Identification of catecholamines in the immune system by desorption/ionization on silicon

Desorption/ionization from porous silicon dioxide (DIOSD), in combination with a standard matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) instrument, was used for the identification of catecholamines in the human peripheral blood lymphocytes. A routine MALDI-TOF analysis does not allow for sensitive detection of low molecular mass compounds (i.e. below 400 Da) due to the pronounced background ions arising from the matrix. Therefore, we have tested DIOSD methodology for the identification of catecholamines in the immune system. Using DIOSD, catecholamines were unambiguously identified in the cell extract of peripheral blood lymphocytes at the femtomolar level. The DIOSD extends the possible use of MALDI-TOF mass spectrometry towards small molecules that were previously detected by other methods.     

Endgroup determination of synthetic polymers by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

Electrospray ionization (ESI) was performed on a Fourier transform ion cyclotron resonance mass spectrometer for the endgroup and monomer mass determination of three poly(oxyalkylene)s in the mass range of 400–8000 Da. A combined use of the multiple charge states observed with ESI, leads to a threefold increase in accuracy of the endgroup and monomer determination. The improvement is attributed to the increased number of datapoints used for the regression procedure, yielding more accurate results. Endgroup masses are determined with a mass error better than 5 and 75 millimass units for the molecular weight range of 400–4200 and 6200–8000 Da, respectively. A mass error of better than 1 millimass unit was observed for all monomer mass determinations. With ESI, endgroup and monomer masses have been determined for poly(ethylene glycol) oligomers with a mass higher than 8000 Da. This is almost two times higher than observed with matrix-assisted laser desorption/ ionization on the same instrument.     

Analysis of tetrapyrrolic pigments and derivatives in sediments by high performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry

A new application is described of the on-line high-performance liquid chromatography/positive and negative ion atmospheric pressure chemical ionization mass spectrometry (APCI(+/–)-MS) to the identification of tetrapyrrolic compounds in sediment samples containing complex mixtures of natural pigments and their diagenetic products. The method relies on the production of radical anions in order to determine the molecular masses of the molecules which yield abundant fragment ions when analyzed in the positive mode. This approach allows to verify the occurrence of coeluting components by examining their negative ion spectra and to complete their identification on the basis of their fragment ions produced in the positive ion spectra. Received: 24 September 1998 / Revised: 23 December 1998 / Accepted: 29 December 1998     

Analysis of tetrapyrrolic pigments and derivatives in sediments by high performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry

A new application is described of the on-line high-performance liquid chromatography/positive and negative ion atmospheric pressure chemical ionization mass spectrometry (APCI(+/–)-MS) to the identification of tetrapyrrolic compounds in sediment samples containing complex mixtures of natural pigments and their diagenetic products. The method relies on the production of radical anions in order to determine the molecular masses of the molecules which yield abundant fragment ions when analyzed in the positive mode. This approach allows to verify the occurrence of coeluting components by examining their negative ion spectra and to complete their identification on the basis of their fragment ions produced in the positive ion spectra. Received: 24 September 1998 / Revised: 23 December 1998 / Accepted: 29 December 1998     

纳米材料辅助负离子激光解吸电离-飞行时间质谱分析小分子研究进展

基质辅助激光解吸电离-飞行时间质谱（MALDI-TOF MS）作为一种软电离质谱技术,目前已被广泛用于蛋白质、多肽、核酸、聚合物等大分子分析。由于传统有机化合物基质在低相对分子质量（小于700 Da）区域的干扰,该技术在小分子物质分析方面受到很大限制。为克服传统有机化合物基质在低相对分子质量区域的干扰,近年来以纳米材料为代表的无机基质材料备受关注。相对传统有机化合物基质或纳米材料正离子模式,基于纳米材料的负离子激光解吸电离（LDI）有效避免了正离子模式下一种化合物会产生多种加合物的问题,具有图谱简单易于解析、灵敏度高、重现性好等优点。该文综述了近5年来纳米材料负离子LDI-TOF MS技术在小分子分析方面的研究进展,以期拓展该技术在小分子分析方面的应用。     

