Expansion Cooling in the Matrix Plume is Under-Recognized in MALDI Mass Spectrometry

Time-of-flight (TOF) mass spectra for a peptide (Y₆) were obtained by utilizing matrix-assisted infrared laser desorption ionization (IR-MALDI) with glycerol as the matrix and by ultraviolet MALDI with α-cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid (SA), and 2,5-dihydroxybenzoic acid (DHB). Collisional activation during ion extraction and exothermicity in the gas-phase proton transfer were found to be unimportant as the driving forces for in-source (ISD) and post-source (PSD) decays, indicating that the thermal energy acquired during photo-ablation is responsible for their occurrence. The temperatures of [Y₆ + H]⁺ in the ‘early’ and ‘late’ matrix plumes were estimated by the kinetic analysis of the ISD and PSD yields, respectively. The order of the temperatures was glycerol < DHB ≈ SA < CHCA in the early plume and glycerol < DHB < SA < CHCA in the late plume. For each matrix, the temperature in the late plume was lower than in the early plume by 300–400 K, which was attributed to expansion cooling. The model (thermalization followed by expansion cooling) proposed to explain the occurrence of both rapid ISD and slow PSD is not only in sharp contrast with but also mutually exclusive with the prevailing explanation that the exothermicity in proton transfer and in-plume collisional activation are the driving forces for ion fragmentation in MALDI. The model also explains why MALDI is more successful for mass spectrometry of labile molecules than other desorption techniques that do not utilize a matrix. Factors affecting the plume temperature are also discussed.     

In-source decay and fragmentation characteristics of peptides using 5-aminosalicylic acid as a matrix in matrix-assisted laser desorption/ionization mass spectrometry

The use of 5-aminosalicylic acid (5-ASA) as a new matrix for in-source decay (ISD) of peptides including mono- and di-phosphorylated peptides in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is described. The use of 5-ASA in MALDI-ISD has been evaluated from several standpoints: hydrogen-donating ability, the outstanding sharpness of molecular and fragment ion peaks, and the presence of interference peaks such as metastable peaks and multiply charged ions. The hydrogen-donating ability of several matrices such as α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (2,5-DHB), 1,5-diaminonaphthalene (1,5-DAN), sinapinic acid (SA), and 5-ASA was evaluated by using the peak abundance of a reduction product [M + 2H + H]⁺ to that of non-reduced protonated molecule [M + H]⁺ of the cyclic peptide vasopressin which contains a disulfide bond (S-S). The order of hydrogendonating ability was 1,5-DAN > 5-ASA > 2,5-DHB > SA = CHCA. The chemicals 1,5-DAN and 5-ASA in particular can be classified as reductive matrices. 5-ASA gave peaks with higher sharpness for protonated molecules and fragment ions than other matrices and did not give any interference peaks such as multiply-protonated ions and metastable ions in the ISD mass spectra of the peptides used. Particularly, 1,5-DAN and 5-ASA gave very little metastable peaks. This indicates that 1,5-DAN and 5-ASA are more “cool” than other matrices. The 1,5-DAN and 5-ASA can therefore be termed “reductive cool” matrix. Further, it was confirmed that ISD phenomena such as N-Cα bond cleavage and reduction of S-S bond is a single event in the ion source. The characteristic fragmentations, which form a− and (a + 2)-series ions, [M + H − 15]⁺, [M + H − 28]⁺, and [M + H − 44]⁺ ions in the MALDI-ISD are described.     

Changes in Dissociation Efficiency and Kinetics of Peptide Ions Induced by Basic Residues and Their Mechanistic Implication

With matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry, total abundance of product ions formed by dissociation inside (in-source decay, ISD) and outside (post-source decay, PSD) the source was measured for peptide ions [Y ₅ X + H]⁺, [XY ₅ + H]⁺, [Y ₂ XY ₃ + H]⁺, and [XY ₄ X + H]⁺ (X = tyrosine (Y), histidine (H), lysine (K), and arginine (R) with H for the ionizing proton). α-Cyano-4-hydroxycinammic acid was used as matrix. Product abundance became smaller in the presence of basic residues (H, K, and R), in the order Y > H ≈ K > R. In particular, product abundances in ISD of peptide ions with R were smaller than those with H or K by an order of magnitude, which, in turn, were smaller than that for [Y ₆ + H]⁺ by an order of magnitude. Product abundance was affected by the most basic residue when more than one basic residue was present. A kinetic explanation for the data was attempted under the assumption of quasi-thermal equilibrium for peptide ions in MALDI plume which undergoes expansion cooling. Dramatic disparity in product abundance was found to arise from small difference in critical energy and entropy. Results indicate similar transition structures regardless of basic residues present, where the ionizing proton keeps interacting with a basic site. Further implication of the results on the dissociation mechanism along b-y channels is discussed.     

