Use of monoatomic and polyatomic projectiles for the characterisation of polylactic acid by static secondary ion mass spectrometry

The application of polyatomic primary ions is a strongly developing branch of static secondary ion mass spectrometry (S-SIMS), since these projectiles allow a significant increase in the secondary ion yields to be achieved. However, the different limitations and possibilities of certain polyatomic primary ions for use on specific functional classes of samples are still not completely known. This paper compares the use of monoatomic and polyatomic primary ions in S-SIMS for thin layers of polylactic acid (PLA), obtained by spin-coating solutions on silicon wafers. Bombardment with Ga+, Xe+ and SF5+ primary ions allowed the contribution of the projectile mass and number of atoms in the gain in ion yield and molecular specificity (relative importance of high m/z and low m/z signals) to be assessed. Samples obtained by spin-coating solutions with increasing concentration showed that optimal layer thickness depended on the primary ion used. In comparison with the use of Ga+ projectiles, the yield of structural ions increased by a factor of about 1.5 to 2 and by about 7 to 12 when Xe+ and SF5+ primary ion bombardment were applied, respectively. A detailed fragmentation pattern was elaborated to interpret ion signal intensity changes for different projectiles in terms of energy deposition and collective processes in the subsurface, and the internal energy of radical and even-electron precursor ions.     

Static secondary ion mass spectrometry (S-SIMS) for the characterization of surface components in mineral particulates

The feasibility of static secondary ion mass spectrometry (S-SIMS) for the detection of molecule specific information from complex materials, such as natural clay and soil samples, has been investigated. Ion trap (IT), as well as triple quadrupole (TQ) instruments, have been used for mass analysis. Secondary ion images have been acquired using time-of-flight (TOF) S-SIMS. The generation of molecular adduct ions from thin and thick layers on the mineral substrates has been investigated using KBr as a simple model system. Results show that molecular adducts of KBr can be indeed detected from the spiked materials. However, the concentrations of the spiking solutions have to be significantly larger than expected from the surface area measured by gas adsorption techniques. In addition imaging analysis has evidenced that the detection of adduct ions in the mass spectra directly relates to the presence of local micro-crystallites.     

Static secondary ion mass spectrometry to monitor solid-phase peptide synthesis

Insights into the direct monitoring of supported peptide synthesis were realized through the design of time of flight static secondary ion mass spectrometry (TOF-S-SIMS) experiments. The mass spectrometric method was carried out at the resin bead level and was found reproducible (intra- and inter-day assays), sensitive (femtomol level) and non-destructive (only 0.01% of the peptides were destroyed by the primary ion beam bombardment). The nature of the peptide-resin linkage governed the recovery of ions characterizing the whole peptide sequence. A S-SIMS cleavable bond was thus required solely in that position to achieve the release of the growing structures from the insoluble support into the gas phase without any fragmentation. Results are presented with standard solid-phase resins allowing linkage through an amide or an ester bond. The latter was orthogonally broken upon the bombardment and thus constituted a convenient S-SIMS cleavable bond.     

Complementary use of Fourier transform laser microprobe mass spectrometry and time-of-flight static secondary ion mass spectrometry for the study of the surface adsorption of organic dyes on silicate materials

The adsorption of organic ionic dyes on different pore size engineered silica materials with potential application for industrial wastewater treatment has been investigated using Fourier transform laser microprobe mass spectrometry (FT-LMMS) and time-of-flight secondary ion mass spectrometry (TOF-S-SIMS). The complementary use of the two methods with different information depth allowed data on the subsurface distribution and pore penetration of the adsorbed organic compounds. Macroscopic methods were employed to determine the amount adsorbed on the particles and the specific external surface area. Local MS analysis allows identification of the organic dyes exclusively at the outer particle surface when the pore size is inferior to the size of the adsorbing molecule, or at the surface of the channels inside the material. Specifically, the monolayer information depth of TOF-S-SIMS causes a signal to refer essentially to the adsorbate at the outer particle surface, which is only a fraction of the total adsorption in mesoporous materials, while FT-LMMS allowed detection of the presence of adsorbates at the outer surface as well as inside the subsurface of 10 to 50 nm depending on the material under study. The observed data open perspectives for the molecular monitoring of the adsorption behaviour of different materials at the (sub) microm scale.     

