Metabolite imaging using matrix-enhanced surface-assisted laser desorption/ionization mass spectrometry (ME-SALDI-MS)

We describe here the use of a hybrid ionization approach, matrix-enhanced surface-assisted laser desorption/ionization mass spectrometry (ME-SALDI-MS) in bioimaging. ME-SALDI combines the strengths of traditional matrix-assisted laser desorption/ionization (MALDI) and SALDI and enables successful MS imaging of low-mass species with improved detection sensitivity. Using 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) as the MS standard, MS performances of MALDI, SALDI, and ME-SALDI are systematically compared. The analyte desorption and ionization mechanism in ME-SALDI is qualitatively speculated based on the observation of significantly reduced matrix background and improved survival yields of molecular ions. Improvements in detection sensitivity of low-mass species using ME-SALDI over MALDI in imaging are demonstrated with mouse heart and brain tissues.     

Functionalized pyrolytic highly oriented graphite polymer film for surface‐assisted laser desorption/ionization mass spectrometry in environmental analysis

The pyrolytic highly oriented graphite polymer film (PGS) was first employed to analyze low‐mass analytes in environmental analysis by surface‐assisted laser desorption/ionization mass spectrometry (SALDI‐MS). PGS is a synthetic uniform and highly oriented graphite polymer film with high thermal anisotropic conductivity. We have found that negative ion mode SALDI‐MS using oxidized PGS (PGS‐SALDI‐MS) can be used to detect [M–H]^? ions from perfluorooctanoic acid (PFOA) and other perfluoroalkylcarboxylic acids when the PGS surface is modified with the cationic polymer polyethyleneimine (PEI). The signal intensity of PFOA when employing the PEI modification showed a ten‐fold increase over that obtained from desorption/ionization on porous silicon (DIOS). PFOA was quantified using PGS‐SALDI‐MS and the calibration curve showed a wide linear dynamic range of response (20–1000 ppb). The combination of atmospheric pressure ionization and PGS (AP‐PGS‐SALDI) showed greater signal intensity than vacuum PGS‐SALDI for deprotonated PFOA. Several other environmentally important chemicals, including perfluoroalkylsulfonic acid, pentachlorophenol, bisphenol A, 4‐hydroxy‐2‐chlorobiphenyl, and benzo[a]pyrene, were also successfully used to evaluate PGS‐SALDI‐MS. In addition, we found that nonafluoro‐1‐butanesulfonic acid was able to produce protonated peptides in positive ion PGS‐SALDI‐MS, but that perfluoropentanoic acid and trifluoroacetic acid were not. It is suggested that perfluoroalkylsulfonic acids are better protonating agents than perfluoroalkylcarboxylic acids in SALDI‐MS. Copyright © 2009 John Wiley & Sons, Ltd.     

On the role of defects and surface chemistry for surface-assisted laser desorption ionization from silicon

The generation of ions from silicon substrates in surface-assisted laser desorption ionization (SALDI) has been studied using silicon substrates prepared and etched by a variety of different methods. The different substrates were compared with respect to their ability to generate peptide mass spectra using standard liquid sample deposition. The desorption/ionization processes were studied using gas-phase analyte deposition. Mass spectra were obtained from compounds with gas-phase basicities above 850 kJmol and with molecular weights up to 370 Da. UV, VIS, and IR lasers were used for desorption. Ionization efficiencies were measured as a function of laser fluence and accumulated laser irradiance dose. Solvent vapors were added to the ion source and shown to result in fundamental laser-induced chemical and physical changes to the substrate surfaces. It is demonstrated that both the chemical properties of the substrate surface and the presence of a highly disordered structure with a high concentration of "dangling bonds" or deep gap states are required for efficient ion generation. In particular, amorphous silicon is shown to be an excellent SALDI substrate with ionization efficiencies as high as 1%, while hydrogen-passivated amorphous silicon is SALDI inactive. Based on the results, a novel model for SALDI ion generation is proposed with the following reaction steps: (1) the adsorption of neutral analyte molecules on the SALDI surface with formation of a hydrogen bond to surface Si-OH groups, (2) the electronic excitation of the substrate to form free electron/hole pairs (their relaxation results in trapped positive charges in near-surface deep gap states, causing an increase in the acidity of the Si-OH groups and proton transfer to the analyte molecules), and (3) the thermally activated dissociation of the analyte ions from the surface via a "loose" transition state.     

