with uniform crystals of approximately 1–2 μm and do not require solvents that may lead to analyte delocalization within a tissue section. We report here the use of matrix pre-coated targets for imaging of lipids, peptides, and pharmaceuticals in tissues.     

MALDI‐based intact spore mass spectrometry of downy and powdery mildews

Fast and easy identification of fungal phytopathogens is of great importance in agriculture. In this context, matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) has emerged as a powerful tool for analyzing microorganisms. This study deals with a methodology for MALDI‐TOF MS‐based identification of downy and powdery mildews representing obligate biotrophic parasites of crop plants. Experimental approaches for the MS analyses were optimized using Bremia lactucae, cause of lettuce downy mildew, and Oidium neolycopersici, cause of tomato powdery mildew. This involved determining a suitable concentration of spores in the sample, selection of a proper MALDI matrix, looking for the optimal solvent composition, and evaluation of different sample preparation methods. Furthermore, using different MALDI target materials and surfaces (stainless steel vs polymer‐based) and applying various conditions for sample exposure to the acidic MALDI matrix system were investigated. The dried droplet method involving solvent evaporation at room temperature was found to be the most suitable for the deposition of spores and MALDI matrix on the target and the subsequent crystallization. The concentration of spore suspension was optimal between 2 and 5 × 10⁹ spores per ml. The best peptide/protein profiles (in terms of signal‐to‐noise ratio and number of peaks) were obtained by combining ferulic and sinapinic acids as a mixed MALDI matrix. A pretreatment of the spore cell wall with hydrolases was successfully introduced prior to MS measurements to obtain more pronounced signals. Finally, a novel procedure was developed for direct mass spectra acquisition from infected plant leaves. Copyright © 2012 John Wiley & Sons, Ltd.     

Laser-Induced Oxidation of Cholesterol Observed During MALDI-TOF Mass Spectrometry

Conditions for the detection of three odd-electron cholesterol oxidation peaks were determined and these peaks were shown to be artifacts of the matrix-assisted laser desorption time of flight (MALDI-TOF) process. Matrix choice, solvent, laser intensity and cholesterol concentration were systematically varied to characterize the conditions leading to the highest signals of the radical cation peaks, and it was found that initial cholesterol solution concentration and resultant density of solid cholesterol on the MALDI target were important parameters in determining signal intensities. It is proposed that hydroxyl radicals, generated as a result of laser irradiation of the employed 2, 5-dihydroxybenzoic acid (DHB) matrix, initiate cholesterol oxidation on the MALDI target. An attempt to induce the odd-electron oxidation peaks by means of adding an oxidizing agent succeeded using an acetonitrile solution of DHB, cholesterol, and cumene hydroperoxide. Moreover, addition of free radical scavengers reduced the abundances of some oxidation products under certain conditions. These results are consistent with the mechanism of oxidation proposed herein involving laser-induced hydroxyl radical production followed by attack on neutral cholesterol. Hydroxyl radical production upon irradiation of dithranol matrix may also be responsible for generation of the same radical peaks observed from cholesterol in dithranol by an analogous mechanism.     

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.     

A solid-phase bioreactor with continuous sample deposition for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

We report the development of a solid‐phase proteolytic digestion and continuous deposition microfluidic chip platform for low volume fraction collection and off‐line matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry. Tryptic peptides were formed in an on‐chip bioreactor and continuously deposited onto a MALDI target plate using a motor‐driven xyz stage. The bioreactor consisted of a 4 cm × 200 µm × 50 µm microfluidic channel with covalently immobilized trypsin on an array of 50 µm diameter micropost structures with a 50 µm edge‐to‐edge inter‐post spacing. A 50 µm i.d. capillary tube was directly attached to the end of the bioreactor for continuous sample deposition. The MALDI target plate was modified by spin‐coating a nitrocellulose solution containing a MALDI matrix on the surface prior to effluent deposition. Protein molecular weight standards were used for evaluating the performance of the digestion and continuous deposition system. Serpentine sample traces 200 µm wide were obtained with a 30 fmol/mm quantity deposition rate and a 3.3 nL/mm volumetric deposition rate. Copyright © 2011 John Wiley & Sons, Ltd.     