Dissociation kinetics of singly protonated leucine enkephalin investigated by time-resolved photodissociation tandem mass spectrometry

The yields of post-source decay (PSD) and time-resolved photodissociation (PD) at 193 and 266 nm were measured for singly protonated leucine enkephalin ([YGGFL + H]⁺), a benchmark in the study of peptide ion dissociation, by using tandem time-of-flight mass spectrometry. The peptide ion was generated by matrix-assisted laser desorption ionization (MALDI) using 2,5-dihydroxybenzoic acid as the matrix. The critical energy (E₀) and entropy (ΔS‡ at 1000 K) for the dissociation were determined by Rice-Ramsperger-Kassel-Marcus fit of the experimental data. MALDI was done for a mixture of YGGFL and Y₆ and the plume temperature determined by the kinetic analysis of [Y₆ + H]⁺ data were used to improve the precision of E₀ and ΔS‡ for [YGGFL + H]⁺. E₀ and ΔS‡ thus determined (E₀ = 0.67 ± 0.08 eV, ΔS‡=−24.4 ± 3.2 eu with 1 eu = 4.184 J K⁻¹mol⁻¹) were significantly different from those determined by blackbody infrared radiative dissociation (BIRD) (E₀ = 1.10 eV, ΔS‡ = −14.9 eu), and by surface-induced dissociation (SID) (E₀ = 1.13 eV, ΔS‡ = −10.3 eu). Analysis of the present experimental data with the SID kinetics (and BIRD kinetics also) led to an unrealistic situation where not only PSD and PD but also MALDI-TOF signals could not be detected. As an explanation for the discrepancy, it was suggested that transition-state switching occurs from an energy bottleneck (SID/BIRD) to an entropy bottleneck (PSD/PD) as the internal energy increases.     

Coumarins as new matrices for matrix-assisted laser-desorption/ionization Fourier transform ion cyclotron resonance mass spectrometric analysis of hydrophobic compounds

Hydrophobic compounds with hydroxyl, aldehyde or ketone groups are generally difficult to detect using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), because these compounds have low proton affinity and are poorly ionized by MALDI. Herein, coumarins have been used as new matrices for MALDI-MS analysis of a variety of hydrophobic compounds with low ionization efficiency, including steroids, coenzyme Q10, a cyclic lipopeptide and cholesterol oleate. Five coumarins, including coumarin, umbelliferone, esculetin, 7-hydroxycoumarin-3-carboxylic acid (HCA) and 6,7-dihydroxycoumarin-3-carboxylic acid (DCA), were compared with the conventional matrices of 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (CHCA). Coumarins with hydroxyl or carboxylic acid groups enabled detection. Taking DCA as an example, this matrix proved to be superior to DHB or CHCA in detection sensitivity, stability, spot-to-spot and sample-to-sample reproducibility, and accuracy. DCA increased the stability of the target compounds and decreased the loss of water. The [M + Na]⁺ peaks were observed for all target compounds by adding NaCl as an additive, and the [M − H₂O + H]⁺ and [M + H]⁺ peaks decreased. DCA was selected for the identification of sterols in yeast cells, and thirteen sterols were detected by Fourier transform ion cyclotron resonance (FT ICR) mass spectrometry. This work demonstrates the potential of DCA as a new matrix for detection of hydrophobic molecules by MALDI-MS and provides an alternative tool for screening sterols in antifungal research.     

Enhanced in-source fragmentation in MALDI-TOF-MS of oligonucleotides using 1,5-diaminonapthalene

The capability to rapidly and confidently determine or confirm the sequences of short oligonucleotides, including native and chemically-modified DNA and RNA, is important for a number of fields. While matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) has been used previously to sequence short oligonucleotides, the typically low fragmentation efficiency of in-source or post-source decay processes necessitates the accumulation of a large number of spectra, thus limiting the throughput of these methods. Here we introduce a novel matrix, 1,5-diaminonapthalene (DAN), for facile in-source decay (ISD) of DNA and RNA molecular anions, which allows for rapid sequence confirmation. d-, w-, and y-series ions are prominent in the spectra, complementary to the (a-B)- and w- ions that are typically produced by MALDI post-source decay (PSD). Results are shown for several model DNA and RNA oligonucleotides, including combinations of DAN-induced fragmentation with true tandem TOF MS (MS/MS) for pseudo-MS³ and “activated-ion PSD.”     