Effects of ZnO nanowire length on surface-assisted laser desorption/ionization of small molecules

The effects of nanowire (NW) length on surface-assisted laser desorption/ionization (SALDI) mass spectrometry (MS) of small molecules were investigated using ZnO NWs of 50 nm diameter with a broad range of lengths ranging from 25 to 1600 nm. Characterization of the ZnO NWs revealed that the length was the only parameter that varied in this study, while other properties of the NWs remained essentially the same as the bulk properties. Experiments on SALDI efficiency exhibited that the SALDI processes on NWs have a certain length window. In the present case of ZnO NWs, the SALDI efficiency was found to be enhanced on the nanowires of 250 nm length, corresponding to an aspect ratio of 5. The roles of NW length in the SALDI processes were discussed from the viewpoint of efficient energy-transfer media as well as physical obstacles screening laser irradiation and preventing the escape of nascent ions from NW surfaces. The existence of the length window may provide valuable insight for tailoring new nanostructures for efficient SALDI of small molecules.     

Surface-assisted laser desorption/ionization mass spectrometry on titania nanotube arrays

Titania nanotube arrays (NTA) generated from anodizing processes are tested as the substrate for surface-assisted laser desorption/ionization mass spectrometry (SALDI MS). The background generated from titania NTA is very low, making the approach suitable for the analysis of small molecules. The upper detectable mass is approximately 29 kDa. Homogeneous sample deposition leads to good shot-to-shot reproducibility and suitability for quantitative analysis. Additionally, phosphopeptides can be selectively trapped on the titania NTA substrate, as illustrated by simply depositing a tryptic digest of beta-casein followed by titania NTA SALDI MS analysis. The detection limit for small organics and peptides is in low fmol.     

Minimization of Fragmentation and Aggregation by Laser Desorption Laser Ionization Mass Spectrometry

Measuring average quantities in complex mixtures can be challenging for mass spectrometry, as it requires ionization and detection with nearly equivalent cross-section for all components, minimal matrix effect, and suppressed signal from fragments and aggregates. Fragments and aggregates are particularly troublesome for complex mixtures, where they can be incorrectly assigned as parent ions. Here we study fragmentation and aggregation in six aromatic model compounds as well as petroleum asphaltenes (a naturally occurring complex mixture) using two laser-based ionization techniques: surface assisted laser desorption ionization (SALDI), in which a single laser desorbs and ionizes solid analytes; and laser ionization laser desorption mass spectrometry (L²MS), in which desorption and ionization are separated spatially and temporally with independent lasers. Model compounds studied include molecules commonly used as matrices in single laser ionization techniques such as matrix assisted laser desorption ionization (MALDI). We find significant fragmentation and aggregation in SALDI, such that individual fragment and aggregate peaks are typically more intense than the parent peak. These fragment and aggregate peaks are expected in MALDI experiments employing these compounds as matrices. On the other hand, we observe no aggregation and only minimal fragmentation in L²MS. These results highlight some advantages of L²MS for analysis of complex mixtures such as asphaltenes.

A novel approach of combining thin-layer chromatography with surface-assisted laser desorption/ionization (SALDI) time-of-flight mass spectrometry

A novel means of combining thin-layer chromatography (TLC) with laser desorption/ionization mass spectrometry using a liquid matrix is proposed. Surface-assisted laser desorption/ionization (SALDI) mass spectrometry, which uses a mixture of a micrometer-sized carbon powder (graphite or activated carbon, the SALDI solid) and 15% sucrose/glycerol, dissolved in an equal volume of methanol (SALDI liquid) as a SALDI matrix, is used for laser desorption mass analysis. The ablation of carbon powder from a pencil drawing was used as an alternative to the SALDI solid. The liquid matrix resembled that used in a conventional SALDI matrix system. A line was drawn before separation with a pencil on the track of the sample developed on the TLC plate. After TLC separation, approximately 0.1 microl of SALDI liquid was directly applied to the chromatographic spots on the TLC plate. Porphyrins were used to demonstrate this combination owing to the visible colors of this type of compound. The analyte signal can be easily detected by irradiating the laser along the pencil line on the TLC plate. An additive, p-toluenesulfonic acid, is added to the SALDI liquid to enhance the signal's intensity. This additive dramatically improves the signal-to-noise ratio. A detection limit of approximately 500 pg is demonstrated for porphines, which is 50 times better than that corresponding to conventional TLC SALDI.     

