Matrix-assisted laser desorption ionization with a magnetic mass spectrometer.

Matrix-assisted laser desorption ionization has been carried out with a high mass double-focusing magnetic mass spectrometer. The pulsed ion signal, generated by irradiation of the sample (substance P, ubiquitin, and cytochrome c) embedded in 2,5-dihydroxybenzoic acid with a XeF excimer laser (353 nm, 12 ns pulse, 10-160 Hz), was recorded with an integrating array detector. Good resolution of 2600 (full width at half maximum) and high sensitivity (a few pmol) were obtained. The loss of small neutral fragments was observed, supporting the notion that the peak broadening observed in the time-of-flight mass spectrometers commonly used for this ionization mode is due to such metastable decomposition.     

Matrix-assisted laser desorption/ionization mass spectrometry using a visible laser

Visible matrix-assisted laser desorption/ionization (VIS-MALDI) was performed using 2-amino-3-nitrophenol as matrix. The matrix is of near-neutral pH, and has an optical absorption band in the near-UV and visible region. A frequency-doubled Nd:YAG laser operated at 532 nm wavelength was used for matrix excitation and comparisons were made with a frequency-tripled Nd:YAG laser (355 nm). Visible and ultraviolet (UV)-MALDI produce similar mass spectra for peptides, polymers, and small proteins with comparable sensitivities. Due to the smaller optical absorption coefficient of the matrix at 532 nm wavelength, the optical penetration depth is larger, and the sample consumption per laser shot in VIS-MALDI is higher than that of UV-MALDI. Nevertheless, VIS-MALDI using 2-amino-3-nitrophenol as matrix may offer a complementary technique to the conventional UV-MALDI method in applications where deeper laser penetration is required.     

Matrix-assisted laser desorption mass spectrometry of oligodeoxythymidylic acids.

Ferulic acid, sinapinic acid and 2,5-dihydroxybenzoic acid (DHBA) have been tested as matrix materials for matrix-assisted laser desorption of the pure oligonucleotide pd(T)12 and a mixture of oligonucleotides pd(T)12 through pd(T)18 using pulsed 337 nm radiation combined with reflecting time-of-flight mass spectrometry. The three matrix materials are compared with respect to obtainable mass resolution, degree of fragmentation, and adduct formation for these oligonucleotides. DHBA was found to produce the least fragmentation and adduct formation, as well as the highest mass resolution.     

基体辅助激光解吸质谱法测定蛋白质分子量

本文叙述用自行研制成功的激光微探针飞行时间质谱仪及采用基体辅助激光解吸的新方法, 对溶菌酶、细胞色素C、肌红蛋白、胰蛋白酶、蛋白酶、白蛋白等多种蛋白质的分子量进行测定, 并对蛋白质混合物进行分析, 得一以满意的结果。此方法测定蛋白质分子量具有速度快(十分钟一个样品), 准确度高(±1%-0.1%), 灵敏度高(10^-^1^2～10^-^1^5mol)等优点, 是传统生物方法难以比拟的。     

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.     

Wide mass range trapping using a 7-T internal source matrix-assisted laser desorption/ionization Fourier transform mass spectrometer

A wide mass range trapping experiment using internal source matrix-assisted laser desorption–Fourier transform mass spectrometry (MALDI–FTMS) was evaluated. In this method, the front trap plate potential is ramped up and the rear trap plate potential is simultaneously decreased using a cubic cell to trap ions over a wide range of mass-to-charge ratios. To apply this to MS/MS experiments, a second ion ejection procedure would remove unwanted ions, with the selected remaining ions then fragmented by collision-induced dissociation. In measurements using a 7.2-T unshielded magnet presented here, an approximately equimolar mixture of a set of poly(ethylene glycol) (PEG) species for the ramped measurements had peak areas of 1.0:1.0:1.0:1.0, as did the previously described integral method which gave peak areas of 1.0:1.1:1.0:1.0, in good agreement with the known composition of the samples deposited on the MALDI probe tip. Comparative MALDI–TOF in reflectron mode results were of similar quality for the equimolar mixture, giving a ratio of 1.0:1.0:1.2:0.9. All methods failed to varying degrees when individual PEG compositions of the trial mixture were changed. However, the previously described integral method showed relatively better results for all but the PEG 8000 doubled mixture.     

