Mass spectrometric analysis of low molecular mass polyesters by laser desorption/ionization on porous silicon

A low molecular mass polyester was analyzed by desorption/ionization on porous silicon (DIOS) mass spectrometry. The results were compared with those of matrix-assisted laser desorption ionization (MALDI) mass spectrometry using matrixes of alpha-cyano-4-hydroxycinnamic acid (CHCA) and 10,15,20-tetrakis(pentafluorophenyl)porphyrin (F20TPP). The CHCA matrix was not suitable for characterization of low molecular mass components of the polyester because the matrix-related ions interfered with the component ions. On the other hand, the F20TPP matrix showed no interference because no matrix-related ions appeared below m/z 822. However, the solvent selection for determining optimal conditions of sample preparation was limited, because F20TPP does not dissolve readily in any of the available organic solvents. In the DIOS spectra, the polymer ions were observed at high sensitivity without a contaminating ion. No matrix is needed for DIOS spectra of low molecular mass polyesters, facilitating sample preparation and selectivity of a precursor ion in post-source decay measurements.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of 4-sulfophenyl isothiocyanate-derivatized peptides on AnchorChip sample supports using the sodium-tolerant matrix 2,4,6-trihydroxyacetophenone and diammonium citrate

The reagent 4-sulfophenyl isothiocyanate (SPITC) is an effective, stable, and inexpensive alternative to commercially available reagents used in the N-terminal sulfonation of peptides for enhanced postsource decay (PSD) in matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analyses. However, suppression of ionization of sulfonated peptides due to sample and matrix contaminants such as sodium can be a problem when using prestructured MALDI target sample supports, such as the Bruker Daltonics AnchorChip. We show that use of the salt-tolerant matrix 2,4,6-trihydroxyacetophenone containing diammonium citrate (THAP/DAC) as an alternative to alpha-cyanohydroxycinnamic acid (HCCA) reduces the need for extensive washing of ZipTip-bound peptides or additional on-target sample clean-up steps. Use of the THAP/DAC matrix results in selective ionization of sulfonated peptides with greater peptide coverage, as well as detection of higher mass derivatized peptides, than was observed for HCCA or THAP alone. The THAP/DAC matrix is quite tolerant of sodium contamination, with SPITC-peptides detectable in preparations containing up to 50 mM NaCl. In addition, THAP/DAC matrix was found to promote efficient PSD fragmentation of sulfonated peptides. We demonstrated the utility of using the THAP/DAC MALDI matrix for peptide sequencing with DNA polymerase beta tryptic peptide mixture, as well as tryptic peptides derived from Xiphophorus maculatus brain extract proteins previously separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE).     

Atmospheric pressure laser desorption/ionization using a 6–7 µm‐band mid‐infrared tunable laser and liquid water matrix

Due to the characteristic absorption peaks in the IR region, various molecules can be used as a matrix for infrared matrix‐assisted laser desorption/ionization (IR‐MALDI). Especially in the 6–7 µm‐band IR region, solvents used as the mobile phase for liquid chromatography have absorption peaks that correspond to their functional groups, such as O–H, CO, and CH₃. Additionally, atmospheric pressure (AP) IR‐MALDI, which is applicable to liquid‐state samples, is a promising technique to directly analyze untreated samples. Herein we perform AP‐IR‐MALDI mass spectrometry of a peptide, angiotensin II, using a mid‐IR tunable laser with a tunable wavelength range of 5.50–10.00 µm and several different matrices. The wavelength dependences of the ion signal intensity of [M + H]⁺ of the peptide are measured using a conventional solid matrix, α‐cyano‐4‐hydroxycinnamic acid (CHCA) and a liquid matrix composed of CHCA and 3‐aminoquinoline. Other than the O–H stretching and bending vibration modes, the characteristic absorption peaks are useful for AP‐IR‐MALDI. Peptide ions are also observed from an aqueous solution of the peptide without an additional matrix, and the highest peak intensity of [M + H]⁺ is at 6.00 µm, which is somewhat shorter than the absorption peak wavelength of liquid water corresponding to the O–H bending vibration mode. Moreover, long‐lasting and stable ion signals are obtained from the aqueous solution. AP‐IR‐MALDI using a 6–7 µm‐band IR tunable laser and solvents as the matrix may provide a novel on‐line interface between liquid chromatography and mass spectrometry. Copyright © 2015 John Wiley & Sons, Ltd.     

