MILESTONES IN

Andrea Mele

2002 The golden year of structural chemistry ANDREA MELE1*, FRANCA CASTIGLIONE2 *Corresponding author 1. Politecnico di Milano, Dipartimento di Chimica, Materiali e Ingegneria Chimica “” Via L. Mancinelli, 7I – 20131 MILANO (Italy) 2. Politecnico di Milano, Dipartimento di Chimica, Materiali e Ingegneria Chimica “Giulio Natta” Via L. Mancinelli, 7I – 20131 MILANO (Italy)

Bumblebee, funded by the Navy, aimed at the design and ABSTRACT: Modern genomics, proteomics and life-science manufacturing of a ramjet-powered anti-aircraft missile for would have not undergone milestones achievements the fleet. Bumblebees are, as a matter of fact, a living without the support of powerful physical methods for the paradox as they would not be able to fly, according to the investigation of molecular structures. Chemistry, laws of aerodynamics. and medical research are in debt with But, as bumblebees ignore aerodynamics, they do fly, as and NMR . The Nobel many years later would molecular elephants have done in Prize in Chemistry for 2002 awarded the fundamental the ionization chamber of modern mass spectrometers. contribution of soft ionization MS methods – electrospray The experience gained by Fenn in thermodynamics of free- and MALDI – and biomolecular NMR. jet expansion of gases played a key role in the effective implementation of the Electrospray Ionization (ESI) source for mass spectrometry, nowadays one of the most used soft INTRODUCTION ionization techniques, along with MALDI, in the research practise. Soft ionization is required to evaporate and ionize Three , John B. Fenn (Yale and V i rg i n ia a with no side-fragmentation or decomposition. Commonwealth University), Koichi Tanaka ( Corp.) This is a severe problem for polar and/or high molecular and Kurt Wüthrich (ETH, Zürich), were awarded of the Nobel weight . Prize in Chemistry for 2002. Fenn and Tanaka gave In the early days of soft ionization techniques, a class of fundamental contribution to the development of modern methods were developed based on the rapid and local soft ionization techniques in mass spectrometry – heating of the sample caused by collision with a variety of electrospray ionization (ESI) and matrix assisted laser/ “bullets”. The rate of desorption/ionization is much higher desorption ionization (MALDI), respectively – pivotal for the than that of unimolecular decomposition, thus leading to structural studies of biomolecules. intact gaseous ready to be analyzed in suitable mass Wüthrich developed multidimensional nuclear magnetic analyzers. This is the ground of the so called “energy resonance (NMR) methods for the three-dimensional sudden” principle. characterization of and nucleic acids in solution. In Different types of colliding agents were used: , ions, this brief note we mainly stress the methodological aspects photons, or radioactive decay products. and the multidisciplinary approach that brought the three These approaches gave rise, in the order, to Fast scientists to the most important scientific award and the Bombardment (FAB), Laser Desorption (LD, the precursor of routes they opened in modern chemical, biochemical and modern MALDI), and 253Cf Plasma Desorption (PD) medical research. ionization sources. Common features shared by these methods were the limited mass range, hampering investigation on polymers and bio-macromolecules, and ELECTROSPRAY IONIZATION (ESI): HOW TO MAKE ELEPHANTS FLY complex experimental set-up, especially for 253Cf PD. A completely different approach consisted in the The metaphor used by John B. Fenn in his Nobel lecture (1), application of an electric field to a solution of non-volatile “Electrospray Wings for Molecular Elephants”, clearly substrate, in turn nebulized and undergoing collision with an indicates the major breakdown that his discoveries inert, dense gas such as N2 at atmospheric pressure. introduced in Chemistry and Molecular Biology research: The inert gas plays the double role of source of enthalpy for the possibility to turn high molecular weight, complex and desolvation of the ions and moderator of the kinetic energy thermally unstable molecules like proteins, polysaccharides, of the ions themselves, useful for an easy analysis (e.g. via nucleic acids or, even better, a non-covalent complex quadrupole or TOF analyzers). among them, into gas-phase ions amenable to mass The breakthrough occurred in 1968, when Dole and analysis and collision induced decomposition for structural co-workers published a paper (2) that opened the way to assessment. Indeed, even a small seems an the modern ESI technology. elephant compared to a small molecule. The basic idea can be summarized as follows: a dilute And the whole story started, actually, around 1945 with a solution containing the analyte is sprayed through a needle research project with a highly evocative name: the Project kept at positive voltage with respect to surroundings.