Speciation analysis of oxides with static secondary ion mass spectrometry

Speciation analysis of inorganic solids, without dissolution of the sample, aims at specific molecular information. Two potentially useful microanalytical techniques emerge, namely, laser microprobe mass spectrometry (LMMS) and static secondary ion mass spectrometry (S-SIMS). This paper focuses on the molecular characterisation of oxides by application of the S-SIMS method. For this purpose, mass spectra of pure oxides were acquired under static conditions. Analytical parameters such as repeatability, accuracy and resolution were assessed. Also, the peak patterns in the mass spectra are discussed in connection with the older Plog model, describing the relative ion yield as a function of the cluster size. Finally, a comparison is made with the mass spectra from a S-SIMS library and with those obtained by Fourier transform LMMS. Copyright 1999 John Wiley & Sons, Ltd.     

Comparison of primary monoatomic with primary polyatomic ions for the characterisation of polyesters with static secondary ion mass spectrometry

Static secondary ion mass spectrometry (S-SIMS) emerges as one of the most adequate methods for the surface characterisation of polymers with an information depth of essentially one monolayer. The continuing search for increased analytical sensitivity and specificity has led to exploring the use of polyatomic primary ions as an alternative to the traditionally applied monoatomic projectiles. As part of a systematic investigation on polyatomic bombardment of organic and inorganic solids, this paper focuses on selected polyesters. Mass spectra and ion yields are compared for layers deposited on silicon wafers by spincoating solutions with different concentrations of poly(epsilon-caprolactone) (PCL), poly(butylene adipate) (PBA) and poly(ethylene adipate) (PEA). Accurate mass measurements have been used to support the assignment of the ions and link the composition of the detected ions to the analyte structure. Use of polyatomic projectiles increases the yield of structural ions with a factor of +/-15, +/-30 and +/-10 for PCL, PBA and PEA, respectively, in comparison to bombardment with Ga+ primary ions, while the molecular specificity is improved by the detection of additional high m/z ions.     

Static secondary ion mass spectrometry for organic and inorganic molecular analysis in solids

The use of mass spectra in secondary ion mass spectrometry (S-SIMS) to characterise the molecular composition of inorganic and organic analytes at the surface of solid samples is investigated. Methodological aspects such as mass resolution, mass accuracy, precision and accuracy of isotope abundance measurements, influence of electron flooding and sample morphology are addressed to assess the possibilities and limitations that the methodology can offer to support the structural assignment of the detected ions. The in-sample and between-sample reproducibility of relative peak intensities under optimised conditions is within 10%, but experimental conditions and local hydration, oxidation or contamination can drastically affect the mass spectra. As a result, the use of fingerprinting for identification becomes compromised. Therefore, the preferred way of interpretation becomes the deductive structural approach, based on the use of the empirical desorption–ionisation model. This approach is shown to allow the molecular composition of inorganic and organic components at the surface of solids to be characterised. Examples of inorganic speciation and identification of organic additives with unknown composition in inorganic salt mixtures are given. The methodology is discussed in terms of foreseen developments with respect to the use of polyatomic primary ions.     

Secondary ion and laser ablation mass spectrometry for the quantitative characterization of styrene-butadiene copolymers

Styrene-butadiene copolymers were analyzed by static secondary ion mass spectrometry (S-SIMS) and laser ablation Fourier transform ion cyclotron resonance mass spectrometry (LA-FTICRMS) to obtain quantitative information based on specific ions. Silver deposition was performed on polystyrene, butadiene rubber and styrene-butadiene rubber. Under these experimental conditions, new secondary ions were detected, in particular silver-cationized butadiene [M(butadiene) - Ag](+) and styrene [M(styrene) - Ag](+) monomers. In contrast, LA-FTICRMS experiments did not require pretreatment. At high laser power density, UV photons (193, 266 and 355 nm) allowed the detection of styrene and butadiene monomers at m/z 104 and 54, respectively. The use of the observed ions by SIMS or LA-FTICRMS ensures that quantitative information on the relative distribution of each monomer is obtained. However, the silver coating thickness in the SIMS experiment seems to have an important influence on the quantitative information obtained. For LA-FTICRMS experiments, the best results are obtained at a wavelength of 355 nm.     