Protein identification with Teflon as matrix-assisted laser desorption/ionization sample support

Protein identification is a critical step in proteomics, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) plays an important role in that identification. Polytetrafluoroethylene (Teflon) was tested as a new MALDI sample support to improve protein identification. The tryptic peptides obtained from a model protein were bound to the surface of a modified MALDI sample holder via the hydrophobic interactions that occur between the Teflon surface and the peptide ion-pairs, and the affinity of alpha-cyano-4-hydroxycinnamic acid for the peptides. During that surface-binding step, the peptide mixture was also desalted and concentrated. A greater number of matched peptides and a larger sequence coverage were obtained for the proteins when Teflon was used as the sample support compared with conventional sample preparation methods and a stainless-steel surface. In addition, the characterization of a small amount of protein was improved with Teflon. Nine silver-stained protein spots obtained from 2-D gel of a human cerebrospinal fluid (CSF) proteome were identified by this method. Among the nine protein spots, peptide 6:c3c fragment and procollagen c-proteinase enhancer were not annotated in any published 2-D map of human CSF. A Teflon MALDI sample support is a low-cost, simple, and effective method that can be used to improve the quality of the MALDI mass spectrum of a complex tryptic peptide mixture, and to achieve a higher level of reliability and success in protein identification.     

Disappearance of interfering alkali-metal adducted peaks from matrix-assisted laser desorption/ionization mass spectra of peptides with serine addition to alpha-cyano-4-hydroxycinnamic acid matrix

It has been described that ion yield in both positive- and negative-ion matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) of peptides is often inhibited by trace amounts of alkali metals and that the MALDI mass spectra are contaminated by the interfering peaks originating from traces of alkali metals, even when sample preparation is carefully performed. Addition of serine to the commonly used MALDI matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) significantly improved and enhanced the signals of both protonated and deprotonated peptides, [M+H](+) and [M-H](-). The addition of serine to CHCA matrix eliminated the alkali-metal ion adducts, [M+Na](+) and [M+K](+), and the CHCA cluster ions from the mass spectra. Serine and serinephosphate as additives to CHCA enhanced and improved the formation of molecular-related ions of phosphopeptides in negative-ion MALDI mass spectra.     

Matrix-free infrared soft laser desorption/ionization

Infrared soft laser desorption/ionization was performed using a 2.94 µm Er : YAG laser and a commercial reflectron time-of-flight mass spectrometer. The instrument was modified so that a 337 nm nitrogen laser could be used concurrently with the IR laser to interrogate samples. Matrix-assisted laser desorption/ionization (MALDI), laser desorption/ionization and desorption/ionization on silicon with UV and IR lasers were compared. Various target materials were tested for IR soft desorption ionization, including stainless steel, aluminum, copper, silicon, porous silicon and polyethylene. Silicon surfaces gave the best performance in terms of signal level and low-mass interference. The internal energy resultant of the desorption/ionization was assessed using the easily fragmented vitamin B₁₂ molecule. IR ionization produced more analyte fragmentation than UV-MALDI analysis. Fragmentation from matrix-free IR desorption from silicon was comparable to that from IR-MALDI. The results are interpreted as soft laser desorption and ionization resulting from the absorption of the IR laser energy by the analyte and associated solvent molecules. Copyright © 2004 John Wiley & Sons, Ltd.     

Identification of formylglycine in sulfatases by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

C(alpha)-Formylglycine, the catalytic amino acid residue in the active site of sulfatases, is generated by post-translational modification of a cysteine or serine residue. We describe a highly sensitive procedure for the detection of C(alpha)-formylglycine-containing peptides in tryptic digests of sulfatase proteins. The protocol is based on the formation of hydrazone derivatives of C(alpha)-formylglycine-containing peptides when using dinitrophenylhydrazine as a matrix for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The hydrazone derivatives desorb and ionize with high efficiency and can be detected in the sub-femtomole range. The presence of C(alpha)-formylglycine is indicated by a mass increment of 180.13 u, corresponding to the hydrazone moiety, and also by a unique C-terminal fragment ion, characteristic of sulfatases, that becomes prominent in MALDI post-source decay mass spectra of the hydrazone derivatives.     

