Electrospray Mass Spectrometry of Biomacromolecular Complexes with Noncovalent Interactions—New Analytical Perspectives for Supramolecular Chemistry and Molecular Recognition Processes

The development of “soft” ionization methods in recent years has enabled substantial progress in the mass spectrometric characterization of macromolecules, in particular important biopolymers such as proteins and nucleic acids. In contrast to the still existing limitations for the determination of molecular weights by other ionization methods such as fast atom bombardment and plasma desorption, electrospray ionization (ESI) and matrix-assisted laser desorption have provided a breakthrough to macromolecules larger than 100 kDa. Whereas these methods have been successfully applied to determine the molecular weight and primary structure of biopolymers, the recently discovered direct characterization by ESI-MS of complexes containing noncovalent interactions (“noncovalent complexes”) opens new perspectives for supramolecular chemistry and analytical biochemistry. Unlike other ionization methods ESI-MS can be performed in homogeneous solution and under nearly physiological conditions of pH, concentration, and temperature. ESI mass spectra of biopolymers, particularly proteins, exhibit series of multiply charged macromolecular ions with charge states and distributions (“charge structures”) characteristic of structural states in solution, which enable a differentiation between native and denatured tertiary structures. In the first part of this article, fundamental principles, the present knowledge about ion formation mechanism(s) of ESI-MS, the relations between tertiary structures in solution and charge structures of macro-ions in the gas phase, and experimental preconditions for the identification of noncovalent complexes are described. The hitherto successful applications to the identification of enzyme–substrate and –inhibitor complexes, supramolecular protein–and protein–nucleotide complexes, double-stranded polynucleotides, as well as synthetic self-assembled complexes demonstrate broad potential for the direct analysis of specific noncovalent interactions. The present results suggest new applications for the characterization of supramolecular structures and molecular recognition processes that previously have not been amenable to mass spectrometry; for example, the sequence-specific oligomerization of polypeptides, antigen–antibody complexes, enzyme–and receptor–ligand interactions, and the evaluation of molecular specificity in combinatorial syntheses and self-assembled systems.     

Laser desorption mass spectrometry on thin liquid jets

A recently developed soft desorption method for mass spectrometry is presented, which is called Laser Induced Liquid Beam Ionization/Desorption (LILBID). Analyte ions are desorbed from a thin jet of analyte solution directly into vacuum by means of an IR laser pulse, which has been tuned to a vibrational resonance of the solvent. A comparative experiment with ammonium chloride and aniline hydrochloride shows that ion formation via proton transfer takes place in the solution. Thermally unstable compounds, as well as supra- and biomolecular complexes, can be detected intact and mass analyzed in a reflectron time-of-flight (Re-TOF) mass spectrometer. During the desorption process, noncovalent interactions and some solvation characteristics are preserved. Three examples for the capacity of LILBID are given in this short overview: (a) ion-solvent interactions with the formation of a clathrate structure Cs⁺(H₂O)₂₀, (b) host-guest interactions with the K⁺ selectivity of valinomycin, and (c) noncovalent interactions with the dimerization of gramicidin. Received: 29 July 1997 / Revised: 4 September 1997 / Accepted: 12 September 1997     

MALDI质谱检测蛋白质与富勒醇的非共价复合物

基质辅助激光解吸电离(MALDI)质谱由于受到酸性基质、样品制备、激光诱导聚合和基质加合物的形成等条件的限制而难以用于非共价复合物的检测.本文以芥子酸为基质,观察到蛋白质与富勒醇的特殊相互作用,一些质谱特征,如质量数迁移、宽的加合峰和定量结合比表明,在蛋白质和富勒醇之间形成了特殊的非共价复合物.其中,血红蛋白与富勒醇的结合比是1:4,而肌红蛋白与富勒醇的结合比是1:1.实验结果表明:富勒醇可用来保护血红蛋白,有在酸性介质中防止其分解的作用.因此,通过在基质组份中添加特性有机化合物保护被测样品,有可能实现用MALDI质谱测定四级结构蛋白质的分子量.     