Static secondary ion mass spectrometry for nanoscale analysis: surface characterisation of electrospun nanofibres

The viability of static secondary ion mass spectrometry (S-SIMS) for selected applications of nanoscale analysis has been investigated, focusing on nanofibres produced by electrospinning (ES) as a test case. The samples consist of non-woven nanowebs of which the individual fibres have diameters in the range of 100 nm. Use of solutions with functionalised polymers or polar additives potentially allows the surface composition to be tailored as a function of the application. So far nanowebs are primarily characterised by morphological examination. This paper describes the first detailed characterisation of molecular composition at the surface of nanofibres electrospun from poly(epsilon-caprolactone) (PCL) solutions in acetone containing 0-15 mol% (relative to PCL) of cetyltrimethylammonium bromide (CTAB). Application of S-SIMS to nanowebs has allowed mass spectra to be recorded containing the major diagnostic ions of both components. Their relative intensities point to surface enrichment and depletion of the polar CTAB additive relative to the PCL matrix for samples electrospun from solution containing low and high CTAB concentrations, respectively.     

Formation of lithiated adducts of glycerophosphocholine lipids facilitates their identification by electrospray ionization tandem mass spectrometry

Electrospray ionization (ESI) tandem mass spectrometry (MS) has simplified analysis of phospholipid mixtures, and, in negative ion mode, permits structural identification of picomole amounts of phospholipid species. Collisionally activated dissociation (CAD) of phospholipid anions yields negative ion tandem mass spectra that contain fragment ions representing the fatty acid substituents as carboxylate anions. Glycerophosphocholine (GPC) lipids contain a quaternary nitrogen moiety and more readily form cationic adducts than anionic species, and positive ion tandem mass spectra of protonated GPC species contain no abundant ions that identify fatty acid substituents. We report here that lithiated adducts of GPC species are readily formed by adding lithium hydroxide to the solution in which phospholipid mixtures are infused into the ESI source. CAD of [MLi⁺] ions of GPC species yields tandem mass spectra that contain prominent ions representing losses of the fatty acid substituents. These ions and their relative abundances can be used to assign the identities and positions of the fatty acid substituents of GPC species. Tandem mass spectrometric scans monitoring neutral losses of the head-group or of fatty acid substituents from lithiated adducts can be used to identify GPC species in tissue phospholipid mixtures. Similar scans monitoring parents of specific product ions can also be used to identify the fatty acid substituents of GPC species, and this facilitates identification of distinct isobaric contributors to ions observed in the ESI/MS total ion current.     

On the risk of false positive identification using multiple ion monitoring in qualitative mass spectrometry: Large-scale intercomparisons with a comprehensive mass spectral library

Analysts involved in qualitative mass spectrometry have long debated the minimum data requirements for demonstrating that signals from an unknown sample are identical to those from a known compound. Often this process is carried out by comparing a few selected ions acquired by multiple ion monitoring (MIM), with due allowance for expected variability in response. In a few past experiments with electron-ionization mass spectrometry (EI-MS), the number of ions selected and the allowable variability in relative abundance were tested by comparing one spectrum against a library of mass spectra, where library spectra served to represent potential false positive signals in an analysis. We extended these experiments by carrying out large-scale intercomparisons between thousands of spectra and a library of one hundred thousand EI mass spectra. The results were analyzed to gain insights into the identification confidence associated with various numbers of selected ions. A new parameter was investigated for the first time, to take into account that a library spectrum with a different base peak than the search spectrum may still cause a false positive identification. The influence of peak correlation among the specific ions in all the library mass spectra was also studied. Our computations showed that (1) false positive identifications can result from similar compounds, or low-abundance peaks in unrelated compounds if the method calls for detection at very low levels; (2) a MIM method's identification confidence improves in a roughly continuous manner as more ions are monitored, about one order of magnitude for each additional ion selected; (3) full scan spectra still represent the best alternative, if instrument sensitivity is adequate. The use of large scale intercomparisons with a comprehensive library is the only way to provide direct evidence in support of these conclusions, which otherwise depend on the judgment and experience of individual analysts. There are implications for residue chemists who would rely on standardized confirmation criteria to assess the validity of a given confirmatory method. For example, standardized confirmation criteria should not be used in the absence of interference testing and rational selection of diagnostic ions.     

Ion detection limitations to mass resolution in matrix-assisted laser desorption time-of-flight mass spectrometry.