Sublimation as a method of matrix application for mass spectrometric imaging

Common organic matrix-assisted laser desorption/ionization (MALDI) matrices, 2,5-dihydroxybenzoic acid, 3,5-dimethoxy-4-hydroxycinnamic acid, and alpha-cyano-4-hydroxycinnamic acid, were found to undergo sublimation without decomposition under conditions of reduced pressure and elevated temperature. This solid to vapor-phase transition was exploited to apply MALDI matrix onto tissue samples over a broad surface in a solvent-free application for mass spectrometric imaging. Sublimation of matrix produced an even layer of small crystals across the sample plate. The deposition was readily controlled with time, temperature, and pressure settings and was highly reproducible from one sample to the next. Mass spectrometric images acquired from phospholipid standards robotically spotted onto a MALDI plate yielded a more intense, even signal with fewer sodium adducts when matrix was applied by sublimation relative to samples where matrix was deposited by an electrospray technique. MALDI matrix could be readily applied to tissue sections on glass slides and stainless steel MALDI plate inserts as long as good thermal contact was made with the condenser of the sublimation device. Sections of mouse brain were coated with matrix applied by sublimation and were imaged using a Q-q-TOF mass spectrometer to yield mass spectral images of very high quality. Image quality is likely enhanced by several features of this technique including the microcrystalline morphology of the deposited matrix, increased purity of deposited matrix, and evenness of deposition. This inexpensive method was reproducible and eliminated the potential for spreading of analytes arising from solvent deposition during matrix application.     

Reverse thin layer method for enhanced ion yield of oligosaccharides in matrix‐assisted laser desorption/ionization

A sample preparation method that is suitable for sensitive detection of underivatized oligosaccharides by matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) has been investigated. As compared with the conventional dried‐droplet or ethanol (EtOH) recrystallization method, superior mass spectra in terms of ion yield and signal‐to‐noise (s/n) ratio were obtained when methanol (MeOH) was used as a solvent for the mixture of matrix and oligosaccharides. Based on these results, a new sample preparation method, named the ‘reverse thin layer method’, was developed. This method comprises two steps: first, complete drying of the oligosaccharide solution on the MALDI target plate; and second, deposition of the matrix dissolved in a small amount of MeOH. Using this method, a relatively homogeneous matrix crystal was generated and higher yields of both positive and negative ions were obtained from oligosaccharides compared with conventional methods. Notably, the method can be applied to various matrices including both solid and liquid matrices. Copyright © 2009 John Wiley & Sons, Ltd.     

The effect of sodium dodecyl sulfate and anion‐exchange silica gel on matrix‐assisted laser desorption/ionization mass spectrometric analysis of proteins

Sodium dodecyl sulfate (SDS), an anionic surfactant, is widely used in peptide and protein sample preparation. When the sample is analyzed by matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS), this surfactant can often cause signal suppression. We have previously reported an on‐probe sample preparation method using a suspension of anion‐exchange silica gel and sinapinic acid (i.e., gel‐SA suspension) as a matrix, thereby greatly improving the MALDI signal detection of the protein solutions containing SDS. In this study, we found that a certain amount of SDS enhanced the MALDI signal intensity for protein samples. This effect was also observed when using sodium decyl sulfate and sodium tetradecyl sulfate instead of SDS. Furthermore, this on‐probe sample preparation method using both SDS and the gel‐SA suspension improved the detection limit of protein samples in the MALDI‐MS analysis by about ten‐fold as compared to that of protein samples without SDS and the gel‐SA suspension. This method can be applied not only to the MALDI‐MS analysis of samples containing SDS, but also to the examination of proteins at femtomole levels or insoluble proteins such as membrane proteins. Copyright © 2009 John Wiley & Sons, Ltd.     

Effective detection of peptides containing cysteine sulfonic acid using matrix-assisted laser desorption/ionization and laser desorption/ionization on porous silicon mass spectrometry