Characterization of some synthetic Ru and Ir complexes by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was applied to the analysis of Ru(OCOCF(3))(2)(CO)(PPh(3))(2), Ru(OCOC(3)F(7))(2)(CO)(PPh(3))(2), Ir(tBuppy)(3) and Ir(ppy)(2)(acac) complexes. A troublesome problem in the MALDI-TOFMS characterization of these metal complexes is the possible replacement of complex ligands by matrix. In this contribution, 10 matrices, ranging from acidic to basic, were investigated: alpha-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), sinapinic acid (SA), dithranol, 2,4,6-trihydroxyactophenone (THAP), 6-azo-2-thiothymine (ATT), norharman, 2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2-enylidene]malononitrile (DCTB), 4-nitroaniline (NA) and 2-amino-5-nitrophyridine (ANP). With most of the matrices, including the neutral and basic ones, matrix substitution of ligand could clearly be detected. Based on the experimental results, possible mechanisms of matrix substitution were discussed. It was demonstrated that the ligand exchange process might also occur through the gas-phase reactions initiated by laser shots. Among the matrices tested, DCTB was found to be the best one for the complexes that are prone to ligand exchange by matrix.     

Internal energy transfer in laser desorption/ionization from silicon nanowires

Laser-induced desorption/ionization from silicon nanowires (SiNW) is an emerging method for mass spectrometry of small to medium-size molecules. In this new technique, we examined the internal energy transfer to seven benzylpyridinium thermometer ions and extracted the corresponding internal energy distributions. To explore the effect of the energy-deposition rate on the internal energy transfer, two lasers with significantly different pulse lengths (4 ns vs 22 ps) were utilized as excitation sources. A comparison of ion yields indicated that the SiNW substrates required 5-8 times less laser fluence for ion production than either matrix-assisted laser desorption/ionization (MALDI) or desorption/ionization on silicon (DIOS). In contrast however, the survival yield (SY) values showed that the internal energy transferred to the thermometer ions was more than (ps laser) or comparable to (ns laser) MALDI but it was significantly less than in DIOS. The internal energy transfer was only slightly dependent on laser fluence and on wire density. These effects were rationalized in terms of the confinement of thermal energy in the nanowires and of unimpeded three-dimensional plume expansion. Unlike in MALDI from CHCA and in perfluorophenyl-derivatized DIOS, for desorption from SiNWs the effect of laser pulse length on the internal energy transfer was found to be negligible.     

Phosphopeptide detection and sequencing by matrix-assisted laser desorption/ionization quadrupole time-of-flight tandem mass spectrometry

A prototype matrix-assisted laser desorption/ionization quadrupole time-of-flight (MALDI-TOF) tandem mass spectrometer was used to sequence a series of phosphotyrosine-, phosphothreonine- and phosphoserine-containing peptides. The high mass resolution and mass accuracy of the instrument allowed the localization of one, three or four phosphorylated amino acid residues in phosphopeptides up to 3.1 kDa. Tandem mass spectra of two different phosphotyrosine peptides permitted amino acid sequence determination and localization of one and three phosphorylation sites, respectively. The phosphotyrosine immonium ion at m/z 216.04 was observed in these MALDI low-energy CID tandem mass spectra. Elimination of phosphate groups was evident from the triphosphorylated peptide but not from the monophosphorylated species. The main fragmentation pathway for the synthetic phosphothreonine-containing peptide and for phosphoserine-containing peptides derived from beta-casein and ovalbumin was the beta-elimination of phosphoric acid with concomitant conversion of phosphoserine to dehydroalanine and phosphothreonine to 2-aminodehydrobutyric acid. Peptide fragment ions of the b- and y-type allowed, in all cases, the localization of phosphorylation sites. Ion signals corresponding to (b-17), (b-18) and (y-17) fragment ions were also observed. The abundant neutral loss of phosphoric acid (-98 Da) is useful for femtomole level detection of phosphoserine-peptides in crude peptide mixtures generated by gel in situ digestion of phosphoproteins.     