Matrix-assisted laser desorption/ionization mass spectrometry of taxanes

Taxanes are biologically active compounds that have been extensively used in pharmacology for their powerful anticancer properties. High specificity and low level sensitivity for analysis of these compounds have been obtained with reversed-phase high-pressure liquid chromatography/mass spectrometry (RP-HPLC/MS), but the number of applications of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for low molecular weight analytes is rapidly growing. A new MALDI-MS approach for the rapid screening of a variety of taxanes and a tandem mass spectrometric (MS/MS) analysis of the most important and diagnostic taxane fragmentation pathways are proposed. A solid-phase extraction method followed by preliminary quantification is also reported.     

Ionisation and fragmentation of complex glycans with a quadrupole time-of-flight mass spectrometer fitted with a matrix-assisted laser desorption/ionisation ion source

This paper reports the use of an experimental matrix-assisted laser desorption/ionisation (MALDI) ion source fitted to a quadrupole time-of-flight (Q-Tof) mass spectrometer for the analysis of carbohydrates, particularly the N-linked glycans from glycoproteins. Earlier work on the Q-Tof instrument, using electrospray ionisation, gave excellent MS/MS spectra, particularly from the [M + Na]+ ions, but suffered from the major disadvantages that the signal was often split between singly and multiply charged ions and that sensitivity fell dramatically as the molecular weight of the carbohydrate rose. The MALDI ion source did not suffer from these problems and the instrument produced excellent MS and MS/MS spectra from small amounts of complex, underivatised glycans as well as those derivatised at the reducing terminus. Positive ion MS spectra of sialylated glycans recorded on the new instrument were much less complex than those recorded with a conventional MALDI-TOF instrument because of the absence of ions resulting from metastable (post-source decay, (PSD)) fragmentations occurring in the flight tube. However, considerable fragmentation by loss of sialic acid still occurred. MS/MS spectra of the [M + Na]+ ions from all compounds were almost identical to those recorded earlier with the electrospray-Q-Tof combination and far superior to MALDI-PSD spectra recorded with reflectron-TOF instruments. Spectra are shown for neutral and sialylated N-linked glycans from chicken ovalbumin, riboflavin binding protein, alpha1-acid glycoprotein, bovine fetuin and ribonuclease B, both as free glycans and as those derivatised at their reducing termini. The technique was applied to the structural determination of N-linked glycans from human secretory IgA and Apo-B 100 from human low-density lipoprotein.     

Matrix-assisted laser desorption/ionization mass spectrometric analysis of polysulfated-derived oligosaccharides using pyrenemethylguanidine

A better understanding of the biological roles of carbohydrates requires the use of tools able to provide efficient and rapid structural information. Unfortunately, highly acidic oligomers-such as polysulfated oligosaccharides-are very challenging to characterize because of their high polarity, structural diversity, and sulfate lability. These features pose special problems for matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis because polysulfated carbohydrates exhibit poor ionization efficiency and usually do not produce any signal. The present report demonstrates how MALDI-MS can be used to derive structural and compositional information from pure and mixed fractions of polysulfated oligosaccharides. Indeed, pyrenemethylguanidine (pmg, a derivatizing agent and ionization efficiency enhancer) was used for the analysis of di- to decasaccharides, carrying from two to nine sulfate groups. The method is applied to various highly sulfated chondroitin and carrageenan oligosaccharides as well as to the analysis of mixtures of compounds. In the mass spectra, the observation of a unique pmg-complexed ladder of peaks in both ionization modes allows an easy and rapid determination of both the number of sulfate groups carried by the analyte and its molecular weight. Moreover, we have developed a software tool for the rapid and automatic structural elucidation of carrageenans based on the mass spectra obtained.     