Cysteine sulfonic acid-containing peptides, being typical acidic peptides, exhibit low response in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. In this study, matrix conditions and the effect of diammonium hydrogencitrate (DAHC) as additive were investigated for ionization of cysteine sulfonic acid-containing peptides in MALDI. A matrix-free ionization method, desorption/ionization on porous silicon (DIOS), was also utilized to evaluate the effect of DAHC. When equimolar three-component mixtures of peptides carrying free cysteine, cysteine sulfonic acid, and carbamidomethyl cysteine were measured by MALDI using a common matrix, alpha-cyano-4-hydroxycinnamic acid (CHCA), no signal corresponding to cysteine sulfonic acid-containing peptide could be observed in the mass spectrum. However, by addition of DAHC to CHCA, the peaks of cysteine sulfonic acid-containing peptides were successfully observed, as well as when using 2,4,6-trihydroxyacetophenone (THAP) and 2,6-dihydroxyacetophenone with DAHC. In the DIOS mass spectra of these analytes, the use of DAHC also enhanced the peak intensity of the cysteine sulfonic acid-containing peptides. On the basis of studies with these model peptides, tryptic digests of oxidized peroxiredoxin 6 were examined as a complex peptide mixture by MALDI and DIOS. In MALDI, the peaks of cysteine sulfonic acid-containing peptides were observed when using THAP/DAHC as the matrix, but this was not so with CHCA. In DIOS, the signal from cysteine sulfonic acid-containing peptides was suppressed; however, the use of DAHC significantly enhanced the signal intensity with an increase in the number of observed peptides and increased signal-to-noise ratio in the DIOS spectra. The results show that DAHC in the matrix or on the DIOS chip decreases discrimination and suppression effects in addition to suppressing alkali-adduct ions, which leads to a beneficial effect on protonation of peptides containing cysteine sulfonic acid.     

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.     

Comparison of Novel Sampling Methods for the Analysis of Capillary Electrophoresis Fractions by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry

Two methods for the collection of capillary electrophoresis fractions for analysis by matrix-assisted laser desorption/ionization have been investigated; the first using a novel ion-porous joint design for dropwise fraction collection and the second, a process of electrospray deposition. Sensitivity, shot-to-shot reproducibility, effect of matrix preparation and influence of buffer additives encountered in micellar electrokinetic capillary chromatography (MECC) have been examined using a standard peptide, methionine enkephalin. The direct collection of droplets from the capillary outlet was found to give a superior signal intensity, whereas the electrospray method, in its first stage of development, suffered through dilution effects and excessively wide sample distribution. The target compound was successfully collected by electrospray deposition and results indicated that signal intensity and sample homogeneity would be enhanced if the sprayed output could be collected into the sample wells in its entirety. The advantage of using electrospray deposition for the preparation of matrix layers followed by direct droplet collection onto the electrosprayed matrix was clearly demonstrated by improvements in both sensitivity and shot-to-shot variance in signal intensity across the sample well compared with collection onto spotted matrix. In addition, compatibility of the droplet collection method with MECC was illustrated by the recovery of the compound of interest from a buffer containing 30 mm sodium dodecyl sulphate. © 1997 John Wiley & Sons, Ltd.     

Characterization of unknown compounds from stainless steel plates in matrix-assisted laser desorption/ionization mass spectrometry

Peaks originating from unknown compounds on stainless steel plates used in matrix-assisted laser desorption/ionization (MALDI) mass spectrometers are observed around m/z 304.3, 332.3, 360.4, and 388.4 regardless of the matrix and/or solvent, and are even observed with bare plates. These peaks were characterized using three different types of MALDI-MS instrumentation: MALDI-TOF MS, MALDI-TOF/TOF MS, and MALDI-FTMS. The fragmentation data from MALDI-TOF/TOF MS and accurate mass determination by MALDI-FTMS enabled identification of the chemical formulae and structures. The unknown compounds are, in fact, likely benzylalkylmethylammonium salts, as confirmed by closely matching fragmentation patterns with a commercially available benzalkonium chloride.     

Enhanced detection of phosphopeptides in matrix-assisted laser desorption/ionization mass spectrometry using ammonium salts

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has been used successfully to detect phosphorylation sites in proteins. Applications may be limited by the low response of phosphopeptides compared to nonphosphorylated peptides in MALDI MS. The addition of ammonium salts to the matrix/analyte solution substantially enhances the signal for phosphopeptides. In examples shown for equimolar mixtures, the phosphorylated peptide peaks become the largest peaks in the spectrum upon ammonium ion addition. This can allow for the identification of phosphopeptides in an unfractionated proteolytic digestion mixture. Sufficient numbers of protonated phosphopeptides can be generated such that they can be subjected to postsource decay analysis, in order to confirm the number of phosphate groups present. The approach works well with the common MALDI matrices such as α-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid, and with ammonium salts such as diammonium citrate and ammonium acetate.     