一种基于肽质量指纹谱技术鉴定蓖麻毒素的新方法

应用基质辅助激光解吸电离飞行时间质谱(MALD I-TOF/MS)法实现了对蓖麻毒素(R ic in)的鉴定。测定蓖麻毒素的分子量为62925Da,实现了蓖麻粗毒的凝胶电泳分离,并通过胶内酶切获得了蓖麻毒素的肽质量指纹谱(PMF);经过数据库检索,在输入检索的22条肽段中有17条获得了匹配。检索结果显示,利用生物质谱技术是鉴定蓖麻毒素的最有效的新方法之一。     

Mass spectrometric study of organocyclosiloxanes

Organocyclosiloxanes of various chemical structures were studied by mass spectrometry using different ionization methods. The electron ionization mass spectra contain no peaks of molecular ions, and the main fragment ions are formed due to complicated rearrangements in a molecular ion, which provides no comprehensive view about the molecular structure. The desorption spectra exhibit peaks of quasimolecular and fragment ions, which characterize both molecular weights and chemical structures of the compounds under study. Dedicated to Academician G. A. Abakumov on the occasion of his 70th birthday. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 9, pp. 1746–1749, September, 2007.     

Using a triple-quadrupole mass spectrometer in accurate mass mode and an ion correlation program to identify compounds

Atomic masses and isotopic abundances are independent and complementary properties for discriminating among ion compositions. The number of possible ion compositions is greatly reduced by accurately measuring exact masses of monoisotopic ions and the relative isotopic abundances (RIAs) of the ions greater in mass by +1 Da and +2 Da. When both properties are measured, a mass error limit of 6-10 mDa (< 31 ppm at 320 Da) and an RIA error limit of 10% are generally adequate for determining unique ion compositions for precursor and fragment ions produced from small molecules (less than 320 Da in this study). 'Inherent interferences', i.e., mass peaks seen in the product ion mass spectrum of the monoisotopic [M+H]+ ion of an analyte that are -2, -1, +1, or +2 Da different in mass from monoisotopic fragment ion masses, distort measured RIAs. This problem is overcome using an ion correlation program to compare the numbers of atoms of each element in a precursor ion to the sum of those in each fragment ion and its corresponding neutral loss. Synergy occurs when accurate measurement of only one pair of +1 Da and +2 Da RIAs for the precursor ion or a fragment ion rejects all but one possible ion composition for that ion, thereby indirectly rejecting all but one fragment ion-neutral loss combination for other exact masses. A triple-quadrupole mass spectrometer with accurate mass capability, using atmospheric pressure chemical ionization (APCI), was used to measure masses and RIAs of precursor and fragment ions. Nine chemicals were investigated as simulated unknowns. Mass accuracy and RIA accuracy were sufficient to determine unique compositions for all precursor ions and all but two of 40 fragment ions, and the two corresponding neutral losses. Interrogation of the chemical literature provided between one and three possible compounds for each of the nine analytes. This approach for identifying compounds compensates for the lack of commercial ESI and APCI mass spectral libraries, which precludes making tentative identifications based on spectral matches.     

Thiaminase I (42 kDa) heterogeneity,sequence refinement,and active site location from high-resolution tandem mass spectrometry

Thiaminase I (E.C. 2.5. 1.2) from Bacillus thiaminolyticus catalyzes the degradation of thiamin (vitamin B₁). Unexpected mass heterogeneity (MW 42,127, 42,197, and 42,254; 1:2:1) in recombinant thiaminase I from Escherichia coli was detected by electrospray ionization Fourier-transform mass spectrometry, resolving power 7×10⁴. Nozzle-skimmer fragmentation data reveal an extra Ala (+71.02; 71.04=theory) and GlyAla (+128.04; 128.06=theory) on the N-terminus, in addition to the fully processed enzyme. However, the fragment ion masses were consistent only with this sequence through 330 N-terminal residues; resequencing of the last 150 bps of the thiaminase I gene yields a sequence consistent with the molecular weight values and all 61 fragment ion masses. Covalently labeling the active site with a 108-Da pyrimidine moiety via mechanism-based inhibition produces a corresponding molecular weight increase in all three thiaminase I components, which indicates that they are all enzymatically active. Inspection of the fragment ions that do and do not increase by 108 Da indicates that the active site nucleophile is located between Pro⁷⁹ and Thr¹⁷⁷ in the 379 amino acid enzyme.     