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This configuration generates positively charged droplets; The latter assumes that the ultimate steps of the process are the evaporation of solvent would increase the charge mediated by the intense electric field at the droplet density of the droplet up to a critical value –the Rayleigh surface, strong enough to cause the “evaporation” of ions limit (3)– at which the Coulomb repulsion equals the surface from the surface into the surrounding gas ( evaporation tension. Further increase of the curvature radius, due to the model, IEM). assumption of continuous evaporation, would cause the At this stage the consolidated experience of Fenn in free-jet disruption of the droplet in smaller ones. expansion gave a significant improvement to the This cascade of evaporation-disintegration episodes is technique. Indeed, one of the major pitfall of Dole’s expected to yield, at the final step, the isolated analyte experimental set-up was in the competitive and extensive retaining some of the positive charges of the precursor resolvation of gaseous ions during the ES process. 2002 droplet. The problem was solved by implementing a counter-current The golden year of structural chemistry nitrogen stream able to flush away the solvent vapour from ANDREA MELE1*, FRANCA CASTIGLIONE2 *Corresponding author 1. Politecnico di Milano, Dipartimento di Chimica, Materiali e Ingegneria Chimica “Giulio Natta” Via L. Mancinelli, 7I – 20131 MILANO (Italy) 2. Politecnico di Milano, Dipartimento di Chimica, Materiali e Ingegneria Chimica “Giulio Natta” Via L. Mancinelli, 7I – 20131 MILANO (Italy)

Figure 1. Top: Scheme of charge residue model (CRM) proposed Figure 2. Scheme of the first ESI source built at Yale in 1982 by Dole for the formation of gaseous ions from nebulized solutions (from ref. 1). undergoing electric field. Bottom: Sketch of the Ion Evaporation the evaporating droplets, thus eliminating the possibility of Model proposed by Iribarne and Thomson (from ref. 1). resolvation during the free-jet expansion. The first “ready to The sketch of this interpretation, commonly referred to as go” mass spectrometer equipped with ESI source and the “charged residue model” (CRM) is displayed in Figure 1, quadrupole mass analyzer was assembled at Yale in 1982 along with the competitive explanation proposed by by John Fenn’s post-doc Masamichi Yamashita. Iribarne and Thomson (4). The ESI source is showed in Figure 2.

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The initial results on simple model molecules like achievable provided a suitable matrix could be found with Gramicidine S pointed out that multiple-charged ions could the purpose of converting the photons energy into thermal be easily obtained, thus opening the avenue to facile mass energy, according to the “energy sudden” principle. The analysis of high molecular weight molecules through the non- eyes watched at Ultra Fine Metal Powder formation of multicharged adducts detected at lower m/z (UFMP) as favourite candidate. The nanometric size of the values. It is amazing how such simple principle raised particles, comparable with the wavelength of the incident sceptical or openly hostile attitudes of part of the scientific light, was expected to increase light scattering with respect community: as a matter of fact, the first Fenn’s paper on ESI to reflection, commonly observed in bulk metal matrixes. detection of intact protein was severely criticised by The effect of radiation scattering was then to retain inside reviewers: in front of what we now recognize as the the sample the whole incident radiation, as depicted in envelope of the multiple charged adducts stemming from a Figure 4. polypeptide, one of the referees argued that “the The reduced heat dissipation ensured the rapid heating of multiplicity of peaks for each species in a mixture would the sample. inevitably make the spectra too congested to be The tests with PEGs were positive, the LD spectra in the interpreted” (5). presence of UFMP allowed the detection of intact macromolecules up to 2000 Da with no significant fragmentation and opened the route for the use of a matrix to assist the laser desorption. However, that was not enough for biomolecules, and further improvement was necessary. At this stage, while searching the best formulation of the matrix by suspending UFMP in a repertoire of organic solvents by trial and error, Tanaka “committed a monumental blunder” (9) followed by a sequence of events that we could simply define a bad laboratory practise: in few words, Tanaka mixed UFMP with glycerol instead of acetone, nevertheless used the “wrong” mixture just because UFMP was rather expensive, speeded up the glycerol evaporation by laser irradiation because he had no time to waste and, icing on the cake, left the detector open to see what happened. Figure 3. Sketch of formation of multi charged ions from ESI of a protein solution. The incredible outcome of this collection of The inset shows the result of peak deconvolution giving the molecular mass of unfair operation was the observation, for the intact protein (adapted from ref. 6). the first time ever, of the intact signal of vitamin B12, of which only the fragments A disconfirm of such a claim was soon provided by Mathias have been detected so far under LD conditions. Mann, post-doc in Fenn’s lab. The development of an After this finding, an increasing number of high mass algorithm for peak deconvolution gave a direct way to biomolecules were successfully tested with the new spectrum interpretation, affording the nominal molecular ionization method, including Carboxypeptidase A (ca. 35 mass of the protein, as sketched in Figure 3. The theory of kDa) and Lysozime clusters up to 100 kDa. multiple charge proposed by Fenn (7) in 1987 definitely consolidated ESI-MS as a fundamental physical method for the next to come “omics research”, genomics, proteomics and metabonomics.

