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.     

Atmospheric pressure matrix-assisted laser desorption/ionisation ion trap mass spectrometry of synthetic polymers: a comparison with vacuum matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry

Atmospheric pressure matrix-assisted laser desorption/ionisation quadrupole ion trap (AP-MALDI/QIT) mass spectrometry has been investigated for the analysis of polyethylene glycol (PEG 1500) and a hyperbranched polymer (polyglycidol) in the presence of alkali-metal salts. Mass spectra of PEG 1500 obtained at atmospheric pressure showed dimetallated matrix/analyte adducts, in addition to the expected alkali-metal/PEG ions, for all matrix/alkali-metal salt combinations. The relative intensities of the desorbed ions were dependent on the matrix, the alkali-metal salt added to aid cationisation and the ion trap interface conditions [capillary temperature, in-source collisionally-induced dissociation (CID)]. These data indicate that the adducts are rapidly stabilised by collisional cooling enabling them to be transferred into the ion trap. Experiments using identical sample preparation conditions were carried out on a vacuum MALDI time-of-flight (ToF) mass spectrometer. In all cases, vacuum MALDI-ToF spectra showed only alkali-metal/PEG ions and no matrix/analyte adducts. The tandem mass spectrometry (MS/MS) capability of the ion trap has been demonstrated for a lithiated polyglycol yielding a rich fragment-ion spectrum. Analysis of the hyperbranched polymer polyglycidol by AP-MALDI/QIT reveals the characteristic ion series for these polymers as also observed under vacuum MALDI-ToF conditions.     

Improved matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry of carboxymethyl cellulose

A refined sample preparation procedure for matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) was developed for the evaluation of the degree of substitution (DS) in partially depolymerised carboxymethyl cellulose (CMC). By adding ammonium sulphate to the sample mixture prior to the analysis, good quality mass spectra could be acquired. The usual time-consuming search for 'sweet-spots' at the crystalline rim of the MALDI target spot was also avoided. This quality improvement made it possible to investigate whether various positions on the target spot generated mass spectra in which the measured DS varied. The accuracy and reproducibility of the sample preparation procedure were tested by applying it on three commercial CMCs. The study shows that the DS values that were calculated from the spectra acquired from the centre region of the MALDI target spot were in better agreement with the DS provided by the supplier than were the values obtained from the large crystals at the target spot rim. This observation could be one reasonable explanation for the higher DS values reported in other publications. By applying our refined MALDI sample preparation procedure DS values that were in good agreement with the values provided by the manufacturer could be obtained. This indicates that MALDI-TOFMS of partially depolymerised CMCs can be used for an estimation of the DS as a complement to the more established methods, e.g. NMR, titrimetry, and chromatographic techniques.     

Identification of carbohydrates by matrix-free material-enhanced laser desorption/ionisation mass spectrometry

Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) is a sensitive mass spectrometric technique which utilises acidic materials as matrices for laser energy absorption, desorption and ionisation of analytes. These matrix materials produce background signals particularly in the low-mass range and make the detection and identification of small molecules difficult and nearly impossible. To overcome this problem this paper introduces matrix-free material-enhanced laser desorption/ionisation mass spectrometry (mf-MELDI-MS) for the screening and analysis of small molecules such as carbohydrates. For this purpose, 4,4'-azo-dianiline was immobilised on silica gel enabling the absorption of laser energy sufficient for successful desorption and ionisation of low molecular weight compounds. The particle and pore sizes, the solvent system for suspension and the sample preparation procedures have been optimised. The newly synthesised MELDI material delivered excellent spectra with regard to signal-to-noise ratio and detection sensitivity. Finally, wheat straw degradation products and Salix alba L. plant extracts were analysed proving the high performance and excellent behaviour of the introduced material.     