Analysis of high mass peptides using a novel matrix-assisted laser desorption/ionisation quadrupole ion trap time-of-flight mass spectrometer

A novel matrix-assisted laser desorption/ionisation quadrupole ion trap time-of-flight (MALDI QIT ToF) mass spectrometer has been used to analyse high mass peptide ions exceeding 2000 Da. Human adrenocorticotropic hormone (fragment 18-39) and oxidised bovine insulin chain B were utilised to evaluate the performance of the instrument both in MS and in MS/MS mode. Its ability to efficiently isolate ions and to fragment them using collisionally activated decomposition (CAD) has been demonstrated using mixtures diluted to the low-femtomole level on target. Additionally, multiple stage mass spectrometry (MS/MS/MS) provides a second-generation product ion spectrum in which new fragment ions are detected and new stretches of amino acids are identified.     

Small molecule matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a polymer matrix

We have employed a light-absorbing electrically conductive polymer as a matrix to determine the molecular mass of small organic molecules using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. This method, which is in contrast to the usual MALDI strategy for matrix selection in which a small molecule matrix is used with a high molecular mass analyte, addresses the problem of matrix interference which limits the usefulness of MALDI-TOF for small molecule analysis. Use of negative ion mode offers advantages for this application. Using this approach, we have obtained clean molecular ion mass spectra of small organic molecules in the mass range 100-300 Da.     

Matrix-assisted laser desorption/ionization, fast atom bombardment and plasma desorption mass spectrometry of polyethylene glycol esters of (2-benzothiazolon-3-yl)acetic acid.

Fast atom bombardment (FAB), matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and plasma desorption (PD) mass spectra of newly synthesized polyethylene glycols (PEGs), (M(w) 600-4000 Da) chemically modified with biologically active (2-benzothiazolon-3-yl)acetyl end-groups are described (products 1-6). The spectra were also used for the determination of the molecular mass characteristics (number average (M(n)) and weight average (M(w)) molecular masses) of the initial and modified PEGs. As expected, M(n) and M(w) of the modified samples are higher than those of the non-modified samples. However, it is shown that molecular mass dispersity (determined by the comparison of the polydispersity indices (PDI = M(w)/M(n)) of both types of PEGs) essentially do not change during this modification. The FAB mass spectra, together with molecular species, show the presence of abundant [M + Na](+) ions of product 1 and [M + Na + H](+) species of 2 and 3, and [M + Na + 2H](+) of product 4. Two main series of fragment ions, derived from the cleavage of the ether bonds, are observed. The number fractions of the molecular adduct ions and fragment adduct ions, determined from the FAB and PD mass spectra of the modified PEGs, are compared. The MALDI-TOF mass spectra of compounds 1-6 show the presence of two series of polymers. The most abundant peaks are due to [M + Na](+) and [M + K](+) ions originating from the polymers, in which the two terminal hydroxyl groups of PEGs are esterified with (2-benzothiazolon-3-yl)acetic acid. The less abundant peaks are due to the monosubstituted polymers.     

Applications of a matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometer. l. Metastable decay and collision-induced dissociation for sequencing peptides

