Michael O. Glocker

Characterization of Specific Noncovalent Protein Complexes by UV Matrix-assisted Laser Desorption Ionization Mass Spectrometry

While electrospray (ESI) mass spectrometry has already established its potential for the characterization of non-covalent protein complexes, matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) seemed not to be applicable hitherto because of limitations in matrix chemistry and sample preparation. In this work, a sample preparation method has been developed in which 6-aza-2-thiothymine (ATT) was used as a matrix without any addition of organic cosolvents, and proteins were dissolved in aqueous buffers such as ammonium hydrogencarbonate, ammonium citrate and ammonium acetate. Under these conditions, the intact non-covalent protein complexes, RNAse S, the non-covalent complex of S-protein and S-peptide and specific dimers of coiled-coil leucine zipper polypeptides were observed by UV-MALDI/MS. The specificity of complex formation was ascertained by admixture of non-specific peptides which did not yield detectable aggregate ions. In addition, on-target tryptic digestion of cytochrome c and leucine zipper peptides was carried out after MALDI/MS molecular mass determination in the presence of the ATT matrix. Mass spectrometric analyses of these tryptic digests yielded spectra that showed complete digestion of the proteins. These results indicate that proteins maintained intact tertiary structures necessary for the formation of specific non-covalent complexes, and that trypsin retained its functional enzymatic structure and full biological activity with the present sample preparation method.

Mass spectrometric approaches to molecular characterization of protein-nucleic acid interactions

The recent development of soft ionization-desorption methods has lead to a breakthrough for the mass spectrometric analysis of biomacromolecules such as proteins and nucleic acids. In particular, the feasibility of electrospray-ionization mass spectrometry (ESI-MS) for the direct characterization of non-covalent supramolecular complexes is opening new analytical perspectives. Examples hitherto analyzed by ESI-MS include enzyme-substrate and -inhibitor complexes, homo- and heterodimers/trimers of leucine zipper polypeptides, and several other DNA- and RNA-binding proteins. Furthermore, the characterization of double-stranded and higher-order oligo- and polynucleotide complexes by negative-ion ESI has been demonstrated. Ions specific of non-covalent protein and oligonucleotide complexes can be selectively dissociated by changing the solution conditions and by increasing the desolvation potential. These results form the basis for the molecular characterization of protein-nucleotide interactions, thus complementing protein-chemical approaches, and other methods of structure determination.

Characterization of the folding pathway of recombinant human macrophage-colony stimulating-factor β (rhM-CSF β) by bis-cysteinyl modification and mass spectrometry

Melarsen oxide [p‐(4,6‐diamino‐1,3,5‐triazin‐2‐yl)aminophenylarsonous acid (MEL)], which selectively bridges spatially neighboring bis‐cysteinyl residues in (reduced) proteins, was used to trap folding intermediates chemically during 1) time‐dependent renaturation of recombinant human macrophage colony‐stimulating factor (rhM‐CSF); by redox refolding in vitro; 2) reductive unfolding in the presence of the trapping reagent; and 3) denaturing unfolding reactions in urea and guanidinium hydrochloride. Characterization of intermediates from folding and unfolding reactions was performed by electrospray ionization mass spectometry (ESI‐MS). In all folding and unfolding reactions a characteristic dimeric intermediate with two attached melarsen oxide (MEL) groups was observed, suggesting that these rhM‐CSF β species were important refolding intermediates. These intermediates presented a characteristic “charge structure” in ESI spectra with a most abundant 26+ charged molecular ion whereas the mature homodimeric rhM‐CSF β showed a most abundant 23+ molecular ion, indicating that the final product was more compact. The major locations of the two MEL groups were identified by mass spectrometric peptide mapping at cysteine residues C157 and C159 from each monomer. Cysteine residues C7 and C90 were minor modification sites. The mass spectrometric results from the in vitro folding reactions of rhM‐CSF β are in agreement with intrinsic tryptophan fluorescence measurements and are consistent with the folding pathway that starts with a fully reduced monomer (R), includes partially folded monomeric intermediates (M) and dimeric intermediates (D), and yields a final product with the native tertiary structure (N): 2Ru2009⇒u20092Mu2009⇒u2009Du2009⇒u2009N. Our results show that selective chemical trapping of bis‐thiol groups of proteins with MEL permits study of folding pathways by mass spectrometric structure characterization of intermediates with otherwise transient conformations. Proteins Suppl. 2:50–62, 1998.