Fragmentation studies of noncovalent sugar-sugar complexes by infrared atmospheric pressure MALDI

An investigation of sugar-sugar noncovalent complex fragmentation was conducted using a 2.94 microm Er:YAG laser for infrared (IR) atmospheric pressure matrix-assisted laser desorption/ionization (AP MALDI) on an ion trap mass spectrometer (ITMS). This approach allowed the analysis of weak noncovalent complexes between a variety of biologically relevant oligosaccharides. The strength of interaction varied with different sugar structures, potentially due to varying strength of hydrogen bonding networks. In some cases, fragmentation of intramolecular sugar bonds preceded breakdown of the noncovalent complex. This result appeared primarily when complexes contained sugars with at least one sialic acid. Globotrios dimers also showed intramolecular fragmentation in preference to breakdown of the noncovalent dimer. This technique will allow further study of sugar-sugar interactions known to play a role in cellular interactions.     

Probing the hydrophobic effect of noncovalent complexes by mass spectrometry

The study of noncovalent interactions by mass spectrometry has become an active field of research in recent years. The role of the different noncovalent intermolecular forces is not yet fully understood since they tend to be modulated upon transfer into the gas phase. The hydrophobic effect, which plays a major role in protein folding, adhesion of lipid bilayers, etc., is absent in the gas phase. Here, noncovalent complexes with different types of interaction forces were investigated by mass spectrometry and compared with the complex present in solution. Creatine kinase (CK), glutathione S-transferase (GST), ribonuclease S (RNase S), and leucine zipper (LZ), which have dissociation constants in the nM range, were studied by native nanoelectrospray mass spectrometry (nanoESI-MS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with chemical cross-linking (XL). Complexes interacting with hydrogen bonds survived the transfer into gas phase intact and were observed by nanoESI-MS. Complexes that are bound largely by the hydrophobic effect in solution were not detected or only at very low intensity. Complexes with mixed polar and hydrophobic interactions were detected by nanoESI-MS, most likely due to the contribution from polar interactions. All noncovalent complexes could easily be studied by XL MALDI-MS, which demonstrates that the noncovalently bound complexes are conserved, and a real “snap-shot” of the situation in solution can be obtained.     

Detection of specific noncovalent interaction of peptide with DNA by MALDI-TOF

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to obtain spectra of peptide-DNA complexes formed by basic domain (BD15) of c-Fos protein and DNA AP-1 site (5'-TGAGTCA-3'). The noncovalent interaction between single stranded DNA and BD15 was observed and confirmed to be an ionic one between the negatively charged sugar-phosphate backbone of DNA and positively charged side chains of Arg- and lys-rich peptides as demonstrated by Vertes and coworkers and Woods and coworkers. But the specific noncovalent interaction between DNA AP-1 site and the dimer of BD15 was firstly detected in this paper. Various different sequence DNAs were studied and it was found that this interaction is a sequence-specific one, and AP-1 site was essential for this interaction. This specific interaction depends on the matrix. It was only observed in the ATT matrix and not in the other two matrixes (CHCA and DHBA).     

MALDI方法研究蛋白质的非共价复合物

飞行时间质谱仪(TOFMS)在理论上无质量范围的限制,可实现大分子蛋白质与核酸的非共价复合物的直接检测.特别是在近中性溶液条件下通过对芥子酸和6-氮杂-2-硫代胸腺嘧啶基质的使用及双层样品制备方法的改善,获得了稳定复合物的高灵敏度质谱检测.肌红蛋白-血红素复合物能够在芥子酸基质的不同pH条件下(pH2.0或pH5.0)同时观察到.而运用双层样品制备方法,获得了核糖核酸酶复合物(RNaseS)在第一次激光照射下的突出质谱峰,但其丰度均随更多的激光打击而减弱.     

RNA-RNA noncovalent interactions investigated by microspray ionization mass spectrometry.

