Kerstin Strupat

2,5-Dihydroxybenzoic acid : a new matrix for laser desorption-ionization mass spectrometry

The performance of 2,5-dihydroxybenzoic acid (DHB) as a matrix for laser desorption—ionization mass spectrometry of proteins is described. Samples from protein/matrix mixtures with mass ratios between 1.5 × 10−2 and 1 × 10−4 were prepared by drying solutions onto metal substrates as well as by growing single crystals which contain the proteins at a concentration comparable to that in the solution in an apparently homogeneous distribution. The detection limit is demonstrated with a spectrum of cytochrome c from a 1 fmol preparation. DHB is insensitive to contaminations by inorganic salts, buffers and detergents, even to a 10% sodium dodecylsulfate admixture. Spectra from single crystal preparations taken at threshold irradiance are free of matrix signals in the low mass range. An upper limit of 1–10 amol is determined for the amount of ablated protein per spectrum.

Principles and applications of matrix-assisted UV-laser desorption/ionization mass spectrometry

Abstract Matrix-assisted laser desorption/ionization mass spectrometry (LDI MS) has shown its potential for desorbing ions of biomolecules with relative molecular masses ( M r ) up to about 300 000 daltons. Determinations of M r with an accuracy of 0.01–0.2% from about 1 pmol of sample are possible. Examples are shown for proteins, glycoproteins, oligonucleotides and carbohydrates. Examples of the combination of LDI MS with biochemical methods are shown, including the determination of the carbohydrate content of glycoproteins, the identification of disulphide-bonded subunits by cleavage of the light and heavy chains of a monoclonal antibody, the detection of the time course of enzymatic reactions and the analysis of proteins bound to a PVDF membrane.

Is the incorporation of analytes into matrix crystals a prerequisite for matrix-assisted laser desorption/ionization mass spectrometry? A study of five positional isomers of dihydroxybenzoic acid

The 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-positional isomers of dihydroxybenzoic (DHB) acid were investigated with respect to their function as matrix-assisted laser desorption/ionization (MALDI) matrices. Optical absorption spectra of solid matrix samples, recorded in diffuse reflection from samples revealed peak broadening and a red shift of the peaks relative to the solution spectra. Single crystals of all isomers were grown from solution and analyzed by x-ray crystallography. Single crystals, as well as standard dried droplet and thin layer preparations with added cytochrome c, were analyzed by UV- (266, 308, 337, 355 nm) and IR- (2.94 μm) MALDI time-of-flight mass spectrometry (MALDI-TOF-MS). A spectrophotometric measurement of the heme absorption around 400 nm of redissolved single crystals showed a quantitative incorporation of the protein into crystals of 2,5-DHB and a partial protein incorporation with large statistical fluctuations into single crystals of 2,4-DHB. No protein incorporation above the detection limit of 10−5 molar analyte-to-matrix ratio was found for the other isomers. Best MALDI spectra from standard preparations were recorded for dried droplet preparations of 2,5-DHB at 337 and 355 nm and for 2,4-DHB at 266 and 308 nm. Both matrices performed well in the IR, too. 2,6-DHB yielded spectra of comparable quality at 337 nm and 2.94 μm, but only when prepared in a thin layer from an acetone solution. The results suggest that protein incorporation into the crystals of solid MALDI matrices is helpful, but not a prerequisite for MALDI. A large surface-to-volume ratio, typical for microcrystalline thin layer preparations supports protein desorption if no measurable incorporation occurs. Undesirable matrix adduct formation to the protein ions was seen for all DHB isomers at the wavelengths of 308 and 266 nm.