The ion detection process in a discrete-dynode electron multiplier can result in significant mass resolution losses in time-of-flight mass spectrometry (TOF-MS) for higher mass-to-charge (m/z) ion species. This resolution loss is attributed to propagation time delays and signal broadening in the ion detector. This is presumed to be due to the generation of a distribution of secondary ion species produced initially upon impact of a primary ion with the first dynode surface of the ion detector. Comparisons are made between the signals produced by a standard discrete dynode ion detector (which amplifies the negatively charged species produced by impact of a primary ion) and a detector modified to respond to only the positively charged secondary ion species produced by a primary ion impact. Ion signals for higher m/z ions with the standard detector geometry are seen to be due to a narrow signal component, most likely due to the generation of secondary electrons and/or very low mass secondary ions (H-), and a broad signal component, apparently due to secondary ions which take significant amounts of time to traverse the low potential fields between the first and second detector dynode. This results in ion signal tailing for higher m/z ion species. Numerical subtraction of the ion signal obtained with the standard and modified detector geometries (singly protonated molecular ion species of equine myoglobin) results in an improvement in mass resolution, such that a new adduct ion species (from trifluoroacetic acid) can be resolved.     

Analysis of tryptic peptides using desorption electrospray ionisation combined with ion mobility spectrometry/mass spectrometry

A novel method is reported for rapid protein identification by the analysis of tryptic peptides using desorption electrospray ionisation (DESI) coupled with hyphenated ion mobility spectrometry and quadrupole time-of-flight mass spectrometry (IMS/Q-ToF-MS). Confident protein identification is demonstrated for the analysis of tryptically digested bovine serum albumin (BSA), with no sample pre-treatment or clean-up. Electrophoretic ion mobility separation of ions generated by DESI allowed examination of charge-state and mobility distributions for tryptic peptide mixtures. Selective interrogation of singly charged ions allowed isobaric peptide responses to be distinguished, along with a reduction in spectral noise. The mobility-selected singly charged peptide responses were presented as a pseudo-peptide mass fingerprint (p-PMF) for protein database searching. Comparative data are shown for electrospray ionisation (ESI) of the BSA digest, without sample clean-up, from which confident protein identification could not be made. Implications for the robustness of the DESI method, together with potential insights into mechanisms for DESI of proteolytic digests, are discussed.     

Ion yield improvement for static secondary ion mass spectrometry by use of polyatomic primary ions

Static secondary ion mass spectrometry (S-SIMS) is one of the potentially most powerful and versatile tools for the analysis of surface components at the monolayer level. Current improvements in detection limit (LOD) and molecular specificity rely on the optimisation of the desorption-ionisation (DI) process. As an alternative to monoatomic projectiles, polyatomic primary ion (P.I.) bombardment increases ion yields non-linearly. Common P.I. sources are Ga+ (liquid metal ion gun (LMIG), SF5+ (electron ionisation) and the newer Au(n)+, Bi(n)q+ (both LMIG) and C60+ (electron ionisation) sources. In this study the ion yield improvement obtained by using the newly developed ion sources is assessed. Two dyes (zwitterionic and/or thermolabile polar functionalities on a largely conjugated backbone) were analysed as a thin layer using Ga+, SF5+, C60+, Bi+, Bi3(2+) and Bi5(2+) projectiles under static conditions. The study aims at evaluating the improvement in LOD, useful and characteristic yield and molecular specificity. The corrected total ion count values for the different P.I. sources are compared for different instruments to obtain a rough estimate of the improvements. Furthermore, tentative ionisation and fragmentation schemes are provided to describe the generation of radical and adduct ions. Characteristic ion yields are discussed for the different P.I. sources. An overview of the general appearances of the mass spectra obtained with the different P.I. sources is given to stress the major improvement provided by polyatomic P.I.s in yielding information at higher m/z values.     