The use of a high-performance orthogonal time-of-flight (o-TOF) mass spectrometer for sequence analysis is described. The mass spectrometer is equipped with a matrix-assisted laser desorption/ionization (MALDI) source that operates at elevated pressure, 0.01-1 Torr. Ion fragmentation is controlled by varying the pressure of the buffer gas, the laser energy, the voltage difference between the MALDI target and the adjacent sampling cone, and between the cone and the quadrupole ion guide. The peptides were analyzed under optimal ionization conditions to obtain their molecular mass, and under conditions that promote ion dissociation via metastable decomposition or collision-induced dissociation (CID). The fragmentation spectra were used to obtain sequence information. Ion dissociation was promoted via three configurations of the ionization parameters. All methods yielded sequencing-grade b- and y-type ions. Two binary mixtures of peptides were used to demonstrate that: (1) external calibration provides a standard deviation (sigma) of 4 ppm with a mode of 9 ppm; and (2) that peptides with molecular masses that differ by a factor of two may be independently fragmented by appropriately choosing the CID energy and the low-mass cut-off. Analyses of tryptic digests employed liquid chromatography (LC), deposition of the eluant on a target, and finally MALDI-TOF mass spectrometry. The mass fingerprint and the (partial) sequence of the tryptic peptides were matched to their precursor protein via database searches.     

Efficient analysis of non-polar environmental contaminants by MALDI-TOF MS with graphene as matrix

In this Application Note, we describe, for the first time, the rapid analysis of hydrophobic compounds present in environmental contaminants, which includes polycyclic aromatic hydrocarbons (PAHs) and estrogen, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with the use of graphene as matrix. MALDI-TOF MS with conventional matrix has limitations in analyzing low-polarity compounds owing to their difficulty in ionization. We demonstrate that compared with conventional matrix, graphene displays higher desorption/ionization efficiencies for PAHs, and no fragment ions are observed. The method also holds potential in quantitative analysis. In addition, the ionization signal increases with the increasing number of benzene rings in the PAHs, suggesting that graphene binds to PAHs via π–π stacking interactions. Furthermore, graphene as adsorbent for solid-phase extraction of coronene from river water sample displays good performance with a detection limit of 10^–7 M. This work provides a novel and convenient method for analyzing low-polarity environmental contaminants by MALDI-TOF MS.     

Peptide structural analysis using continuous Ar cluster and C60 ion beams

A novel application of time-of-flight secondary ion mass spectrometry (ToF-SIMS) with continuous Ar cluster beams to peptide analysis was investigated. In order to evaluate peptide structures, it is necessary to detect fragment ions related to multiple neighbouring amino acid residues. It is, however, difficult to detect these using conventional ToF-SIMS primary ion beams such as Bi cluster beams. Recently, C₆₀ and Ar cluster ion beams have been introduced to ToF-SIMS as primary ion beams and are expected to generate larger secondary ions than conventional ones. In this study, two sets of model peptides have been studied: (des-Tyr)-Leu-enkephalin and (des-Tyr)-Met-enkephalin (molecular weights are approximately 400 Da), and [Asn¹ Val⁵]-angiotensin II and [Val⁵]-angiotensin I (molecular weights are approximately 1,000 Da) in order to evaluate the usefulness of the large cluster ion beams for peptide structural analysis. As a result, by using the Ar cluster beams, peptide molecular ions and large fragment ions, which are not easily detected using conventional ToF-SIMS primary ion beams such as Bi₃ ⁺, are clearly detected. Since the large fragment ions indicating amino acid sequences of the peptides are detected by the large cluster beams, it is suggested that the Ar cluster and C₆₀ ion beams are useful for peptide structural analysis.     

Field desorption mass spectrometry of large multiply branched saturated hydrocarbons

Large multiply branched saturated hydrocarbons containing 67-103 carbon atoms (molecular masses 941.8-1446.8 Da) were analyzed by field desorption mass spectrometry (FD-MS) with a double-focusing mass spectrometer. FD-MS was found to have detection limits in the 100 fmol range. The FD mass spectra exhibited molecular ions of astonishingly low abundance. However, the fragment ions formed were closely related to the proposed molecular structure, allowing us to set up rules for straightforward structure elucidation of unknowns. In detail, (i) dehydrogenation, (ii) alkyl losses from molecular ions and (iii) subsequent alkene losses were observed. The influence of the electric field strength on dehydrogenation and C-C cleavages was examined by variation of the emitter potential. Additionally, ion dissociations in the ion source and in the first and second field-free regions, respectively, were compared to study the relative importance of field-induced and thermally induced processes.     