Calcium-induced noncovalently linked tetramers of MRP8 and MRP14 detected by ultraviolet matrix-assisted laser desorption/ionization mass spectrometry

MRP8 and MRP14 are members of the S100 family of calcium-binding proteins which play an important role during calcium-induced activation of phagocytes. Both proteins form noncovalently associated complexes as a prerequisite for biological functions. The exact stoichiometric composition of these complexes, however, has not been completely clarified yet. In the present study we show for the first time by ultraviolet matrix-assisted laser desorption/ionization mass spectrometry (UV-MALDI-MS) the calcium-induced formation of noncovalently associated (MRP8/MRP14)2 tetramers. Furthermore, we could determine posttranslational modifications of MRP8 and MRP14, the stoichiometric proportion of the two known MRP14 isoforms in the complexes as well as the number of calcium ions bound to the single MRP8 and MRP14 monomers and tetramers. MRP14 showed a higher affinity for calcium than MRP8. Upon complex formation the calcium binding increased to maximal saturation of the known EF hands in the complexed forms. Calcium-induced stabilization of the MRP8/MRP14 complexes was confirmed by DSC studies. Our results extend scope and application of UV-MALDI-MS by allowing identification of noncovalent protein complexes, the identification of minor alterations of subunits in such complexes as well as the determination of bound calcium ions.

Mechanisms in MALDI analysis: surface interaction or incorporation of analytes?

Abstract Since the early days of matrix-assisted laser desorption/ionization (MALDI), the definition of a unified theory on the MALDI process is a major challenge. The new results presented in this paper clearly show that the idea of a uniform MALDI mechanism that accounts for the spreading applications has to be given up. Based on different preparation protocols distinct differences in the desorption/ionization process for carbohydrates in contrast to peptides/proteins are elucidated. Although isolated/incorporated and cluster-desorbed peptides/proteins are effectively entrained and cooled within the expanding plume of matrix clusters, as shown by a low degree of metastable analyte-ion fragmentation, a laser desorption and gas-phase cationization mechanism can be confirmed as the dominant part in ionization for neutral oligosaccharides which can be initiated even for particulate analyte material or deposits onto a matrix surface. The previously presented “lucky survivor-model” on cluster desorption of preformed ions thus needs this extension.

Calcium-induced noncovalently linked tetramers of MRP8 and MRP14 are confirmed by electrospray ionization-mass analysis

Proteins of the S100- family such as MRP8 (S100A8) and MRP14 (S100A9)—and its isoform MRP14*—show two calcium-binding sites (EF hands) per protein chain. MRP8, MRP14*, and MRP14, isolated from human granulocytes or monocytes, are known to form noncovalently associated complexes; the exact stoichiometries of these complexes in the presence of calcium are still controversially discussed in the literature. The present electrospray ionization-mass spectrometry (ESI-MS) study shows that MRP8, MRP14*, and MRP14 exist as heterodimers MRP8/14* and MRP8/14, respectively, in the absence of calcium confirming both a recent nuclear magnetic resonance study and a biochemical study on this topic. Furthermore, this ESI-MS study confirms the previously published matrix-assisted laser desorption ionization (MALDI)-MS study, which states that the MRP8/14* and MRP8/14 heterodimeric complexes tetramerize to heterotetramers (MRP8/14*)2, (MRP8/14*)(MRP8/14), and (MRP8/14)2, respectively, in the presence of calcium. The number of Ca2+ ions bound to the individual tetramer is determined to be eight for nonphosphorylated fractions; this is in agreement with the previously reported MALDI study on these fractions. About 1.2 Ca2+ ions more are bound to the phosphorylated form; it is speculated that the additional Ca2+ ions are bound to the phosphate groups in the tetramers. This study is, therefore, convincing proof of the reliability of MALDI-MS in studying noncovalent protein-protein interactions.

Analysis of Quaternary Protein Ensembles by Matrix Assisted Laser Desorption/Ionization Mass Spectrometry

The intact noncovalent structure of the homo-oligomeric complexes of streptavidin (52 kDa), alcohol dehydrogenase (150 kDa), and beef liver catalase (240 kDa) have been observed using the matrix 2,6-dihydroxyacetophenone in an organic solvent. Intact streptavidin tetramers could also be observed with ferulic acid and other hydroxyacetophenone derivatives. Intact complexes are observed only for the first shot at a given position, which may be due to physical segregation or precipitation of the noncovalent complexes at the crystal surface. This effect is independent of the macroscopic crystal structure or the type of substrate (hydrophobic versus hydrophilic). Observation of intact complexes is not affected by addition of less than 10 mM salts or buffers, and appears to be independent of the pH stability range of the protein samples investigated.