MATRIX ASSISTED LASER DESORPTION IONIZATION

The surprising biography of Koichi Tanaka points out that, sometimes, a disenchanted glance at complicated scientific problems may end up with incredible discoveries (8). Tanaka was awarded of the Noble Prize in Chemistry at the age of 43, after an education in electrical engineering, a technical position at Shimadzu Corporation and basically no knowledge of Chemistry above undergraduate level. At Shimadzu, he was in charge of the development of innovative and efficient sample preparation and ionization technologies. Figure 4. The use of ultrafine metal to improve the conversion of The way followed at the company was based on pulsed photon energy into thermal energy. Left: bulk metal matrix: the nitrogen laser shots (330 nm) in the ns time-range onto the light reflection on the metal surface limits the conversion of analyte in order to achieve high energy density release in radiant energy to thermal. Right: in the UFMP matrix the short time intervals to get the rapid heating of the sample. scattering phenomena contribute to decrease reflection and The intact desorption of macromolecules seemed to be heat dispersion (from ref. 8).

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FT-NMR allowed the investigators to manipulate the sample magnetization by designing pulse sequences with different purposes. This “spin engineering” provided hundreds of different NMR experiments useful to disentangle the tremendous spectral complexity of biomolecular NMR spectra and gaining detailed structural information –like interatomic distances– so far available only by diffraction methods in the crystal state. Two different types of correlation experiments are of pivotal importance in the assessment of the structure of proteins in solution: correlations through bonds via scalar coupling and correlation through space via NOE. The basic experiments related to those correlations are COSY and NOESY, respectively. The latter, in particular, provides distance information on nuclei close in space but not necessarily in the backbone. The NOE intensity decreases rapidly with the sixth power of the internuclear distance r of the interacting nuclei, thus setting the limit for NOE detection at the threshold distance of 5 Å. Moreover, the NOE intensity is also modulated by the chaotic tumbling of the molecule in

Figure 5. Protein images from a glioblastoma section. (a) solution, as described by the correlation function f(tC), with Reference image of the tissue coated with MALDI matrix. Othe tC the rotational correlation time. images are generated by the spatial distribution of different The combined use of COSY and NOESY data, especially proteins in the specimen: (b) Actin, (c) S100A4, and (d) Thymosin short- medium- and long-range NOE, allowed Wüthrich to b4. (Adapted from ref. 13) establish a standard protocol for the determination of the 3D-structure of proteins in solution. An assay of what the method might look like is sketched in Despite the fact that these results represented milestone Figure 6. achievements in instrumental analysis and chemistry A quite severe limitation of the use of NMR for massive research, it was only after two conference communications investigation on proteins relies upon the size of the (one of which in Japanese) that the international molecule, in turn affecting the tumbling rate and the community could share the discoveries by a fundamental relaxation rates, giving the final results of uselessly broad paper published in Rapid Communication in Mass NMR lines. Spectrometry (10). On a parallel track, M. Karas and F. Hillenkamp developed an approach based on suspension or co-crystallization of analyte and organic matrix (2,5-dihydroxybenzoic acid, ferulic acid, nicotinic acid, hydroxycinnamic acid etc) that, combined with the use of YAG laser at 266 nm showed excellent performance in desorption/ionization of intact p rotei ns (11). The research during the ‘90s provided the scientists with optimized experimental conditions and protocols for chemical and biochemical substrates, and made MALDI a powerful and experimentally friendly technique. The most recent applications exploit the facile protein identification and spatial localization in medical specimens, e.g. tissues, for frontier application in diagnostics, such as MALDI imaging (12, 13). An example is reported in Figure 5.