Electrospray ionization mass spectrometry is playing an increasing role in the study of noncovalent interactions involving biomolecules. RNA-RNA complexes are important in many areas of biology, including RNA catalysis, RNA splicing, ribosome function, and gene regulation. Here, microelectrospray mass spectrometry (microESI-MS) is used to study noncovalent base-pairing interactions between RNA oligonucleotides, an area not previously explored by this technique. Using a set of complementary RNA oligonucleotides, we demonstrate the formation of the expected double-helical RNA complexes composed of three distinct oligonucleotides. The ability to study specific RNA noncovalent interactions by microESI-MS has the potential to provide a unique method by which to analyze and assign precise molecular masses to RNA-RNA complexes.     

Characterization of specific noncovalent complexes between guanidinium derivatives and single-stranded DNA by MALDI

Noncovalently bound complexes between highly basic sites of 12 guanidinium compounds and single-stranded DNA were studied using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. 6-Aza-2-thiothymine (ATT) was used as the matrix in the presence of ammonium citrate, and spectra were recorded in the positive ion mode. Detailed control experiments confirmed unambiguously the high selectivity and specificity of the guanidinium moiety for phosphate groups of DNA. The results verify the binding stoichiometry and show preferential binding of hydrophobic binders (pyrene and anthracene guanidinium derivatives) to all sequences examined. In addition, we demonstrate that electrostatic noncovalent interactions are strengthened with phosphorothioate analogs of DNA. These results clearly highlight the structure-directing role of the self-assembling organic species and strongly emphasize the significance of concentration, hydrophobicity, hydrogen-bonding, and pi-pi interactions of the artificial receptor in the formation of these noncovalent complexes. Because of the ability of DNA-binding compounds to influence gene expression, and therefore cell proliferation and differentiation, the interactions described above could be important in providing a better understanding of the mechanism of action of these noncovalent genetic regulators.     

Investigation of the first shot phenomenon in MALDI mass spectrometry of protein complexes

Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) of noncovalent protein complexes is difficult, due to the disruptive nature of processes occurring during MALDI sample preparation and ion formation. Sometimes the observation of intact noncovalent protein complexes with MALDI is only possible if data are acquired from the first laser shot fired at a fresh sample; this is called the 'first shot phenomenon'. To study the origin of the first shot phenomenon, we used MALDI-MS and confocal laser scanning microscopy (CLSM) to examine typical MALDI sample preparations with embedded protein complexes, labeled with fluorophores. Fluorescence energy transfer techniques allowed the differentiation between intact and dissociated protein complexes with CLSM. In cases where a first shot behavior was observed by MALDI-MS, it was found to be accompanied by localization of protein complexes at the exterior of the sample crystals. Segregation of the large protein complexes to the exterior and dissociation of the complexes in the crystal interior during sample crystallization can rationalize this observation.     

Sequence confirmation of chemically modified RNAs using exonuclease digestion and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

A broadly applicable, robust, and rapid method for complete sequence confirmation of highly modified oligonucleotides containing a mixture of 2′‐deoxy, 2′‐fluoro, 2′‐o‐methyl, abasic and ribonucleotides is presented. The passenger (sense) and guide (antisense) strands from synthetic short interfering RNA duplexes (siRNA) were digested individually using both 5′‐ and 3′‐exonucleases and the resulting ladders were analyzed using matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry. Conditions for enzymatic digestion and MALDI‐TOF mass analysis were investigated and optimized, and the digestion pattern and sequence coverage of each strand was discussed. Complete sequence confirmation for the antisense strands of four synthetic RNA duplexes was obtained, whereas a three‐base sequence gap in the 5′‐end was observed for all four sense strands. A general strategy is proposed for routine sequence confirmation of highly modified oligonucleotides, and the potential for complete automation of the method is also discussed. Copyright © 2009 John Wiley & Sons, Ltd.     