Coffee-ring effects in laser desorption/ionization mass spectrometry

This report focuses on the heterogeneous distribution of small molecules (e.g. metabolites) within dry deposits of suspensions and solutions of inorganic and organic compounds with implications for chemical analysis of small molecules by laser desorption/ionization (LDI) mass spectrometry (MS). Taking advantage of the imaging capabilities of a modern mass spectrometer, we have investigated the occurrence of “coffee rings” in matrix-assisted laser desorption/ionization (MALDI) and surface-assisted laser desorption/ionization (SALDI) sample spots. It is seen that the “coffee-ring effect” in MALDI/SALDI samples can be both beneficial and disadvantageous. For example, formation of the coffee rings gives rise to heterogeneous distribution of analytes and matrices, thus compromising analytical performance and reproducibility of the mass spectrometric analysis. On the other hand, the coffee-ring effect can also be advantageous because it enables partial separation of analytes from some of the interfering molecules present in the sample. We report a “hidden coffee-ring effect” where under certain conditions the sample/matrix deposit appears relatively homogeneous when inspected by optical microscopy. Even in such cases, hidden coffee rings can still be found by implementing the MALDI-MS imaging technique. We have also found that to some extent, the coffee-ring effect can be suppressed during SALDI sample preparation.     

Affinity-based mass spectrometry using magnetic iron oxide particles as the matrix and concentrating probes for SALDI MS analysis of peptides and proteins

Silane-immobilized magnetic iron oxide particles were used as the assisting material in surface-assisted laser desorption/ionization (SALDI) mass spectrometric analysis. This approach can be used to analyze small proteins and peptides. The upper detectable mass range is approximately 16 kDa. The detection limit for peptides is about 20 fmol. Silanized iron oxide particles with negatively charged functionalities can also be used as the affinity probes to selectively trap oppositely charged species from sample solutions by adjusting the pH of the solution. A tryptic digest product of cytochrome C at a concentration as low as 10 nM can be enriched by the particles and directly analyzed by iron oxide SALDI MS without the need for elution steps. Affinity-based mass spectrometry using the bifunctional silanized magnetic iron oxide particles as the SALDI matrix and concentrating probe is demonstrated in this study.     

Silver-coated gold nanoparticles as concentrating probes and matrices for surface-assisted laser desorption/ionization mass spectrometric analysis of aminoglycosides

A novel method for the determination of aminoglycosides by surface-assisted laser desorption/ionization mass spectrometry (SALDI MS) with the aid of silver-coated gold nanoparticles (Au@AgNPs) has been developed. The Au@AgNPs with surface capped by anionic citrate were used as concentrating probes as well as matrices in SALDI MS. Adsorption of aminoglycosides onto the nanoparticles was mainly through electrostatic attraction. The aminoglycoside-adsorbed nanoparticles were directly characterized by SALDI MS after a simple washing. Using Au@AgNPs to preconcentrate the aminoglycosides from 500 μL buffer solution, the limits of detection (LODs) at signal-to-noise ratio of 3 were 3, 25, 15, 30, and 38 nM for paromomycin, kanamycin A, neomycin, gentamicin, and apramycin, respectively. This method was successfully applied to the determination of aminoglycosides in human plasma samples. The LODs of aminoglycosides in plasma samples were 9, 130, 81, and 180 nM for paromomycin, kanamycin A, neomycin, and gentamicin, respectively. Recoveries of aminoglycosides in plasma samples were about 80%.     