Matrix‐free laser desorption/ionization mass spectrometry using silicon glancing angle deposition (GLAD) films

Glancing angle deposition (GLAD) was used to fabricate nanostructured silicon (Si) thin films with highly controlled morphology for use in laser desorption/ionization mass spectrometry (DIOS‐MS). Peptides, drugs and metabolites in the mass range of 150–2500 Da were readily analyzed. The best performance was obtained with 500 nm thick films deposited at a deposition angle of 85°. Low background mass spectra and attomole detection limits were observed with DIOS‐MS for various peptides. Films used after three months of dry storage in ambient conditions produced mass spectra with negligible low‐mass noise following a 15 min UV‐ozone treatment. The performance of the Si GLAD films was as good as or better than that reported for electrochemically etched porous silicon and related materials, and was superior to matrix‐assisted laser desorption/ionization (MALDI)‐MS for analysis of mixtures of small molecules between 150–2500 Da in terms of background chemical noise, detection limits and spot‐to‐spot reproducibility. The spot‐to‐spot reproducibility of signal intensities (100 shots/spectrum) from 21 different Si GLAD film targets was ±13% relative standard deviation (RSD). The single shot‐to‐shot reproducibility of signals on a single target was ±19% RSD (n = 7), with no indication of sweet spots or mute spots. Copyright © 2010 John Wiley & Sons, Ltd.     

Matrix‐free thin‐layer chromatography/laser desorption ionization mass spectrometry for facile separation and identification of medicinal alkaloids

Quaternary protoberberine alkaloids belong to a pharmaceutically important class of isoquinoline alkaloids associated with bactericidal, fungicidal, insecticidal and antiviral activities. As traditional medicine gains wider acceptance, quick and robust analytical methods for the screening and analysis of plants containing these compounds attract considerable interest. Thin‐layer chromatography (TLC) combined with matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) is a powerful technique but suffers from dilution of the TLC bands resulting in decreased sensitivity and masking of signals in the low‐mass region both due to addition of matrix. This study integrates for the first time conventional silica gel TLC and laser desorption ionization mass spectrometry (LDI‐MS) thus eliminating the need for any external matrix. Successful separation of berberine (R_f = 0.56) and palmatine (R_f = 0.46) from Berberis barandana including their identification by MS are demonstrated. Furthermore, a robust electrospray ionization (ESI)‐MS method utilizing residual sample from TLC for quantification of berberine applying selected reaction monitoring and standard addition method is presented. The amount of berberine in the plant root prepared for the study was determined to be 0.70% (w/w). Copyright © 2009 John Wiley & Sons, Ltd.     

Study of latent fingermarks by matrix‐assisted laser desorption/ionisation mass spectrometry imaging of endogenous lipids

Identification of suspects via fingermark analysis is one of the mainstays of forensic science. The success in matching fingermarks, using conventional fingermark scanning and database searching, strongly relies on the enhancement method adopted for fingermark recovery; this in turn depends on the components present in the fingermarks, which will change over time. This work aims to develop a robust methodology for improved analytical detection of the fingermark components. For the first time, matrix‐assisted laser desorption/ionisation mass spectrometry imaging (MALDI‐MSI) has been used to image endogenous lipids from fresh and aged, groomed and ungroomed fingermarks. The methodology was initially developed using oleic acid which was detected along with its degradation products over a 7‐day period, at three different temperatures in a time‐course experiment. The optimised methodology was then transferred to the imaging analysis of real fingermark samples. Fingermark patterns were reconstructed by retrieving the m/z values of oleic acid and its degradation products. This allowed the three aged fingermarks to be distinguished. In order to prove that MALDI‐MSI can be used in a non‐destructive way, a simple washing protocol was adopted which returned a fingermark that could be further investigated with classical forensic approaches. The work reported here proves the potential and the feasibility of MALDI‐MSI for the forensic analysis of fingermarks, thus making it competitive with other MSI techniques such as desorption electrospray ionisation (DESI)‐MS. The feasibility of using MALDI‐MSI in fingermark ageing studies is also demonstrated along with the potential to be integrated into routine fingermark forensic analysis. Copyright © 2009 John Wiley & Sons, Ltd.     