Automated coupling of capillary-HPLC to matrix-assisted laser desorption/ionization mass spectrometry for the analysis of small molecules utilizing a reactive matrix

This study describes the application of a novel, reactive matrix for the mass spectral analysis of steroids by capillary-high performance liquid chromatography (capillary-HPLC) coupled to matrix-assisted laser desorption/ionization (MALDI). The mass spectral analysis of steroids was accomplished after fully automated peak deposition of chromatographic peaks onto MALDI targets. The seven corticosteroids used as test compounds were: triamcinolone, prednisone, cortisone, fludrocortisone, dexamethasone, deoxycorticosterone, and budesonide. They were separated using a PepMap C₁₈ (3 m particle size, 100 Å pore width) column at five different concentration levels of 25, 15, 7.5, 2.5 and 1 ng/L, and the peaks were detected at a wavelength of 237 nm. The column effluent was mixed with 2,4-dinitrophenylhydrazine (DNPH) directly following the UV detector. The chromatographic peaks were then deposited onto the MALDI target with a robotic micro-fraction collector triggered by the UV detector signals. A special hydrophobic surface coating allowed the deposition of up to 4 L (up to 90 % of the chromatographic peak volume) onto one sample spot. The compounds were then identified by MALDI mass spectrometry. Depending on the nature of the analyte, radical cations ([M]^+.) and sodium adduct ions ([M+Na]⁺) of the steroids as well as protonated steroid-dinitrophenylhydrazone derivatives ([M_D+H]⁺) were detected in positive ion mode. The detection limits were between 0.5 and 15 ng injected with capillary-HPLC-MALDI-TOF-MS and between 0.3 and 3 ng on target with MALDI-TOF when deposited manually.     

Direct sample fraction deposition using electrospray in narrow-bore size-exclusion chromatography/matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for polymer characterization

A direct sample fraction deposition method was developed for off-line size-exclusion chromatography (SEC)/matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. By using electrospray, the SEC eluent, together with a suitable matrix solution added coaxially, was directly deposited on the MALDI plate. Owing to the formation of very small droplets in electrospray, solvent evaporation is much faster. The fractionation volume in narrow-bore SEC, which can directly be collected in one MALDI spot, can easily be optimized in the range of a few microlitres. In addition, fairly homogeneous sample spots were obtained. The possible influence of composition variation of the SEC effluent on the analytical results using direct fraction deposition was investigated; no substantial effects were observed. The applicability of the method was demonstrated by characterizing a broad poly(methyl methacrylate) sample. Copyright 2000 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.     

Silica nanoparticles pre-spotted onto target plate for laser desorption/ionization mass spectrometry analyses of peptides

We report on the simple deposition of Stöber silica nanoparticles (SiO₂ NPs) on conventional MALDI target plate for high throughput laser desorption/ionization mass spectrometry (LDI-MS) analyses of peptide mixtures with sensitivity in the femtomolar range. This low-cost easily prepared material allowed straightforward LDI experiments by deposition of the studied samples directly onto a pre-spotted MALDI plate. This analytical strategy can be performed in any laboratory equipped with a MALDI-TOF instrument. All key benefits of organic matrix-free technologies were satisfied while maintaining a high level of detection performances (sensitivity and reproducibility/repeatability). In particular, sample preparation was simple and detection in the low mass range was not hampered by matrix ions. Imaging studies were undertaken to query sample dispersion into the inert SiO₂ NPs and to help into the search of the best experimental conditions producing homogeneous analyte distribution within the deposit. In contrast to commercial disposable LDI targets designed for single use and requiring an adaptor such as NALDI™, the proposed SiO₂ NPs pre-spotting on a MALDI target plate allowed very easily switching between MALDI and LDI experiments. They can be conducted either simultaneously (positions with an organic matrix or SiO₂ NPs) or in the row (support prepared in advance, stored and washed after use). The overall cost and versatility of the methodology made it very attractive to MALDI users in many domains (peptidomics, proteomics, metabolomics).     

