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.

Amino-acetylation and -succinylation reactions in combination with mass spectrometric peptide mapping of tryptic peptide mixtures have been employed for surface topology-probing of lysine residues in bovine ribonuclease A, lysozyme, and horse heart myoglobin as model proteins of different surface structures. Direct molecular weight determinations identifying the precise number of acyl groups in partially modified proteins were obtained by electrospray and 252Cf-plasma desorption mass spectrometry. Electrospray mass spectra of multiply protonated molecular ions and deuterium exchange experiments provided a relative conformational characterization of protein derivatives and enabled the direct determinations of intact, partially acylated heme-myoglobin derivatives. Tryptic peptide mapping analysis, using plasma desorption and fast atom bombardment mass spectrometry, ascertained by mass spectrometric characterization of HPLC-separated modified peptides, yielded the exact identification of acylation sites. Relative reactivities of the amino acylation were derived from the peptide mapping data and from quantitative estimations of modified peptides upon acetylation/trideuteroacetylation and provided direct correlations with the relative surface accessibilities of lysine-epsilon-amino groups taken from X-ray crystallographic structure data of the proteins. The reactive lysine-41 residue in ribonuclease A which is part of the substrate binding site was directly identified from the mass spectrometric data. These results indicate tertiary structure-selective acylation combined with mass spectrometric peptide mapping as an efficient approach for the molecular characterization of surface topology and reactive fundamental lysine residues in proteins.

Mass spectrometric epitope mapping has become a versatile method to precisely determine a soluble antigen's partial structure that directly interacts with an antibody in solution. Typical lengths of investigated antigens have increased up to several 100 amino acids while experimentally determined epitope peptides have decreased in length to on average 10-15 amino acids. Since the early 1990s more and more sophisticated methods have been developed and have forwarded a bouquet of suitable approaches for epitope mapping with immobilized, temporarily immobilized, and free-floating antibodies. While up to now monoclonal antibodies have been mostly used in epitope mapping experiments, the applicability of polyclonal antibodies has been proven. The antibody's resistance towards enzymatic proteolysis has been of key importance for the two mostly applied methods: epitope excision and epitope extraction. Sample consumption has dropped to low pmol amounts on both, the antigen and the antibody. While adequate in-solution sample handling has been most important for successful epitope mapping, mass spectrometric analysis has been found the most suitable read-out method from early on. The rapidity by which mass spectrometric epitope mapping nowadays is executed outperforms all alternative methods. Thus, it can be asserted that mass spectrometric epitope mapping has reached a state of maturity, which allows it to be used in any mass spectrometry laboratory. After 25 years of constant and steady improvements, its application to clinical samples, for example, for patient characterization and stratification, is anticipated in the near future. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 37:229-241, 2018.

The heat shock protein Hsp33 is a very potent molecular chaperone with a distinctive mode of functional regulation; its activity is redox-regulated. In its reduced form all six cysteinyl residues of Hsp33 are present as thiols, and Hsp33 displays no folding helper activity. This indicates a significant conformational change during the activation process of Hsp33. Mass spectrometry, thus, unraveled a novel molecular mechanism by which alteration of the disulfide bond structure, as a result of changes in the cellular redox potential, results in the activation of a molecular chaperone.Hsp33 is a newly discovered heat shock protein that functions as a highly efficient molecular chaperone (1). Hsp33 protects bacterial cells from deleterious effects caused by oxidants like H 2 O 2 showing that it plays a role in the bacterial defense system against oxidative stress. Hsp33 is distinguished from all other known chaperone proteins by the finding that Hsp33 is functionally regulated at posttranslational level, by the redox conditions of the environment (1) (reviewed in Refs. 2 and 3). Elevated H 2 O 2 concentrations induce the chaperone functions of Hsp33 (1), but the precise molecular mechanism that translates the changes of the cellular redox environment into differences in chaperone activity is not yet understood.The Hsp33 amino acid sequence contains a novel conserved motif consisting of four cysteinyl residues near the C terminus of the protein. These cysteinyl residues form a C-X-C motif and a C-Y-Z-C motif (Fig. 1), separated by 27-30 amino acid residues in Escherichia coli Hsp33 and its homologues. These four cysteinyl residues are present in all 27 known Hsp33 homologues suggesting that they play an important role in the function of Hsp33. In addition, two further cysteinyl residues, Cys 141 and Cys 239 , are present in Hsp33. Cys 239 is very poorly conserved occurring in only 4 of the 27 known Hsp33 homologues. Cys 141 is moderately conserved and is present in 10 of the 27 Hsp33 homologues known so far. We have postulated that disulfide bond formation is involved in the activation process of Hsp33. Thus, analysis of the disulfide bond status and connectivities in inactive and in active Hsp33 seemed very important to further understand the regulation of this efficient molecular chaperone.Two complementary mass spectrometry-based strategies are suitable for determining the presence and locations of disulfide bonds in proteins. Which one is superior is dependent on the proximity of the involved cysteinyl residues in the amino acid sequence. Both approaches involve cleavage of the protein by enzymatic or chemical means under conditions that minimize disulfide bond scrambling (4 -6). The first approach can be used when the proteolytically derived peptides contain zero or one cysteinyl residue. The peptide mixtures are directly analyzed by mass spectrometric peptide mapping. Molecular mass analyses identify the peptides and disulfide-bonded dipeptides by assigning the observed ion signals to the corresponding calculat...

Hypoxia has a profound influence on progression and metastasis of malignant tumors. In the present report, we used the oligonucleotide microarray technique to identify new hypoxia-inducible genes in malignant melanoma with a special emphasis on angiogenesis factors.