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.     

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.     

A quantitative model of ultraviolet matrix-assisted laser desorption/ionization

A quantitative model of primary ionization in ultraviolet matrix-assisted laser desorption/ionization (UV-MALDI) is presented. It includes not only photochemical processes such as exciton pooling, but also the effects of the desorption event. The interplay of these two is found to be a crucial aspect of the MALDI process. The desorbing plume is modeled as an adiabatic expansion with entrained clusters. The parameters in the model are defined as much as possible via experiment or by analogy with known effects. The model was applied to the matrix 2,5-dihydroxybenzoic acid and found to reproduce the fluence dependence of the fluorescence yield and key features of the picosecond two-pulse ion generation efficiency curves. In addition, the model correctly predicts a fluence rather than irradiance threshold, the magnitude of the threshold, the magnitude of the ion yield, laser wavelength effects, plume temperatures, plume expansion velocities and the spot size effect.     

Rapid screening of anthocyanins in berry samples by surfactant-mediated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Surfactant-mediated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been used for the identification of flavonoids from three berry extracts: lowbush blueberry (Vaccinium angustifolium), lingonberry (Vaccinium vitis-idaea), and blackberry (Rubus armeniacus). The addition of the surfactant led to suppression of matrix ions from both alpha-cyano-4-hydroxycinnamic acid (CHCA) and 2',4',6'-trihydroxyacetophenone (THAP). This is the first case of this method being successfully employed with a matrix other than CHCA. It was observed that CHCA led to a great deal of fragmentation of the sugar moiety from glycosides, whereas THAP produced more intact glycoside molecules, and thus leads to better characterization of the flavonoids in a berry sample. The flavonoids were characterized and quantified by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) with UV detection. Although MALDI-TOF-MS did not lead to the identification of as many flavonoids, it did enable us to identify many anthocyanin glycosides. Quantification was achieved and demonstrated that use of the THAP matrix can enable quantification of the intact glycosides with relative standard deviation (RSD) values of less than 10% with surfactant addition. These results are comparable with LC results. MALDI-TOF-MS with THAP matrix thus provided a rapid method for the qualitative screening of these compounds. It took only a few minutes, greatly reducing the analysis time from that in traditional LC/MS methods.     

Reduction of organic dyes in matrix-assisted laser desorption/ionization and desorption/ionization on porous silicon

Reduction of analytes in matrix-assisted laser desorption/ionization (MALDI) often obscures the actual determination of molecular structure. To address the redox reactions in laser desorption/ionization processes, the organic dyes Methylene Blue, Janus Green B, Crystal Violet and Rhodamine B were analyzed by MALDI or by desorption/ionization on porous silicon (DIOS). Susceptibility to reduction in MALDI was dependent on both the reduction potentials of analytes and the molar ratio of analyte to matrix molecules. Addition of Cu(II) ions as an electron scavenger suppressed the reduction of Methylene Blue in MALDI. The results suggested that electron transfer to analytes from the sample target and/or from the matrix contributed to the reduction. In DIOS, the reductions of organic dyes were more prominent than in MALDI, and were not prevented by Cu(II) ion doping, probably due to direct contact of the analytes with silicon which had little electric resistance.     

Two-laser infrared and ultraviolet matrix-assisted laser desorption/ionization

Matrix-assisted laser desorption/ionization (MALDI) was performed using two pulsed lasers with wavelengths in the IR and UV regions. A 10.6 micro m pulsed CO(2) laser was used to irradiate a MALDI target, followed after an adjustable delay by a 337 nm pulsed nitrogen laser. The sample consisted of a 2,5-dihydroxybenzoic acid matrix and bovine insulin guest molecule. The pulse energy for both of the lasers was adjusted so that the ion of interest, either the matrix or guest ion, was not produced by either of the lasers alone. The delay time for maximum ion yield occurs at 1 micro s for matrix and guest ions and the signal decayed to zero in approximately 400 micro s. A mechanism is presented for enhanced UV MALDI ion yield following the IR laser pulse based on transient heating.     

