UV-MALDI-TOF Mass Spectrometry Analysis of Carbohydrates Combining Doping Salts, GA and nor- Harmane

ROSA ERRA-BALSELLS ; SILVANA L. GIUDICESSI; FRANCO M. CABRERIZO; DAISUKE HIROTA; HIROSHI NONAMI

Sapporo

Congreso; Joint Conference on Environmental Engineering in Agriculture; 2006

Environmental Control in Biology

UV-MALDI- and ESI-MS are a valuable techniques for the determination of high molecular mass when they are used with proteins while for carbohydrates still the techniques faces serious experimental problems. This limitation is probably related, at least in part, to the structural complexity and to the molecular interactions often found in the oligosaccharide moieties of these biopolymers. Furthermore, the isolated oligosaccharides include in its structure salts naturally present in cells, which can modify the results of the MS analysis. Salt impurities that may originate from the matrix, the analyte, solvents, glassware, surface of sample carriers, etc. may result in alkali metal adducts appearing in the UV-MALDI-MS. This is sometimes undesirable and can complicate the mass spectra. However, some species such as synthetic polymers and carbohydrates are primarily ionized by the formation of metal ion adducts (cationization). For this reason metal salts are often added to samples in order to enhance the analyte signals. Furthermore, cationization is somewhat matrix dependent. Understanding cationization mechanisms and the combined salt-matrix effects on it, are obviously required for better control of ion formation in UV-MALDI process. UV-MALDI-TOF-MS measurements were performed with Shimadzu Kratos, Kompact MALDI 4, laser desorption time-of-flight mass spectrometer, equipped with pulsed nitrogen laser (em=337 nm), tunable PDE and PSD (MS/MS) device. All mass spectra were taken in positive and negative ion modes, in the linear and reflectron modes. Stock matrix solutions 1 mg/0.5 mL and analyte solutions 0.05 mg/0.025 mL in the appropriate solvent were prepared. For MALDI-MS two different sample preparation methods were used: Sandwich method, 0.5 μL of the matrix solution was placed on the sample probe tip and the solvent was removed by blowing air. Subsequently, 0.5 μL of the analyte solution was placed on the same probe tip covering the matrix. Then, two additional portions (0.5 μL) of the matrix solution were deposited on the same sample probe tip. Mixture method, mixtures 10/1 to 1/1 (v/v) of the corresponding matrix and analyte solutions were prepared and transferred (0.5 μL x2) on the probe tip. Salt impurities that may originate from the matrix, the analyte, solvents, glassware, surface of sample carriers, etc. may result in alkali metal adducts appearing in the UV-MALDI-MS. This is sometimes undesirable and can complicate the mass spectra. However, some species such as synthetic polymers and carbohydrates are primarily ionized by the formation of metal ion adducts (cationization). For this reason metal salts are often added to samples in order to enhance the analyte signals. Furthermore, cationization is somewhat matrix dependent. Understanding cationization mechanisms and the combined salt-matrix effects on it, are obviously required for better control of ion formation in UV-MALDI process. We have recently described the analysis of neutral polysaccharides, a family of xylans, that could only be efficiently desorved/ionizated by using nor-harmane as UV-MALDI matrix. It was quite surprisingly that although by adding extra NaCl to the sample the spectra were quite similar after addition of CaCl2 practically all the signals of the oligomers disappeared. At first sight a problem in the formation and stability of the analyte adducts [M+CaCl]+ / [M+Ca]++ could be the cause. Thus, in order to shed some light to this problem a comparative study of the UV-MALDI-MS was conducted with a neutral sugar (b-cyclodextrin) and three potential matrices (GA, nor-harmane and picric acid (PA)), using as doping salts NaCl, KaCl, NH4Cl and CaCl2, in linear positive and negative ion modes, preparing the samples by using the sandwich and the mixture methods. The critical effect observed on the experiments conducted by using nor-harmane as matrix encourage us to perform additional thermal and photophysical studies. Thus, in parallel experiments the thermal stability of the selected doping salts, the used UV-MALDI matrices and b-cyclodextrin, each compound alone and in different binary and ternary mixtures, were studied by classical melting point analysis, thermogravimetric analysis and differential scanning calorimetry as well as the effect of the salts on the UV-vis absorption, fluorescence emission and excitation spectra and acid-base character of nor-harmane in ground and electronic excited states.