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

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

H/D exchange kinetics: Experimental evidence for formation of different b fragment ion conformers/isomers during the gas-phase peptide sequencing

Electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) combined with H/D exchange reactions was utilized to explore the existence of different b₅⁺ and b₄⁺ fragment ion conformers/isomers of hexapeptide WHWLQL in the gas phase. Distinct H/D exchange trends for protonated WHWLQL ([M + H]⁺) and its b₅⁺ and b₄⁺ fragment ions (with ND₃) were observed. Isolated ¹²C_all isotopomers of both b₅⁺ and b₄⁺ fragment ions yielded bimodal distributions of H/D exchanged product ions. The H/D exchange reaction kinetics also confirmed that b₅⁺ and b₄⁺ fragment ions exist as combination of slow-exchanging (“s”) and fast-exchanging (“f”) species. The calculated rate constant for the first labile hydrogen exchange of [M + H]⁺ (k_{[M + H]} ⁺ = 3.80 ± 0.7 × 10⁻¹⁰ cm³ mol⁻¹ s⁻¹) was ∼30 and ∼5 times greater than those for the “s” and “f” species of b₅⁺, respectively. Data from H/D exchange of isolated “s” species at longer ND₃ reaction times confirmed the existence of different conformers or isomers for b₅⁺ fragment ions. The sustained off-resonance irradiation collision-activated dissociation (SORI-CAD) of WHWLQL combined with the H/D exchange reactions indicate that “s” and “f” species of b₅⁺ and b₄⁺ fragment ions can be produced in the ICR cell as well as the ESI source. The significance of these observations for detailed understanding of protein sequencing and ion fragmentation pathways is discussed.     

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

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

Why do the Abundances of Ions Generated by MALDI Look Thermally Determined?

In a previous study (J. Mass Spectrom. 48, 299–305, 2013), we observed that the abundance of each ion in a matrix-assisted laser desorption ionization (MALDI) spectrum looked thermally determined. To find out the explanation for the phenomenon, we estimated the ionization efficiency and the reaction quotient (Q_A) for the autoprotolysis of matrix, M + M → [M + H]⁺ + [M ? H]^?, from the temperature-controlled laser desorption ionization spectra of α-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB). We also evaluated the equilibrium constants (K_A) for the autoprotolysis at various temperatures by quantum chemical calculation. Primary ion formation via various thermal models followed by autoprotolysis-recombination was compatible with the observations. The upper limit of the effective temperature of the plume where autoprotolysis-recombination occurs was estimated by equating Q_A with the calculated equilibrium constant.

1H-Pteridine-2,4-dione (lumazine): a new MALDI matrix for complex (phospho)lipid mixtures analysis

Nowadays, matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry represents an emerging and versatile tool for analysis of lipids. However, direct (i.e., with no previous separation of lipid classes) analysis of crude extracts containing a complex mixture of lipids (a problem typically encountered in shotgun lipidomics) is still a quite challenging task using a conventional MALDI matrix such as 2,5-dihydroxybenzoic acid (DHB). Indeed, in the presence of phospholipids containing quaternary ammonium groups, such as phosphatidylcholines and sphingomyelins, strong ionization-suppression effects are experienced especially in positive ion mode. To overcome this limitation, lumazine (1H-pteridine-2,4-dione) was evaluated as an alternative matrix. Lumazine in the solid state showed an absorption maximum at 350 nm, ionizes/desorbs without appreciable decomposition and extensive cluster formation, and can be used in both ion modes. In positive ion mode, the main species were M ^•+ and 2M ^•+ radical cations and cationized species ([M+H]⁺, [M+Na]⁺, [M+2Na+2Li-3H]⁺). In negative ion mode, the main signals observed were the deprotonated molecular ion and the radical anion. The signal-to-noise ratio for phosphatidylglycerols and phosphatidylethanolamines using lumazine was almost 1 order of magnitude higher than that observed for DHB. Lumazine was successfully used for MALDI analysis (positive and negative ion modes) of crude lipid extracts of milk, soymilk, and hen egg, where phosphatidylethanolamines, phosphatidylserines, and phosphatidylinositols could additionally be detected.     