Matrix-assisted laser desorption ionization mass spectrometry for identification of shrimp

Matrix-assisted laser desorption ionization (MALDI) time of flight mass spectrometry was used to identify shrimp at the species level using commercial mass spectral fingerprint matching software (Bruker Biotyper). In the first step, a mass spectrum reference database was constructed from the analysis of six commercially important shrimp species: Litopenaeus setiferus, Farfantepenaeus aztecus, Sicyonia brevirostris, Pleoticus robustus, Pandalopsis dispar and Pandalus platyceros. This step required a desalting procedure for optimum performance. In the second step, the reference database was tested using 74 unknown shrimp samples from these six species. Correct identification was achieved for 72 of 74 samples (97%): 72 samples were identified at the species level and 2 samples were identified at the genus level using the manufacturer's log score specifications. The MALDI fingerprinting method for the identification of shrimp species was found to be reproducible and accurate with rapid analysis.     

Ultramark 1621 as a reference compound for positive and negative ion fast-atom bombardment high-resolution mass spectrometry

Ultramark 1621, a commercially available mixture of fluorinated phosphazines, was found to bea a useful calibration compound for negative and positive ion fast-atom bombardment (FAB) high-resolution mass spectrometry. Ultramark 1621 worked very well with the most widely used matrices such as glycerol, nitrobenzyl alcohol, and triethanolamine. The negative and positive ion FAB mass spectra of Ultramark include a series of intense peaks extending from 700 to 1900 u.     

Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry of luteinizing hormone releasing hormone-metal ion complexes

Complexes of luteinizing hormone releasing hormone (LHRH) with divalent metal ions (Ni, Zn, Cu) have been studied by matrix-assisted laser desorption ionization (MALDI) and Fourier transform mass spectrometry. LHRH-metal complexes were detected in high abundance for all three metals from synthesized samples, particularly in negative ion mode. The mixture of the apopeptide with the metal salts yielded in most cases a very minor signal of metal-complex ions. As opposed to Ni and Zn, copper complex ions were mostly observed as Cu(I) adducts. This can be partly attributed to plume reactions of Cu(I) with the apopeptide. the Cu(II) complexes appeared only for the synthetic complex. We show how to distinguish between the contribution to the overall signal from desorbed complexes and from Cu(I) complexes formed in the MALDI plume.     

Matrix-assisted laser desorption/ionisation mass spectrometry of oligosaccharides and glycoconjugates

The technique of matrix-assisted laser desorption/ionization mass spectrometry (MALDI) is described and examples are given of its use for the examination of glycoproteins, glycopeptides, glycolipids and oligosaccharides. Abundant [M + H]⁺ ions are produced by the glycoproteins and glycopeptides, whereas glycolipids and oligosaccharides give mainly [M + Na]⁺ ions. Resolution on time-of-flight (TOF) instruments is poor but improved resolution can be obtained by use of ion cyclotron resonance or magnetic sector instruments. Although the technique gives mainly [M + Na]⁺ ions from neutral, underivatised oligosaccharides, with little fragmentation when implemented on TOF systems, the use of a reflectron enables fragment ions produced by post-source decay to be obtained. Acidic sugars give less satisfactory positive ion spectra with TOF analysers. but generally produce abundant negative ions. Extensive fragmentation is observed with these compounds when the spectra are recorded with magnetic sector instruments. Neutral glycolipids produce strong spectra from several matrices but acidic glycolipids show extensive fragmentation as the result of sialic acid loss.     

Analysis of long-chain fatty acyl coenzyme a thioesters by negative ion fast-atom bombardment mass spectrometry and tandem mass spectrometry

Long-chain acyl Coenzyme A (CoA) is essentially composed of three major chemical groups, fatty acyl-, phosphopantetheino-, and 3′, 5′,-adenosine diphospho-moieties. The negative ion fast-atom bombardment mass spectrometry spectra of long-chain acyl CoA thioesters were characterized by the formation of abundant [M ? H]^? and two distinct classes of fragment ions, one class which retained the acyl group and another class which is related to CoA that contains the phosphopantethene and adenine. The ions which retained the acyl group in the spectrum of palmitoyl CoA appeared at m/z 675, 657, 595, and 577 and were found to decompose by loss of alkylketene observed at m/z 357 and 339. Those ions which retained the adenine group were observed at m/z 426 and 408. In contrast to these ions observed following fast-atom bombardment ionization, tandem mass spectrometry of the [M ? H]^?, from palmitoyl CoA (m/z 1004), yielded the adenine-containing ions as major products and the acyl-containing ions were of low abundance or not detected. These results suggested that the formation of many characteristic ions observed in direct FAB analysis occurred during the desorption process. The unique relationship between ions which involved the transition from acyl-containing ions to only CoA-containing ions by the loss of alkylketene allowed the development of tandem mass spectrometry protocols for the analysis of acyl CoA mixtures. Precursor scans of either m/z 357 or 339 yielded the identification of each species in a complex mixture. Identification of specific species was obtained with a neutral loss scan of the mass for a specific alkylketene.     