Matrix-assisted laser desorption / ionisation^mass spectrometry applied to biological macromolecules

Abstract Since its invention in the late 1980s, matrix-assisted laser desorption/ionisation–mass spectrometry (MALDI-MS) has been applied to a large number of analytical problems such as the analysis of proteins, glycoconjugates, polynucleotides and synthetic polymers. This article explains briefly the principle of MALDI and the instrumentation required. The preparation and purification of samples on a microlitre scale prior to mass analysis are described. Examples are given of MALDI mass analysis using infrared and ultraviolet lasers (IR– and UV–MALDI-MS). Analytical applications of MALDI-MS to mass analysis of electroblotted proteins, to unambiguous protein identification, the de novo sequencing of polynucleotides, DNA mutant analysis for diagnostic purposes, and the analysis of non-covalent complexes, are addressed.

Investigations of 2,5-DHB and succinic acid as matrices for IR and UV MALDI. Part: I UV and IR laser ablation in the MALDI process

Abstract UV and IR laser ablation under MALDI conditions is described for two typical solid UV and IR matrices. 2,5-Dihydroxy benzoic acid (2,5-DHB) as a UV and IR matrix and succinic acid (SA) as an IR matrix were investigated systematically by light and electron microscopy, and by mass spectrometry. Large single crystals of 2,5-DHB and SA with and without protein incorporated were used for the experiments. The UV MALDI experiments were performed with a flat-top laser beam profile. Within a limited fluence range, these exposures resulted in the formation of typical cone structures, occurring after several hundred laser shots onto a given spot. Such structures had originally been described for materials processing of polymers and ceramics with excimer lasers. For the IR exposures, a Gaussian laser beam profile was used. The much lower absorption of matrix compounds at IR wavelengths compared with the absorption of matrix compounds at UV wavelengths results in a much larger penetration depth of the IR laser light into the matrix solid and consequently in a much higher ablation depth and amount of ablated material. This large volume of material, ablated per single exposure, prevents the formation of specific surface structures in IR MALDI. The amount of matrix material ablated per laser shot was measured with a laser profilometer to about 10 000 μm3. This determines the amount of consumed protein per laser shot to about 1 fmol under typical IR MALDI conditions.

Investigations of 2,5-DHB and succinic acid as matrices for UV and IR MALDI. Part II: Crystallographic and mass spectrometric analysis

Abstract Crystallographic, mass spectrometric and spectrophotometric data for the two MALDI-matrices 2,5-dihydroxybenzoic acid (2,5-DHB; UV and IR MALDI) and succinic acid (SA, IR MALDI) are presented. It is shown by absorption spectroscopy that SA single crystals incorporate protein molecules quantitatively up to a molar ratio of protein to matrix of about 2 × 10−5. This result is comparable with that obtained earlier for 2,5-DHB single crystals. MALDI-MS data obtained from protein-doped single crystals were comparable with standard preparation results (dried droplet method) for both matrices. Threshold fluences as well as quality of spectra are equal for all accessable crystal faces of the 2,5-DHB crystals. Crystallographic data taken from the literature indicate that the crystals of both matrices have no major hydrophobic domains. The observed efficient protein incorporation must, therefore, be based on other than hydrophobic interactions. X-ray crystallography of protein-doped SA single crystals reveals the conserved crystal data of neat SA; results showed that their structure is not altered by the protein incorporation, which is in good agreement with earlier X-ray crystallography results for protein-doped 2,5-DHB single crystals. This contribution is the second in a row published in this issue about systematic investigations using UV (2,5-DHB) and IR (2,5-DHB and SA) matrices. Most of the relevant experimental details are given in Part I, this issue.