Sol-Gel Microsphere Pelletisation Process for Fabrication of (U,Pu)O2, (U,Pu)C and (U,Pu)N Fuel Pellets for the Prototype Fast Breeder Reactor in India

A “dust-free” sol-gel microsphere pelletisation (SGMP) process has been developed for fabrication of (U,Pu)O₂, (U,Pu)C and (U,Pu)N fuel pellets containing around 15% plutonium for the forthcoming prototype fast breeder reactor (PFBR) in India. The objective was to produce homogeneous sintered pellets of ∼85% T.D. with a predominantly open-pore structure. Hydrated gel-microspheres of UO₃+PuO₂ and UO₃+PuO₂+C have been prepared from nitrate solutions of uranium and plutonium by the “ammonia internal gelation” process, using hexamethylene tetramine (HMTA) as an ammonia generator and silicone oil at 90±1°C as gelation bath. For oxide fuel pellets, the hydrated UO₃+PuO₂ gel-microspheres were calcined at around 700°C in Ar+8% H₂ atmosphere to produce “non-porous”, “free-flowing” and coarse (around 400 micron) microspheres which could be directly pelletised at 550 MPa to green pellets. The mixed oxide pellets were subjected either to low temperature (∼1100°C) oxidative sintering (LTS) in N₂+air containing ∼1500 ppm O₂ or to high temperature (1650°C) sintering, (HTS) in Ar+8% H₂. For monocarbide and mononitride pellets, hydrated gel-microspheres of UO₃+PuO₂+C were subjected to carbothermic synthesis in vacuum (∼1 Pa) and flowing nitrogen (flow rate: 1.2 m³/h) in the temperature range of 1450–1550°C respectively. The monocarbide and mononitride microspheres thus produced were relatively hard and required higher compaction pressure (∼1200 MPa) for making reen pellets which could be sintered to 85% T.D. in Ar+8% H₂ at 1700°C. The sintered oxide, monocarbide and mononitride pellets had a “blackberry” “open” pore microstructure with fine grain size. The microspheres retained their individual identity in the sintered pellets because during sintering densification took place mainly within and not between the microspheres.     

A new method for analysis of disulfide-containing proteins by matrix-assisted laser desorption ionization (MALDI) mass spectrometry

A simple and high-throughput method for the identification of disulfide-containing peptides utilizing peptide-matrix adducts is described. Some commonly used matrices in MALDI mass spectrometry were found to specifically react with sulfhydryl groups within peptide, thus allowing the observation of the peptide-matrix adduct ion [M+n+n′ matrix+H]⁺ or [M+n+n′ matrix+Na]⁺ (n = the number of cysteine residues, n′=1, 2,…, n) in MALDI mass spectra after chemical reduction of disulfide-linked peptides. Among several matrices tested, α-cyano-4-hydroxycinnamic acid (CHCA, molecular mass 189 Da) and α-cyano-3-hydroxycinnamic acid (3-HCCA) were found to be more effective for MALDI analysis of disulfide-containing peptides/proteins. Two reduced cysteines involved in a disulfide bridge resulted in a mass shift of 189 Da per cysteine, so the number of disulfide bonds could then be determined, while for the other matrices (sinapinic acid, ferulic acid, and caffeic acid), a similar addition reaction could not occur unless the reaction was carried out under alkaline conditions. The underlying mechanism of the reaction of the matrix addition at sulfhydryl groups is proposed, and several factors that might affect the formation of the peptide-matrix adducts were investigated. In general, this method is fast, effective, and robust to identify disulfide bonds in proteins/peptides.     

A study on mass spectrometry of methylated [60] fullerenes using the “in-beam” electron impact technique

Mass spectra of the methylated [60]fullerenes were obtained by EI mass spectrometry using “desorption” or “in-beam” technique. The mass spectra of the methylated fullerenes, C₆₀Me_n, have the molecular ion peak M⁺ indicating that the product is stable under the MS (EI) conditions. The appearance of an intense peak at m/z 360 was assigned to the formation of fullerene dication C₆₀⁺⁺. The remaining peaks were assigned to successive loss of methyl groups from molecular monocation and dication.     