NMR OF BIOLOGICAL MACROMOLECULES: FROM NOESY TO TROSY

When reading the early NMR studies of proteins reported in the literature during the ‘60s, the question how such results could have been achieved with stone age instrumentations, comes suddenly to our mind. With our laid-back attitude generated by the routine use of high field, fully automated FT-NMR machines, the structural details on Cytochrome c published by K. Wüthrich in 1969 (14, 15) look amazing. The fundamental NMR parameters –chemical shift, scalar coupling constants, relaxation rates– were fully exploited, but the real breakthrough occurred when both theory and technology made nuclear Overhauser enhancement (NOE) and Fourier transform NMR available for systematic studies.

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biological macromolecules in solution” (16). The dramatic improvement in spectral sensitivity and resolution lead to structural studies on biomolecular complexes up to several hundreds of kDa, an unthinkable limit till the end on ‘90s.

CONCLUSIONS

The biography and scientific production of the three outstanding personalities briefly outlined here point out, beyond the skill, intelligence and managing ability, the key role of multidisciplinary approach in breakthrough discoveries. And a little bit of serendipity.

REFERENCES AND NOTES

1. J.B. Fenn, Angew. Chem. Int. Ed., 42, pp. 3871-3894 (2003). 2. M. Dole, L.L. Mach et al., J. Chem. Phys., 49, pp. 2240-2247 (1968). 3. J.W. Strutt (Lord Rayleigh), Philos. Mag., 14, p. 184 (1882). 4. J.V. Iribarne, B.A. Thomson, J. Chem. Phys., 64, p. 2287 (1976). 5. From ref. 1, p. 3892. 6. Advanced information on the in Chemistry 2002. Figure 6 Scheme of the combined use of correlation via From: http://www.kva.se/Documents/Priser/Nobel/2002/sciback_ke_ chemical bonds (COSY) and through space (via NOE) for a en_02.pdf model peptide chain. Inter-residues NOEs (long range NOEs) can 7. J.B. Fenn, M. Mann et al., Science, 246, pp. 64-71 (1989). be used to identify the helical regions. (Adapted from ref. 6) 8. K. Tanaka, Angew. Chem. Int. Ed., 42, pp. 3861-3870 (2003). 9. Ibidem, p. 3867. This limitation seemed to exclude NMR from investigation 10. K. Tanaka, H. Waki et al., Rapid Commun. Mass Spectrom., 2, pp. fields covering large proteins and, more interestingly, 151-153 (1988). 11. M. Karas, F. Hillenkamp, Anal. Chem., 60, pp. 2299-2301 (1988). supramolecular complexes of proteins like protein-nucleic 12. L.A. McDonnell, R.M.A. Heeren, Mass Spectrom Rev., 26, pp. 606-643 acids. The limitation was successfully challenged by (2007). Wüthrich’s group, putting together the availability of isotope 13. M. Stoeckli, P. Chaurand et al., Nature Medicine, 7, pp. 493-496 labelled proteins and novel pulse sequences designed on (2001). purpose. In 1997 the key idea of smoothing T2 relaxation by 14. K. Wüthrich, Angew. Chem. Int. Ed., 42, pp. 3340-3363 (2003). acting on dipole-dipole coupling and chemical shift 15. K. Wüthrich, Proc. Natl. Acad. Sci. USA, 63, pp. 1071-1078 (1969). anisotropy was translated into the TROSY pulse sequence and 16. K. Pervushin, R. Riek et al., Proc. Natl. Acad. Sci. USA, 94, pp. 12366- opened the “avenue to NMR structures of very large 12371 (1997).

16 chimica oggi/Chemistry Today - vol 29 n 1 January/February 2011 Two kinds of ingenuity for one-of-a-kind projects.

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