Accurate mass measurement of low molecular weight compounds by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

We report the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the accurate measurement of mass of low molecular weight compounds (smaller than 1500 Da), a linear peptide, two types of cyclic depsipeptides, a polyhydroxy-macrocyclic lactone, and two prenylated flavonoids, with delayed extraction in the reflector mode. The performance of the MALDI-TOF instrument was less than those of fast atom bombardment and Fourier-transform ion cyclotron resonance mass spectrometry instruments and insufficient to give acceptable accuracy for literature reporting. Nevertheless, when combined with NMR spectrometry and/or amino acid analysis to give information on the numbers of carbon atoms and index of hydrogen deficiency, MALDI was useful for determination of the elemental composition of the low molecular weight compounds available in small quantities.     

Simultaneous measurement of flight time and energy of large matrix-assisted laser desorption ionization ions with a superconducting tunnel junction detector

We evaluated a cryogenically cooled superconducting Nb-Al₂O₃-Nb tunnel junction (STJ) for use as a molecular ion detector in a matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer. The STJ responds to ion energy and theoretically should detect large molecular ions with a velocity-independent efficiency approaching 100%. The STJ detector produces pulses whose heights are approximately proportional to ion energy, thus the height of a pulse generated by the impact of a doubly charged ion is about twice the height of a singly charged ion pulse. Measurements were performed by bombarding the STJ with human serum albumin (HSA) (66,000 Da) and immunoglobulin (150,000 Da) ions. We demonstrate that pulse height analysis of STJ signals provides a way to distinguish with good discrimination HSA⁺ from 2HSA²⁺, whose flight times are coincident. The rise time of STJ detector pulses allows ion flight times to be determined with a precision better than 200 ns, which is a value smaller than the flight time variation typically observed for large isobaric MALDI ions detected with conventional microchannel plate (MCP) detectors. Deflection plates in the flight tube of the mass spectrometer provided a way to aim ions alternatively at a MCP ion detector.     

MALDI-TOF imaging mass spectrometry of artifacts in “dried droplet” polymer samples

Matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) imaging of polystyrenes with various molecular masses was applied to study spatial molecular mass distribution of polymers in sample spots prepared by the “dried droplet” method. When different solvents and target surfaces were examined, a segregation of single homologous polymers was observed depending upon the evaporation rate of the solvent. For the observed patterns left by the evaporating droplet, a hypothesis is offered taking into account different hydrodynamic interactions and diffusion. The results illustrate that spot preparation using the conventionally “dried droplet” method is prone to artifacts and should be avoided for reliable and reproducible MALDI mass spectrometry experiments with regards to the determination of molecular masses and mass distributions.     

Gold Ion–Angiotensin Peptide Interaction by Mass Spectrometry

Stimulated by the interest in developing gold compounds for treating cancer, gold ion–angiotensin peptide interactions are investigated by mass spectrometry. Under the experimental conditions used, the majority of gold ion–angiotensin peptide complexes contain gold in the oxidation states I and III. Both ESI-MS and MALDI-TOF MS detect singly/multiply charged ions for mononuclear/multinuclear gold-attached peptides, which are represented as [peptide + a Au(I) + b Au(III) + (e - a -3b) H]^e+, where a,b ≥ 0 and e is charge. ESI-MS data shows singly/multiply charged ions of Au(I)-peptide and Au(III)-peptide complexes. This study reveals that MALDI-TOF MS mainly detects singly charged Au(I)-peptide complexes, presumably due to the ionization process. The electrons in the MALDI plume seem to efficiently reduce Au(III) to Au(I). MALDI also tends to enhance the higher polymeric forms of gold-peptide complexes regardless of the laser power used. Collision-induced dissociation experiments of the mononuclear and dinuclear gold-attached peptide ions for angiotensin peptides show that the gold ion (a soft acid) binding sites are in the vicinity of Cys (a soft ligand), His (a major anchor of peptide for metal ion chelation), and the basic residue Arg. Data also suggests that the abundance of gold-attached peptides increases with higher gold concentration until saturation, after which an increase in gold ion concentration leads to the aggregation and/or precipitation of gold-bound peptides.