Analysis of deprotonated acids with silicon nanoparticle-assisted laser desorption/ ionization mass spectrometry

Chemically modified silicon nanoparticles were applied for the laser desorption/negative ionization of small acids. A series of substituted sulfonic acids and fatty acids was studied. Compared to desorption ionization on porous silicon (DIOS) and other matrix-less laser desorption/ionization techniques, silicon nanoparticle-assisted laser desorption/ionization (SPALDI) mass spectrometry allows for the analysis of acids in the negative ion mode without the observation of multimers or cation adducts. Using SPALDI, detection limits of many acids reached levels down to 50 pmol/μl. SPALDI of fatty acids with unmodified silicon nanoparticles was compared to SPALDI using the fluoroalkyl silylated silicon powder, with the unmodified particles showing better sensitivity for fatty acids, but with more low-mass background due to impurities and surfactants in the untreated silicon powder. The fatty acids exhibited a size-dependent response in both SPALDI and unmodified SPALDI, showing a signal intensity increase with the chain length of the fatty acids (C12-C18), leveling off at chain lengths of C18-C22. The size effect may be due to the crystallization of long chain fatty acids on the silicon. This hypothesis was further explored and supported by SPALDI of several, similar sized, unsaturated fatty acids with various crystallinities. Fatty acids in milk lipids and tick nymph samples were directly detected and their concentration ratios were determined by SPALDI mass spectrometry without complicated and time-consuming purification and esterification required in the traditional analysis of fatty acids by gas chromatography (GC). These results suggest that SPALDI mass spectrometry has the potential application in fast screening for small acids in crude samples with minimal sample preparation.     

Matrix-assisted laser desorption/ionization mass spectrometry of collected bioaerosol particles

A method was developed for collection and analysis of bioaerosols by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry using a modified Andersen N6 bioaerosol collector. The overall goal of the study was to develop methods for obtaining mass spectra with minimal reagents and treatment steps for potential use in remote collection and analysis systems. Test bioaerosol particles were generated from a nebulized E. coli bacterial suspension and collected on MALDI targets placed in an Andersen N6 single-stage aerosol impactor. The bioaerosols were mixed with matrix either by deposition on a bare target with the matrix solution added later, or by deposition on a target pre-coated with matrix. The matrix compounds alpha-cyano-4-hydroxycinnamic acid (CHCA) and sinapic acid (SA) were tested and the SA matrix was found to give the best results in number of peaks, resolution, and signal-to-noise ratio. Deposition of bioaerosol particles onto the matrix pre-coated target did not produce signal in the m/z region above 1000, but the signal could be recovered with the addition of a 1:1 (v/v) acetonitrile/water solvent. Addition of solvent by pipette to the pre-coated targets after particle deposition recovered signal comparable to the dried-droplet sample preparations, whereas solvent sprayed into the impactor recovered fewer peaks. Deposition on pre-coated targets with post-collection solvent addition was superior to deposition on bare target followed by post-collection addition of matrix solution.     

Decomposition of protein nitrosothiolsin matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry

The S-nitrosylation of proteins is involved in the trafficking of nitric oxide (NO) in intra- and extracellular milieus. To establish a mass spectrometric method for identifying this post-translational modification of proteins, a synthetic peptide and transthyretin were S-nitrosylated in vitro and analyzed by electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The intact molecular ion species of nitrosylated compounds was identified in the ESI mass spectrum without elimination of the NO group. However, the labile nature of the S-NO bond was evident when the in-source fragmentation efficiently generated [M + H - 30](+) ions. The decomposition was prominent for multiply charged transthyretin ions with high charge states under ordinary ESI conditions, indicating that the application of minimum nozzle potentials was essential for delineating the stoichiometry of nitrosylation in proteins. With MALDI, the S-NO bond cleavage occurred during the ionization process, and the subsequent reduction generated [M + H - 29](+) ions.     

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.