A Mass Spectrometric Approach to the Characterization of Protein Folding Reactions

The arsonous acids melarsen oxide and pyridinyl-3-arsonous acid were successfully applied for chemical trapping of otherwise transient (un)folding intermediates. The trapped and arsonous acid-modified peptides and proteins were amenable to direct molecular weight determination by mass spectrometry, e.g. by nanoelectrospray ionization mass spectrometry or matrix-assisted laser desorption/ionization mass spectrometry. Arsonous acids exhibited several advantageous features such as (i) cross-linking two closely spaced thiol groups providing detailed tertiary structure information, (ii) high solubility as monomeric and oligomeric derivatives in aqueous solution, (iii) adding a relatively large mass increment to proteins upon single modification and (iv) performing fast and specific modification of bis-thiol groups in proteins to form stable structures without any side reactions even with a high molar excess of arsonous acids. Bis-cysteine thiol-selective modification of vasopressin with pyridinyl-3-arsonous acid was confirmed by Fourier transform ion cyclotron resonance mass spectrometry measurements. The major product obtained by denaturing unfolding of bovine pancreatic trypsin inhibitor (BPTI) and trapping with melarsen oxide eluted like folded BPTI in high-performance liquid chromatography analyses, indicating a “native-like” structure of this folding intermediate. The results obtained from the investigated model compounds suggest a general applicability of arsonous acids for selective bis-thiol modifications, enabling the study of protein folding reactions by mass spectrometry.

Approaches to the Characterisation of Tertiary and Supramolecular Protein Structures by Combination of Protein Chemistry and Mass Spectrometry

Soft-ionisation methods, particularly electrospray (ESI) and matrix-assisted laser desorption (MALDI) have enabled a breakthrough in the mass spectrometric structure analysis of proteins. Whereas ESI-MS provides direct information about solution structures and non-covalent interactions, the combination of mass spectrometry with structure-specific protein chemistry is emerging presently as a powerful tool for characterising tertiary structures and structure-function relations. Recent developments of chemical modification reactions are summarised which are suitable to the mass spectrometric analysis of reactive sites, surface topology and antigenic determinants in protein-tertiary structures, and can be efficiently employed in x-ray crystallographic structure determinations. Applications to the structure elucidation, and functional characterisation of porin-channel proteins and to leucine zipper protein-nucleotide complexes illustrate their efficiency in the analysis of two most important topics of structural biology, molecular recognition structures and biomacromolecular interaction.

Characterization of Tertiary Structure States and Specific Noncovalent Complexes of Proteins by UV-Matrix-Assisted Laser-Desorption / Ionization Mass Spectrometry

Hydrogen/deuterium exchange reactions (H/D exchange) in solution have been used for probing protein structures and dynamics and have been monitored using a broad spectrum of analytical methods including mass spectrometry. In order to study the effects of Matrix-Assisted Laser-Desorption/Ionization Mass Spectrometry (MALDIMS) preparation techniques on protein structures, we applied on-target H/D exchange reactions. Samples were prepared for MALDI-MS by mixing aqueous protein and peptide solutions with acidic matrix solutions (pH 2) that contained organic cosolvents. H/D exchange for labile protons of peptides and proteins prepared in this manner was found to be > 90%, indicating that proteins were denatured. By subsequent laser irradiation of one single spot on the MALDI-target, depth-profiles of the deposited matrix/sample mixtures were recorded and revealed that on-target reactions provide exchange only in the top crystal layers. In deeper crystal layers molecular ions of proteins and peptides with unexchanged hydrogens were observed. No peptides with intermediate H/D exchange were detected. By contrast, the results from H/D-exchange reactions in-solution, which were monitored by electrospray/ionization mass spectrometry (ESI-MS), were consistent with folded globular protein structures. Subsequently, conditions for MALDI-MS sample preparations were altered substantially. 6aza-2-thiothymine (ATT) was used as water soluble matrix and was dissolved in 5mM ammonium citrate (pH 5.5). Proteins and peptides were dissolved in aqueous buffers such as ammonium bicarbonate and were mixed with the matrix solution. Under these conditions non-covalent protein complexes could be analyzed and specificity of complex formation was ascertained by admixture of non-specific peptides which did not yield detectable aggregate ions. Also, trypsin maintained its active structure in the presence of the matrix, as was shown by on-target digests of proteins after UV-MALDI-MS molecular weight analyses.