MALDI‐MS detection of noncovalent interactions of single stranded DNA with Escherichia coli single‐stranded DNA‐binding protein

The Escherichia coli single‐stranded DNA binding protein (SSB) selectively binds single‐stranded (ss) DNA and participates in the process of DNA replication, recombination and repair. Different binding modes have previously been observed in SSB?ssDNA complexes, due to the four potential binding sites of SSB. Here, chemical cross‐linking, combined with high‐mass matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry (MS), is used to determine the stoichiometry of the SSB?ssDNA complex. SSB forms a stable homotetramer in solution, but only the monomeric species (m/z 19 100) can be detected with standard MALDI‐MS. With chemical cross‐linking, the quaternary structure of SSB is conserved, and the tetramer (m/z 79 500) was observed. We found that ssDNA also functions as a stabilizer to conserve the quaternary structure of SSB, as evidenced by the detection of a SSB?ssDNA complex at m/z 94 200 even in the absence of chemical cross‐linking. The stability of the SSB?ssDNA complex with MALDI strongly depends on the length and strand of oligonucleotides and the stoichiometry of the SSB?ssDNA complex, which could be attributed to electrostatic interactions that are enhanced in the gas phase. The key factor affecting the stoichiometry of the SSB?ssDNA complex is how ssDNA binds to SSB, rather than the protein‐to‐DNA ratio. This further suggests that detection of the complex by MALDI is a result of specific binding, and not due to non‐specific aggregation in the MALDI plume. Copyright © 2012 John Wiley & Sons, Ltd.     

A novel approach to analyze membrane proteins by laser mass spectrometry: From protein subunits to the integral complex

A novel laser-based mass spectrometry method termed LILBID (laser-induced liquid bead ion desorption) is applied to analyze large integral membrane protein complexes and their subunits. In this method the ions are IR-laser desorbed from aqueous microdroplets containing the hydrophobic protein complexes solubilized by detergent. The method is highly sensitive, very efficient in sample handling, relatively tolerant to various buffers, and detects the ions in narrow, mainly low-charge state distributions. The crucial experimental parameter determining whether the integral complex or its subunits are observed is the laser intensity: At very low intensity level corresponding to an ultrasoft desorption, the intact complexes, together with few detergent molecules, are transferred into vacuum. Under these conditions the oligomerization state of the complex (i.e., its quaternary structure) may be analyzed. At higher laser intensity, complexes are thermolyzed into subunits, with any residual detergent being stripped off to yield the true mass of the polypeptides. The model complexes studied are derived from the respiratory chain of the soil bacterium Paracoccus denitrificans and include complexes III (cytochrome bc(1) complex) and IV (cytochrome c oxidase). These are well characterized multi-subunit membrane proteins, with the individual hydrophobic subunits being composed of up to 12 transmembrane helices.     

The detection of intact double-stranded DNA by MALDI

DNA fragments have been analyzed by matrix-assisted laser desorption ionization (MALDI) and electrospray mass spectrometry. In many cases, only the single-stranded oligonucleotides have been detected. Recently, spectra of intact double-stranded DNA have been obtained in both electrospray and massive cluster impact ionization. We show here the first MALDI spectra of intact double-stranded DNA (EcoR1 adaptor 12/16) that is clearly not due to nonspecific dimer formation. 6-Aza-2-thiothymine was used as the matrix in the presence of ammonium citrate. Via the same procedure but with other matrices commonly employed for oligonucleotide analysis, the intact DNA duplex was not detected. No sign of the homodimer of either of the single strands is observed. Although the spectrum also shows peaks attributable to each of the single strands, these are demonstrated to arise from the DNA solution and not the sample preparation or desorption process.     

Detection of intact hemoglobin from aqueous solution with laser desorption mass spectrometry

Laser induced liquid beam ionization/desorption mass spectrometry (LILBID-MS) is a new desorption method recently developed in our laboratory. This method allows ions to be desorbed directly from the liquid phase into the high-vacuum region of a mass spectrometer. This method has now been applied to the detection of noncovalent protein-protein complexes. The example given in this paper is the quartenary complex of human hemoglobin. For the first time, the intact hemoglobin could be detected by laser desorption mass spectrometry. Furthermore, evidence for the specificity of the complex is given.     

A general mass spectrometry-based assay for the quantitation of protein-ligand binding interactions in solution

A new method that utilizes matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and exploits the hydrogen/deuterium (H/D) exchange properties of proteins was developed for measuring the thermodynamic properties of protein-ligand complexes in solution. Dissociation constants (Kd values) determined by the method for five model protein-ligand complexes that included those with small molecules, nucleic acids, peptides, and other proteins were generally in good agreement with Kd values measured by conventional methods. Important experimental advantages of the described method over existing methods include: the ability to make measurements in a high-throughput and automated fashion, the ability to make measurements using only picomole quantitities of protein, and the ability to analyze either purified or unpurified protein-ligand complexes.     

