Alkali Adducts Research Articles

Gas phase stripping of alkali cations from biomolecules via reaction with crown ethers

Electrospray mass spectra of biomolecules produced from salty solutions can exhibit a large number of alkali adducts. Often the number of attached alkali metal atoms exceeds the net charge state of the ion. For example, the spectral intensity of the 2+ charge state of gramicidin S, cyclo[Val-Orn-Leu- d-Phe-Pro] 2, (GS), withdrawn from a sodium-containing water:methanol solution, is distributed over (GS + 2H) 2+, (GS + H + Na) 2+, (GS + 2Na) 2+, (GS−H + 3Na) 2+, (GS−2H + 4Na) 2+, (GS−3H + 5Na) 2+, and (GS−4H + 6Na) 2+. Each additional metal cation displaces a proton without affecting the net charge of the biomolecule. Hence, in (GS−2H + 4Na) 2+, at least two of the alkali metal adducts must involve displacement of protons from non-traditional basic sites. The character of these coordination sites must be either “salt-like” or zwitterionic. Alkali adducts of trapped polypeptide ions may be removed via gas phase ion-molecule “stripping” reactions with crown ethers 12-crown-4, 15-crown-5, or 18-crown-6. Both products of the stripping reaction, the desalted biomolecule of reduced charge and the alkali-metal-attached crown, are observed in the FTICR mass spectra. For gas phase, “hyper-metallated” peptides, in which the number of adducted alkali metal ions exceeds the net charge, we suggest that some of the attached cations replace amide protons by coordinating as a salt or zwitterion to a tautomerized, deprotonated amide link in the backbone of the peptide. Alkali adduction to these non-traditional base sites leads to slightly higher stripping rates, probably by removing the secondary constraints of transannular NH···O  C hydrogen bonds, which makes attached alkali cations more accessible to the stripping reagent. Stripping of K + is faster than stripping of Na +, as may be expected for electrostatically bound cations.

The utility of nonspecific proteases in the characterization of glycoproteins by high-resolution time-of-flight mass spectrometry

Degradation of glycoproteins with the nonspecific protease pronase was examined by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS). High mass resolution (in excess of 10 000 at FWHM) and mass accuracy on monoisotopic species (better than 10 ppm) obtained with the combination of delayed extraction and the reflector mode of analysis enabled the successful interpretation of very complex mixtures resulting from extensive hydrolysis. After 48 h of degradation of glycoproteins, the glycopeptides were well separated from small peptides based on their molecular weight. Accurate monoisotopic masses of the glycopeptides permitted the determination of the glycan composition and the (nonspecific) peptide segment the glycans were attached to. Ribonuclease B and ovalbumin were used to demonstrate feasibility of the method. Unless the peptide sequence of the glycopeptide contains a basic residue, the negative-ion mode is preferred over the positive-ion mode. The absence of alkali adducts reduce the complexity of the mass spectra and the relative sensitivities of the glycopeptides were found better in the negative-ion mode. A highly complex sample, α-acid glycoprotein was also analyzed. Multiply sialylated glycopeptides had to be run in the linear mode where the metastable loss of sialic acid residues did not interfere with the detection of other components. From the linear mode mass spectrum, five glycosylation sites were identified along with the more abundant glycoforms. Comparison with the literature indicated that several minor components were undetected. The presented approach also permitted the determination of the sialylation pattern of the complex type glycans.

Ultraviolet laser desorption ionisation mass spectrometry of selected calixarenes with and without matrix assistance

Ultraviolet (UV) matrix-assisted laser desorption/ionisation (MALDI) and laser desorption/ionisation (LDI) mass spectrometry have been applied to study the macrocyclic, basket-like compound class of calixarenes for rapid characterisation. Three different types of cyclic oligomeric alkyl phenols—underivatised calixarenes and calixarenes carrying one or several acyl moieties as additional functional groups, as well as so-called resorcinarenes—have been investigated. It is shown that relative molecular mass determination of nitrogen-free calixarenes is possible by detecting alkali adduct ions in the positive-ion mode and sometimes deprotonated molecular ions in the negative-ion mode by both MALDI and LDI. Best results with the MALDI technique have been obtained with the matrices α-cyano-4-hydroxy-cinnamic acid and 3-hydroxy picolinic acid. Depending on the applied laser energy, fragmentation can be induced to some degree in MALDI as well as LDI. Since positive-ion MALDI mass spectrometry gave mainly sodiated molecular ions for the selected calixarenes, post source decay (PSD) experiments were carried out applying a coaxial curved-field reflectron in order to generate fragment ions. The positive-ion PSD spectra contained diagnostic fragment ions, which enabled, to some extent, the kind and number of ester or ether groups on the hydrophilic rim of the bowl-shaped macrocyclic compound to be established unambiguously.

The Matrix Suppression Effect and Ionization Mechanisms in Matrix-assisted Laser Desorption/Ionization

At appropriate matrix:analyte mixing ratios, small to moderate sized analyte ions (1000–20 000 u) can fully suppress positively charged matrix ions in matrix-assisted laser desorption/ionization (MALDI) mass spectra. This is true for all matrix species, including radical cations and adducts with protons or alkali-metal ions. Full matrix suppression is also observed, regardless of the preferred analyte ion form, be it protonated or an alkali adduct. These facts lead us to propose a mechanism for prompt, primary (not secondary gas-phase) MALDI ionization in which excited matrix molecules are the key species. At least two such excited molecules are believed to be necessary for free ion generation. This model is found to be consistent with the available data, as well as making several predictions which are confirmed by new observations. The model also predicts that the matrix suppression effect will not be observable with heavy analytes because their large excluded volume precludes desorption at the necessary mixing ratios.

Alkali-containing molecular ions in organic sims: factors influencing the emission yields

The presence of metal-containing molecular ions in the secondary ion mass spectra of organic compounds is an important feature which can be helpful in identifications and structural assignments. Alkali salts have proved to be suitable for the enhancement of cationization phenomena. Factors influencing the emission of alkali adducts (M + C) +, (M + 2C - H) +, … (C = alkali atom) have been systematically studied using a pyrimidine derivative, 4-(2′-hydroxyphenyl)-5-methyl-2-cyanaminopyrimidine. Deposition of 1–10 monolayer equivalent of organic compound onto a metal substrate previously coated with the alkali salt is shown to be very efficient for the formation of alkali adducts. Simple alkali ion fixation does not depend on the nature of the anion and is favoured by small cations (Li + > Na + > K + > Rb + > Cs +). In contrast, alkali/hydrogen substitution strongly depends on the nature of the anion; it is favoured by large anions, e.g. iodide, tetraphenylboron, diethyldithiocarbamate, suggesting reactions occurring between close species in the solid state. Such experiments can reveal differences in the acidity of hydrogen atoms in the molecule. It must be pointed out that, for less stable compounds, the tendencies observed here, in particular the scaling of the alkali-containing secondary molecular ion intensities (Li + > Na + > K + > Rb + > Cs +), could be partially altered.