Immobilized carbon nanotubes as matrix for MALDI-TOF-MS analysis: Applications to neutral small carbohydrates

In this work, we reported on the advantages of immobilized carbon nanotubes as a novel MALDI-matrix. Recently, carbon nanotubes have been reported to be an effective MALDI matrix for small molecules (Anal. Chem.2003, 75, 6191), as it can eliminate the interfering matrix peaks as well as form a web morphology to fully disperse the analyte and allow strong ultraviolet absorption for enhanced pulsed laser desorption and ionization. In our study, to overcome the problem that the carbon nanotube matrix may fly off from the target, a type of polyurethane adhesive, NIPPOLAN-DC-205, is introduced to immobilize carbon nanotubes on the target, which enables widespread application of carbon nanotubes as matrix for MALDI-MS analysis. At the same time, the properties of the carbon nanotubes as an efficient matrix remained after immobilization. The presence of NIPPOLAN-DC-205 increases the time for analysis at a particular desorption spot by minimizing the time-consuming search for "hot spots" and facilitating experiments such as post source decay (PSD) which need longer-lasting signals. Moreover, NIPPOLAN-DC-205 produces no interference peaks and can easily be cleaned with acetone. Fast evaporation technology may be used to enhance signal reproducibility in MALDI analysis using carbon nanotubes as matrix. Consequently, the applicability of the carbon nanotube as matrix for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of low molecular mass analytes is highly improved. The feasibility of the method employing polyurethane is demonstrated by comparison of the results produced from the carbon nanotube matrix with and without immobilization. In addition, neutral small carbohydrates, which are difficult to be ionized normally, can be cationized with high efficiency by MALDI-TOF-MS using the immobilized carbon nanotube matrix. The method was further applied to analyze peptides and detect urine glucose successfully.     

Study of matrix and polymer substrate in MALDI-TOF mass spectrometry of DNA

Protein matrices such as 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid, SA) and a-cyano-4-hydroxycinnamic acid (CHCA) tend to yield homogeneous dried spots. However, well known MALDI matrices for single- and double-stranded DNA such as 3-hydroxy picolinic acid (HPA) and picolinic acid (PA) forms the crystals at the rim of their spots with uneven distribution of matrix and DNA. This inhomogeneous deposition of DNA-doped matrix crystals at the MALDI spot requires a search for sweet spots. It is important to obtain homogeneous MALDI spots that yield signals not only from the periphery but the entire spot for automated, high throughput MALDI-TOF analysis of short DNA fragments. We have investigated the characteristics of MALDI matrices for DNA and presented a method for improving the homogeneity of MALDI samples by using polymer substrates such as linear polyacrylamide (LPA), poly(ethylene oxide) (PEO), methyl cellulose (MC) and Nafion.     

Covalent attachment and dissociative loss of sinapinic acid to/from cysteine-containing proteins from bacterial cell lysates analyzed by MALDI-TOF-TOF mass spectrometry

We report covalent attachment via a thiol ester linkage of 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid or SA) to cysteine-containing protein biomarkers from bacterial cell lysates of E. coli analyzed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry when using SA as the matrix. Evidence to support this conclusion is the appearance of additional peaks in the MS spectra when using SA, which are absent when using α-cyano-4-hydroxycinnamic acid (HCCA). The additional peaks appear at a mass-to-charge (m/z) ∼208 greater to the m/z of a more abundant protein ion peak. Protein biomarkers were identified by tandem mass spectrometry (MS/MS) using a MALDI time-of-flight/time-of-flight (TOF-TOF) mass spectrometer and top-down proteomics. Three protein biomarkers, HdeA, HdeB, and homeobox or YbgS (each containing two cysteine residues) were identified as having reactivity to SA. Non-cysteine-containing protein biomarkers showed no evidence of reactivity to SA. MS ions and MS/MS fragment ions were consistent with covalent attachment of SA via a thiol ester linkage to the side-chain of cysteine residues. MS/MS of a protein biomarker ion with a covalently attached SA revealed fragment ion peaks suggesting dissociative loss SA. We propose dissociative loss of SA is facilitated by a pentacyclic transition-state followed by proton abstraction of the β-hydrogen of the bound SA by a sulfur lone pair followed by dissociative loss of 3-(4-hydroxy-3,5-dimethoxyphenyl)prop-2-ynal. The apparent reactivity of SA to cysteine/disulfide-containing proteins may complicate identification of such proteins, however the apparent differential reactivity of SA and HCCA toward cysteine/disulfide-containing proteins may be exploited for identification of unknown cysteine-containing proteins.