Matrix-assisted laser desorption/ionization mass spectrometry using porous silicon and silica gel as matrix

Porous silicon powder and silica gel particles have been applied as inorganic matrices for the analysis of small molecules in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOFMS). In contrast to conventional MALDI-TOFMS, the signal interference of low-molecular analytes by the matrix has been eliminated. Almost no fragmentations of the analytes were observed. Effects of various factors, such as the particle and pore size, the suspending solution, and sample preparation procedures, on the intensity of mass spectra have been investigated. The pore structure of the inorganic matrix and penetration of the analytes into the pores must be optimized for effective desorption and ionization of the analytes. Matrices (DHB and HCCA) were covalently bound to silica gel for improvement of spectrum intensity. Copyright © 2001 John Wiley & Sons, Ltd.     

Fragmentation of N-linked glycans with a matrix-assisted laser desorption/ionization ion trap time-of-flight mass spectrometer

N-Linked glycans were ionized from several matrices with a Shimadzu-Biotech AXIMA-QIT matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometer. [M+Na]+ ions were produced from all matrices and were accompanied by varying amounts of in-source fragmentation products. The least fragmentation was produced by 2,5-dihydroxybenzoic acid and the most by alpha-cyano-4-hydroxycinnamic acid and 6-aza-2-thiothymine. Sialic acid loss was extensive but could be prevented by formation of methyl esters. Fragmentation produced typical low-energy-type spectra dominated by ions formed by glycosidic cleavages. MS(n) spectra (n = 3 and 4) were used to probe the pathways leading to the major diagnostic ions. Thus, for example, an ion that was formed by loss of the core GlcNAc residues and the 3-antenna was confirmed as being formed by a B/Y rather than a C/Z mechanism. The proposed structures of several cross-ring cleavage ions were confirmed and it was shown that MS3 spectra could be obtained from as little as 10 fmol of glycan.     

Matrix-assisted laser desorption/ionization of high-mass molecules by Fourier-transform mass spectrometry.

Following the first demonstrations of high-mass analysis using time-of-flight matrix-assisted laser desorption/ionization (MALDI) techniques by Hillenkamp, Tanaka and their co-workers, there have been significant efforts in a number of laboratories to adapt the new methodology to Fourier-transform mass spectrometry (FTMS). The motivation for this research is obvious. Namely, it would be desirable to couple the unparalleled high mass resolution of FTMS with the extended mass range provided by MALDI, particularly for analysis of polymers and biomolecules. Unfortunately, prior to the present work, attempts to mate FTMS and MALDI have met with limited success. The highest mass matrix-assisted laser-desorption-FTMS result previously obtained appears to be the unpublished low resolution spectrum of bovine insulin recently reported by Russell and co-workers. We, Campana and co-workers, and Hettich and Buchanan have had some success with MALDI-FTMS of biomolecules with masses lower than 3000 Da, including melittin, a variety of lower mass peptides, and oligonucleotides with masses lower than 1800 Da. Furthermore, with the single exception of Campana's report of obtaining mass resolution of 5000 for the molecular ion of melittin, such spectra have not displayed high resolution. Here, we report successful development of MALDI-FTMS, demonstrated with spectra obtained from a variety of high-mass polymer and biomolecule samples, using 355 nm radiation from an excimer-pumped dye laser for desorption/ionization and sinapinic acid as matrix. Some of these spectra are of much higher mass resolution than is possible with current time-of flight mass spectrometers.