Determination of rhenium and osmium complexes by surface-assisted laser desorption/ionization coupled to Orbitrap mass analyzer

A surface-assisted laser desorption/ionization (SALDI) source is coupled to the Orbitrap mass analyzer; the instrumental approach is tested for the analysis of rhenium (Re) and osmium (Os) complexes with 8-mercaptoquinoline. Silicon (Si) material obtained by laser treatment of monocrystalline Si is used as SALDI substrate. All studied complexes are detected as radical cations, with no protonated molecules. The comparison of SALDI, matrix-assisted laser desorption/ionization (MALDI), and direct laser desorption/ionization (LDI) on metal plates in the same instrumental setup demonstrated that the detection of the studied complexes using SALDI provides the highest sensitivity. The ability to analyze samples rapidly, high purity of spectra, and good analytical parameters make SALDI coupled to the Orbitrap mass analyzer a potentially powerful tool for the detection of Re and Os complexes and related organic, UV-absorbing compounds.

Detection of aminothiols through surface‐assisted laser desorption/ionization mass spectrometry using mixed gold nanoparticles

We have employed mixtures of two differently sized (average diameters: 3.5 and 14 nm) gold nanoparticles (Au NPs) as selective probes and matrices for the determination of aminothiols using surface‐assisted laser desorption/ionization mass spectrometry (SALDI‐MS). When using 38 and 150 pM solutions of the 3.5‐ and 14‐nm Au NPs, respectively, as the probe and matrix, SALDI‐MS provided limits of detection (signal‐to‐noise ratio = 3) of 2, 20, and 44 nM for 1.0 mL solutions of glutathione (GSH), cysteine (Cys), and homocysteine, respectively. The signal intensities of these analytes varied by less than 20% for SALDI‐MS analyses recorded over 50 sample spots; in contrast, they varied by as much as 60% when using a conventional matrix (2,5‐dihydroxybenzoic acid). We validated the practicality of this approach – with its advantages of sensitivity, reproducibility, rapidity, and simplicity – through the analysis of GSH in MCF‐7 cell lysates and Cys in plasma. Copyright © 2009 John Wiley & Sons, Ltd.     

Surface‐Assisted Laser Desorption/Ionization Mass Spectrometric Detection of Biomolecules by Using Functional Single‐Walled Carbon Nanohorns as the Matrix

A surface‐assisted laser desorption/ionization time‐of‐flight mass spectrometric (SALDI‐TOF MS) method was developed for the analysis of small biomolecules by using functional single‐walled carbon nanohorns (SWNHs) as matrix. The functional SWNHs could transfer energy to the analyte under laser irradiation for accelerating its desorption and ionization, which led to low matrix effect, avoided fragmentation of the analyte, and provided high salt tolerance. Biomolecules including amino acids, peptides, and fatty acids could successfully be analyzed with about 3‐ and 5‐fold higher signals than those obtained using conventional matrix. By integrating the advantages of SWNHs and the recognition ability of aptamers, a selective approach was proposed for simultaneous capture, enrichment, ionization, and MS detection of adenosine triphosphate (ATP). This method showed a greatly improved detection limit (1.0 μM ) for the analysis of ATP in complex biological samples. This newly designed protocol not only opened a new application of SWNHs, but also offered a new technique for selective MS analysis of biomolecules based on aptamer recognition systems.     

Platinum Nanoflowers on Scratched Silicon by Galvanic Displacement for an Effective SALDI Substrate