A new copper containing MALDI matrix that yields high abundances of [Peptide + Cu]+ ions

The dinuclear copper complex (α-cyano-4-hydroxycinnamic acid (CHCA) copper salt (CHCA)₄Cu₂), synthesized by reacting CHCA with copper oxide (CuO), yields increased abundances of [M + xCu − (x−1)H]⁺ (x = 1–6) ions when used as a matrix for matrix-assisted laser desorption ionization (355 nm Nd:YAG laser). The yield of [M + xCu − (x−1)H]⁺ (x = 1∼6) ion is much greater than that obtained by mixing peptides with copper salts or directly depositing peptides onto oxidized copper surfaces. The increased ion yields for [M + xCu − (x−1)H]⁺ facilitate studies of biologically important copper binding peptides. For example, using this matrix we have investigated site-specific copper binding of several peptides using fragmentation chemistry of [M + Cu]⁺ and [M + 2Cu − H]⁺ ions. The fragmentation studies reveal interesting insight on Cu binding preferences for basic amino acids. Most notable is the fact that the binding of a single Cu⁺ ion and two Cu⁺ ions are quite different, and these differences are explained in terms of intramolecular interactions of the peptide-Cu ionic complex.     

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.     

Comparison of mass spectra of peptides in different matrices using matrix-assisted laser desorption/ionization and a multi-turn time-of-flight mass spectrometer, MULTUM-IMG.

The mass spectra of peptides obtained with different matrices were compared using a matrix-assisted laser desorption/ionization (MALDI) ion source and a multi-turn time-of-flight (TOF) mass spectrometer, MULTUM-IMG, which has been developed at Osaka University. Two types of solid matrices, alpha-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB), and a liquid matrix made from a mixture of 3-aminoquinoline and CHCA were used. When measuring the peak signal intensity of human angiotensin II [M+H]+ from a fixed sample position, the liquid matrix produced a stable signal over 1000 laser shots, while the signal obtained with CHCA and DHB decayed after about 300 and 100 shots, respectively. Significant differences in the mass resolving power were not observed between the spectra obtained with the three matrices. Signal peak areas were measured as a function of the cycle number in a multi-turn ion trajectory, i.e., the total flight time over a millisecond time scale. For both [M+H]+ of human angiotensin II and bovine insulin, the decay of the signal peak area was the most significant with CHCA, while that measured with DHB was the smallest. The results of the mean initial ion velocity measurements suggested that the extent of metastable decomposition of the analyte ions increased in order of DHB, the liquid matrix, and CHCA, which is consistent with the difference in the decay of the signal peak area as the total flight time increased.     

Spectral Reproducibility and Quantification of Peptides in MALDI of Samples Prepared by Micro-Spotting

Previously, we reported that MALDI spectra of peptides became reproducible when temperature was kept constant. Linear calibration curves derived from such spectral data could be used for quantification. Homogeneity of samples was one of the requirements. Among the three popular matrices used in peptide MALDI [i.e., α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), and sinapinic acid (SA)], homogeneous samples could be prepared by conventional means only for CHCA. In this work, we showed that sample preparation by micro-spotting improved the homogeneity for all three cases.

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

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

Structural Characterization of Neutral Oligosaccharides by Laser-Enhanced In-Source Decay of MALDI-FTICR MS

MALDI in-source decay (ISD) technique described to date has proven to be a convenient and rapid method for sequencing purified peptides and proteins. However, the general ISD still can not produce adequate fragments for the detailed structural elucidation of oligosaccharides. In this study, an efficient and practical method termed the laser-enhanced ISD (LEISD) technique of MALDI-FTICR MS allows highly reliable and abundant fragmentation of the neutral oligosaccharides, which was attributed to the ultrahigh irradiation laser of mJ level. The yield of ISD fragmentation was evaluated under different laser powers for 7 neutral oligosaccharides using DHB as matrix. Better quality ISD spectra including fragment ions in low-mass region were obtained at higher laser power. Results from the LEISD of oligosaccharides demonstrated that a significantly better signal-to-noise ratio (S/N) and more structural information could be obtained in comparison to the conventional CID. It was also suggested that the valuable A ions derived from cross-ring cleavage of the linear oligosaccharides allowed the distinction among α(1 → 4)-, α(1 → 6)-, β(1 → 4)-, and β(1 → 3)-linked isobaric structures according to fragment types and intensities. In addition, ideal fragmentation ions observed by LEISD method facilitated the determination of the sequences and branched points of complex oligosaccharides from human milk.