On-tissue protein identification and imaging by MALDI-Ion mobility mass spectrometry

MALDI imaging mass spectrometry (MALDI-IMS) has become a powerful tool for the detection and localization of drugs, proteins, and lipids on-tissue. Nevertheless, this approach can only perform identification of low mass molecules as lipids, pharmaceuticals, and peptides. In this article, a combination of approaches for the detection and imaging of proteins and their identification directly on-tissue is described after tryptic digestion. Enzymatic digestion protocols for different kinds of tissues—formalin fixed paraffin embedded (FFPE) and frozen tissues—are combined with MALDI-ion mobility mass spectrometry (IM-MS). This combination enables localization and identification of proteins via their related digested peptides. In a number of cases, ion mobility separates isobaric ions that cannot be identified by conventional MALDI time-of-flight (TOF) mass spectrometry. The amount of detected peaks per measurement increases (versus conventional MALDI-TOF), which enables mass and time selected ion images and the identification of separated ions. These experiments demonstrate the feasibility of direct proteins identification by ion-mobility-TOF IMS from tissue. The tissue digestion combined with MALDI-IM-TOF-IMS approach allows a proteomics “bottom-up” strategy with different kinds of tissue samples, especially FFPE tissues conserved for a long time in hospital sample banks. The combination of IM with IMS marks the development of IMS approaches as real proteomic tools, which brings new perspectives to biological studies.     

Feasibility of static secondary ion mass spectrometry to study physicochemical interactions between organic components and silver in thermographic systems

Chemical engineering of high-technology products requires elucidation of intermolecular interactions in complex materials. As part of an extensive study on thermographic systems, static secondary ion mass spectrometry (S-SIMS) was used to probe the physicochemical behaviour of active compounds, such as different tone modifiers and stabilisers, on silver. In particular, the feasibility of detecting adsorption and/or binding of individual additives and mixtures to silver was examined. Substrates prepared by sputter coating silver on silicon wafers were exposed to solutions of the studied compounds in 2-butanone. The signal intensities measured with S-SIMS for the ad-layers showed reproducibility to within 10%. Radical ions containing silver such as [M-H+Ag]+ * were used as evidence for the formation of bonds in the solid. Also the [M-H+2Ag]+ ions could be assigned to chemisorbed species while [M+Ag]+ ions could be formed by adduct ionisation of molecules with co-ejected Ag+ ions. The signal intensities of [M-H+Ag]+ * and [M-H+2Ag]+ ions were used to monitor the adsorption quantitatively as a function of time.     

Arsenosugar identification in seaweed extracts using high-performance liquid chromatography/electrospray ion trap mass spectrometry

The development of analytical techniques suitable for providing structural information on a wide range of elemental species is a growing necessity. For arsenic speciation a variety of mass spectrometric techniques, mainly inductively coupled plasma mass spectrometry (ICP-MS) and electrospray tandem mass spectrometry (ES-MS/MS) coupled on-line with high-performance liquid chromatography (HPLC), are in use. In this paper we report the identification of arsenic species present in samples of marine origin (seaweed extracts) using ES ion trap mass spectrometry (IT) multistage mass spectrometry (MS(n)). Both reversed-phase and anion-exchange HPLC have been coupled on-line to ES-ITMS. Product ion scans with multiple stages of tandem MS (MS(n); n=2-4) were used to acquire diagnostic data for each arsenosugar. The spectra contain structurally characteristic fragment ions for each of the arsenosugars examined. In addition it was observed that upon successive stages of collision-induced dissociation (CID) a common product ion (m/z 237) was formed from all four arsenosugars examined. This product ion has the potential to be used as an indicator for the presence of dimethylated arsenosugars (dimethylarsinoylribosides). The HPLC/ES-ITMS(n) method developed allows the sensitive identification of arsenosugars present in crude seaweed extracts without the need for extended sample preparation. In fact, sample preparation requirements are identical to those typically employed for HPLC/ICP-MS analysis. Additionally, the resulting product ions are structurally diagnostic of the arsenosugars examined, and tandem mass spectra are reproducible and correspond well to those obtained using other low-energy CID techniques. As a result, the HPLC/ES-ITMS(n) approach minimises the potential for arsenic species misidentification and has great potential as a means of overcoming the need for characterised standards.     

Fragmentation of peptide disulfides under conditions of negative ion mass spectrometry: Studies of oxidized glutathione and contryphan

The fragmentation of positive and negative ions of peptide disulfides under mass spectrometric conditions yields distinctly different product ion distributions. A negative ion upon collision induced dissociation yields intense product ions, which correspond to cleavage at the disulfide linkage. The complete assignment of the product ions obtained upon fragmentation of oxidized glutathione in an ion trap is presented. The cleavage at the disulfide site is mediated by abstraction of CalphaH and CbetaH protons resulting in product ions derived by neutral loss of H2S2 and H2S. The formation of peptide thioaldehydes and persulfides at the cysteine sites is established. Dehydroalanine formation at the Cys residue is predominant. The case of a contryphan, a cyclic peptide disulfide derived from Conus snail venom, illustrates the utility of negative ion mass spectrometry in disulfide identification. Complementary information is derived by combining the fragmentation patterns obtained from positive and negative ions of disulfide containing peptides.     