We report a new and facile method for synthesizing 3D platinum nanoflowers (Pt Nfs) on a scratched silicon substrate by electroless galvanic displacement and discuss the applications of the Pt Nfs in surface‐assisted laser desorption/ionization‐mass spectrometry (SALDI‐MS). Surface scratching of n‐type silicon is essential to induce Pt Nf growth on a silicon substrate (to obtain a Pt Nf silicon hybrid plate) by the galvanic displacement reaction. The Pt Nf silicon hybrid plate showed excellent SALDI activity in terms of the efficient generation of protonated molecular ions in the absence of a citrate buffer. We propose that the acidity of the Si? OH moieties on silicon increases because of the electron‐withdrawing nature of the Pt Nfs; hence, proton transfer from the Si? OH groups to the analyte molecules is enhanced, and finally, thermal desorption of the analyte ions from the surface occurs. Signal enhancement was observed for protonated molecular ions produced from a titania nanotube array (TNA) substrate on which Pt nanoparticles had been photochemically deposited. Moreover, surface modification of the Pt Nf silicon hybrid plate by perfluorodecyltrichlorosilane (FDTS) (to obtain an FDTS‐Pt Nf silicon hybrid plate) was found to facilitate soft SALDI of labile compounds. More interestingly, the FDTS‐Pt Nf silicon hybrid plate acts 1) as a high‐affinity substrate for phosphopeptides and 2) as a SALDI substrate. The feasibility of using the FDTS‐Pt Nf silicon hybrid plate for SALDI‐MS has been demonstrated by using a β‐casein digest and various analytes, including small molecules, peptides, phosphopeptides, phospholipids, carbohydrates, and synthetic polymers. The hybridization of Pt Nfs with a scratched silicon substrate has been found to be important for achieving excellent SALDI activity.     

Exploring the interactions between gold nanoparticles and analytes through surface‐assisted laser desorption/ionization mass spectrometry

Surface‐assisted laser desorption/ionization mass spectrometry (SALDI‐MS) is applied to provide strong evidence for the chemical reactions of functionalized gold nanoparticles (Au NPs) with analytes – Hg²⁺ ions induced MPA?Au NPs aggregation in the presence of 2,6‐pyridinedicarboxylic acid (PDCA) and H₂O₂ induced fluorescence quenching of 11‐MUA?Au NDs. PDCA‐Hg²⁺‐MPA coordination is responsible for Au NPs aggregation, while the formation of 11‐MUA disulfide compounds that release into the bulk solution is responsible for H₂O₂‐induced fluorescence quenching. In addition to providing information about the chemical structures, SALDI‐MS is also selective and sensitive for the detection of Hg²⁺ ions and H₂O₂. The limits of detection (LODs) for Hg²⁺ ions and H₂O₂ by SALDI‐MS were 300 nM and 250 µM, respectively. The spot‐to‐spot variations in the two studies were both less than 18% (50 sample spots). Our results reveal that SALDI‐MS can be used to study analyte‐induced changes in the surface properties of nanoparticles. Copyright © 2010 John Wiley & Sons, Ltd.     

Effect of urea surface modification and photocatalytic cleaning on surface‐assisted laser desorption ionization mass spectrometry with amorphous TiO2 nanoparticles

We have investigated the effect of urea surface modification and the photocatalytic cleaning on surface‐assisted laser desorption ionization mass spectrometry (SALDI‐MS) with amorphous TiO₂ nanoparticles for the reduction of the background noise and the improvement of the sensitivity. In the use of nanoparticles of high surface area, chemical background signals arising from ambient environments and organic contaminants can frequently be serious problems below 500 Da, possibly reducing the advantages of the matrix‐free approach. In this study, removal of contaminants and enhanced SALDI efficiency were easily achieved with UV irradiation via the photocatalyst effect of TiO₂ before SALDI‐MS measurements. The surface cleaning achieved by the UV photocatalytic procedure reduced the background noise and increased the peak intensities of peptides. In addition, we found that urea surface modification of TiO₂ nanoparticles increased the performance of the TiO₂‐SALDI‐MS. (1) The urea‐surface modification of TiO₂ made it possible to produce proton‐adduct forms without citrate buffer, resulting in low background noises below 500 Da, in contrast to the essential use of a citrate buffer in the bare TiO₂‐SALDI‐MS. (2) The detection sensitivity of angiotensin I increased to 0.3 fmol with the urea‐surface modification, as compared to the use of bare TiO₂ nanoparticles (6 fmol). The urea‐TiO₂ could ionize proteins of more than 20 000 Da such as trypsinogen (600 fmol). (3) The urea modification of TiO₂ had the advantage of selective detection of phosphopeptides without sample clean up, or prefractionation in tryptic digest products of bovine hemoglobin. Copyright © 2009 John Wiley & Sons, Ltd.     