Matrix-suppressed laser desorption/ionisation mass spectrometry and its suitability for metabolome analyses

Matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry was investigated for the simultaneous detection of several metabolites, as applicable to global metabolite analysis (metabolomics). The commonly employed organic matrices alpha-cyano-4-hydroxycinnamic acid and 3,5-dihydroxybenzoic acid, in both the crystalline and ionic liquid forms, were investigated. The employment of a low matrix-to-analyte molar ratio suppressed matrix peaks and was effective in detecting all the metabolites with a unique mass in a 30-metabolite synthetic cocktail, albeit to varying degrees. These matrix-suppressed laser desorption/ionisation (MSLDI) analyses were performed in the positive ion mode, and metabolites were detected as the protonated [M+H]+, sodiated [M+Na]+ or potassiated [M+K]+ species. The spectral signals were dominated by basic metabolites. It was possible to detect components of a synthetic cocktail when it was spiked quantitatively into a microbial extract, demonstrating the feasibility of using the technique for detecting metabolite signals in a complex biological matrix. However, analyte suppression effects were noted when the relative proportion of one analyte was allowed to increasingly dominate the others in a mixture. The implications of the findings with respect to applications in metabolomic investigations are discussed.     

Two-dimensional gel electrophoresis/matrix-assisted laser desorption/ionisation mass spectrometry of commercial bovine milk

Proteins in commercial bovine milk have been separated by two-dimensional gel electrophoresis and examined by matrix-assisted laser desorption/ionisation mass spectrometry. Gel separation was conducted in two different pH gradients, 3-10 and 6-11; the latter range resulted in a higher spot resolution and favoured the basic proteins. We have limited the time-of-flight mass spectrometry analysis to the linear mode to examine the capability of reliable relative molecular masses of the intact proteins in their characterisation. The present study draws attention to the difficulty of identifying basic proteins with low molecular masses (below 12000 Da) that are commonly encountered in milk samples.     

Two-dimensional gel electrophoresis/matrix-assisted laser desorption/ionisation mass spectrometry of a milk powder

Proteins in a commercial milk powder have been separated by two-dimensional gel electrophoresis and analysed by matrix-assisted laser desorption ionisation mass spectrometry. The mass spectrometric analyses were conducted in two steps: analysis of the intact proteins following their passive extraction into a suitable solvent mixture and analysis in reflectron mode of in situ digests of a number of gel spots. The combination of the two methods allowed a reliable identification of a number of proteins, including nine caseins as well as certain protein modifications including single/multiple phosphorylation, lactose-protein conjugates and Coomassie Brilliant Blue adducts. Analyses of the intact proteins prior to their in situ digestion contributed to a more efficient and reliable consultation of protein databases.     

Identification of dinucleoside polyphosphates by matrix-assisted laser desorption/ionisation post-source decay mass spectrometry

Dinucleoside polyphosphates are a group of intra- and extracellular mediators controlling numerous physiological functions. In this study dinucleoside polyphosphates were examined by positive ion matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MADLI-TOFMS). 3-Hydroxypicolinic acid was used as UV-absorbing matrix. For the individual dinucleoside polyphosphates Ap(n)A (n = 2-7), Ap(n)G (n = 2-6) and Gp(n)G (n = 2-6), MALDI post-source decay (PSD) mass spectra were measured. Each mass peak in the MALDI-PSD mass spectra could be assigned to individual fragments of dinucleoside polyphosphates. The comparison of the fragmentation patterns of the dinucleoside polyphosphates presented here demonstrates that dinucleoside polyphosphates preferably cleave to fragment ions consisting of the corresponding mononucleoside polyphosphates as well as the corresponding nucleosides and bases during flight in the field-free drift path of the MALDI mass spectrometer. Therefore, the MALDI-PSD approach described here is suitable for identification of other dinucleoside polyphosphates. The present MALDI-PSD mass spectra may be used as MALDI-PSD mass reference spectra for future identification of dinucleoside polyphosphates and other nucleotides.     

Species identification by analysis of bone collagen using matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry

Species identification of fragmentary bone, such as in rendered meat and bone meal or from archaeological sites, is often difficult in the absence of clear morphological markers. Here we present a robust method of analysing genus‐specific collagen peptides by mass spectrometry simply by using solid‐phase extraction (a C18 ZipTip®) for peptide purification, rather than liquid chromatography/mass spectrometry (LC/MS). Analysis of the collagen from 32 different mammal species identified a total of 92 peptide markers that could be used for species identification, for example, in processed food and animal feed. A set of ancient (>100 ka@10°C) bone samples was also analysed to show that the proposed method has applications to archaeological bone identification. Copyright © 2009 John Wiley & Sons, Ltd.