Ion activation methods for tandem mass spectrometry

This tutorial presents the most common ion activation techniques employed in tandem mass spectrometry. In-source fragmentation and metastable ion decompositions, as well as the general theory of unimolecular dissociations of ions, are initially discussed. This is followed by tandem mass spectrometry, which implies that the activation of ions is distinct from the ionization step, and that the precursor and product ions are both characterized independently by their mass/charge ratios. In collision-induced dissociation (CID), activation of the selected ions occurs by collision(s) with neutral gas molecules in a collision cell. This experiment can be done at high (keV) collision energies, using tandem sector and time-of-flight instruments, or at low (eV range) energies, in tandem quadrupole and ion trapping instruments. It can be performed using either single or multiple collisions with a selected gas and each of these factors influences the distribution of internal energy that the activated ion will possess. While CID remains the most common ion activation technique employed in analytical laboratories today, several new methods have become increasingly useful for specific applications. More recent techniques are examined and their differences, advantages and disadvantages are described in comparison with CID. Collisional activation upon impact of precursor ions on solid surfaces, surface-induced dissociation (SID), is gaining importance as an alternative to gas targets and has been implemented in several different types of mass spectrometers. Furthermore, unique fragmentation mechanisms of multiply-charged species can be studied by electron-capture dissociation (ECD). The ECD technique has been recognized as an efficient means to study non-covalent interactions and to gain sequence information in proteomics applications. Trapping instruments, such as quadrupole ion traps and Fourier transform ion cyclotron resonance instruments, are particularly useful for the photoactivation of ions, specifically for fragmentation of precursor ions by infrared multiphoton dissociation (IRMPD). IRMPD is a non-selective activation method and usually yields rich fragmentation spectra. Lastly, blackbody infrared radiative dissociation is presented with a focus on determining activation energies and other important parameters for the characterization of fragmentation pathways. The individual methods are presented so as to facilitate the understanding of each mechanism of activation and their particular advantages and representative applications.     

Comparative study of organic dyes with time-of-flight static secondary ion mass spectrometry and related techniques

A series of cationic, zwitterionic and anionic fluorinated carbocyanine dyes, spin-coated on Si substrates, were measured with time-of-flight static secondary ion mass spectrometry (TOF-S-SIMS) under Ga(+) primary ion bombardment. Detailed fragmentation patterns were developed for all dyes measured. In the positive mode, the resulting spectra showed very intense signals for the precursor ions of the cationic dyes, whereas the protonated signals of the anionic dyes were hardly detected. Differences of three orders of magnitude were repeatedly observed for the secondary ion signal intensities of cationic and anionic dyes, respectively. All measured dyes yielded mass spectra containing several characteristic fragment ions. Although the secondary ion yields were still higher for the cationic than the anionic dye fragments, the difference was reduced to a factor of < or =10. This result and the fact that M(+), [M + H](+) or [M + 2H](+) are even-electron species make it very likely that the recorded fragments were not formed directly out of the (protonated) parent ions M(+), [M + H](+) or [M + 2H](+). In the negative mode, none of the recorded spectra contained molecular information. Only signals originating from some characteristic elements of the molecules (F, Cl), the anionic counter ion signal and some low-mass organic ions were detected. A comparative study was made between TOF-S-SIMS, using Ga(+) primary ions, and other mass spectrometric techniques, namely fast atom bombardment (FAB), electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). The measurements showed that MALDI, ESI and FAB all give rise to spectra containing molecular ion signals. ESI and FAB produced M(+) and [M + H](+) signals, originating from the cationic and zwitterionic dyes, in the positive mode and M(-) and [M - H](-) signals of the anionic and zwitterionic dyes in the negative mode. With MALDI, molecular ion signals were measured in both modes for all the dyes. Structural fragment ions were detected for FAB, ESI and MALDI in both the positive and negative modes. Compared with the other techniques, TOF-S-SIMS induced a higher degree of fragmentation.