Using surfactants to enhance the analyte signals in activated carbon, surface-assisted laser desorption/ionization (SALDI) mass spectrometry

The effect of surface activity in surface-assisted laser desorption/ionization (SALDI) mass spectrometry was examined. Several surfactants, including p-tolunensulfonic acid (PTSA), sodium dodecyl sulfate and alkyltrimethylammonium bromide, were used as analytes or additives in the SALDI matrix to demonstrate the surface activity effect. The experimental results demonstrate that analytes that have good surface activity have good sensitivity. Adding suitable amounts of surfactants to the SALDI matrix can dramatically enhance the sensitivity of analytes lacking surface activity. We propose that the enhancement of analyte signals is due to the ionic interaction between ionic surfactants and analytes because non-ionic surfactant additives in the SALDI matrix do not affect the analyte signals. The detection limit of methylephedrine can be as low as 100 pg in the SALDI analysis of 0.5 M PTSA additive in the SALDI matrix. Although other surfactants can also be used as matrix additives to enhance the analyte signal, they do not improve the ion abundance as much as PTSA does.     

Detection of melamine in infant formula and grain powder by surface‐assisted laser desorption/ionization mass spectrometry

We have developed a method for the determination of melamine (MEL), ammeline (AMN), and ammelide (AMD) by surface‐assisted laser desorption/ionization mass spectrometry (SALDI‐MS) using gold nanoparticles (Au NPs). The major peaks for MEL, AMN, and AMD at m/z 127.07, 128.05, and 129.04 are assigned to the [MEL + H]⁺, [AMN + H]⁺, and [AMD + H]⁺ ions. Because the three tested compounds adsorb weakly onto the surfaces of the Au NPs through Au–N bonding, they can be easily concentrated from complex samples by applying a simple trapping/centrifugation process. The SALDI‐MS method provides limits of detection of 5, 10, and 300 nM for MEL, AMN, and AMD, respectively, at a signal‐to‐noise ratio of 3. The signal variation for 150‐shot average spectra of the three analytes within the same spot was 15%, and the batch‐to‐batch variation was 20%. We have validated the practicality of this approach by the analysis of these three analytes in infant formula and grain powder. This simple and rapid SALDI‐MS approach holds great potential for screening of MEL in foods. Copyright © 2012 John Wiley & Sons, Ltd.     

基体辅助激光解吸电离飞行时时质谱和热裂解色谱/质谱研究聚酯型聚氨酯链结构

聚酯型聚氨酯样品用基体辅助激光裂解离子化质谱（ＭＡＬＤＩ－ＴＯＦ／ＭＳ）和热裂解色谱质谱（ＰＹＧＣ／ＭＳ）方法进行了鉴定,ＰＹＧＣ／ＭＳ方法可检出ＰＵ的一些组成单元的化学结构,但ＰＹＧＣＩＭＳ中大多数的峰,无法从常规的标准化合物的质谱数据库中检索到,而ＭＡＬＤＩ－ＭＳ方法,可明确测出ＰＵ试样各种单元的组合、聚合度及ＰＵ长链的序列分布,质量数范围可达2300.     

A “soft” ionization mass spectrometric study of organic sulfides as sulfonium salts

Preliminary conversion of nonpolar organic sulfides into sulfonium salts was proposed for their study and analysis by laser desorption/ionization (MALDI and SALDI) and electrospray/ionization (ESI) mass spectrometry. General possibilities of the methodology proposed were demonstrated on examples of dialkyl sulfides, substituted thiacyclanes, dibenzothiophene, and methionine methyl ester. Various alkyl and aralkyl halides, as well as trimethyl- and triethyloxonium salts were tested as alkylating agents. S-alkylation was shown to proceed quantitatively under mild conditions. MALDI and SALDI mass spectra (a matrix-free nanostructurized target was used in SALDI experiments) displayed only ion peaks corresponding to sulfonium cations whose mass numbers were equal to the sum of molecular weights of sulfides and weight increments of the introduced alkyl and aralkyl groups. Trans-alkylation was observed for benzyl-substituted sulfides. Tandem mass spectrometry provided preliminary data on the fragmentation of ESI-generated sulfonium cations and demonstrated differences in the MS/MS spectra of regioisomers.