A study of enhanced ion formation from metal‐semiconductor complexes in atmospheric pressure laser desorption/ionization mass spectrometry

The study of the key parameters impacted surface‐assisted laser desorption/ionization‐mass spectrometry is of broad interest. In previous studies, it has been shown that surface‐assisted laser desorption/ionization‐mass spectrometry is a complex process depending on multiple factors. In the presented study, we showed that neither porosity, light absorbance nor surface hydrophobicity alone influence the enhancement phenomena observed from the hybrid metal‐semiconductor complexes versus individual targets, but small changes in the analyte attaching to the target significantly affect laser desorption ionization‐efficiency. By means of Raman spectroscopy and scanning electron microscopy, it was revealed that the formation of an amorphous analyte layer after drying on a solid substrate was essential for the enhanced laser desorption ionization‐signal observed from the hybrid metal‐semiconductor targets, and the crystallization properties of the analyte appeared as a function of the substrate. Obtained results were used for the screening of regular and lactose‐free milk samples through the hybrid metal‐semiconductor target. Copyright © 2016 John Wiley & Sons, Ltd.     

Localization of noncovalent complexes in MALDI-preparations by CLSM

The unambiguous detection of noncovalent complexes (NCCs) by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is still a far cry from being routine. For protein NCCs such as their quaternary structure it has been reported that signals of the intact complex are only obtained for the first or at most the first few laser exposures of a given sample area. This observation was called the first-shot phenomenon. In the present study, this first-shot phenomenon has been investigated for the hexameric protein complex allophycocyanine (APC) by two independent methods, MALDI-MS with a (nearly) pH-neutral matrix 6-aza-2-thiothymine (6-ATT) and by imaging the fluorescence of the complex in APC-6-ATT preparations by confocal laser scan microscopy (CLSM). The intact APC heterohexamer loses its visible fluorescence upon dissociation into its subunits. Both methods consistently show that intact APC complexes are precipitated at the matrix crystal surface, but dissociate upon incorporation into the matrix crystals.     

Multiplexed hybridizations of positively charge-tagged peptide nucleic acids detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Peptide nucleic acid (PNA) is a novel class of DNA analogues in which the entire sugar-phosphate backbone is replaced by a pseudopeptide counterpart. Owing to its neutral character and the consequent lack of electrostatic repulsion, PNA exhibits very stable heteroduplex formation with complementary nucleic acid that is essentially ionic strength independent and enables hybridization under minimum salt conditions. This feature as well as its superior ion stability and easy ionization compared to DNA renders PNA very attractive for hybridization-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) applications. We have developed an approach to DNA characterization that takes advantage of multiplexed PNA hybridizations analyzed by MALDI-TOFMS. Our motivation was the further development of oligonucleotide fingerprinting, an efficient technique for cDNA and genomic DNA library characterization. Through positive 'charge-tagging' of PNA the efficiency of detection in MALDI-TOFMS was considerably enhanced permitting an unparalleled degree of multiplexing. Results from the simultaneous hybridization of 21 charge-tagged PNA hexamer oligonucleotides showed that genomic DNA and cDNA clones are successfully characterized on the basis of their hybridization profiles. The degree of multiplexing achieved may render a significant increase in throughput and hence efficiency of oligonucleotide fingerprinting possible.     

Preservation and detection of specific antibody—peptide complexes by matrix-assisted laser desorption ionization mass spectrometry

The direct detection of an antibody-peptide complex is reported by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Experimental conditions have been found in which specific, noncovalent interactions in solution are maintained throughout the sample preparation and ionization process. Mass measurements based on the ion signals for the intact antibody and 1:1 antibody-peptide complex reveal that specific noncovalent associations between a monoclonal antibody and a peptide, which comprises the determinant of the corresponding antigen, are maintained in the gas phase. These results support the wider application of MALDI-MS to studies of the structure and specificity of macromolecular complexes important to immune and other biological function.