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The 2007 Benjamin Franklin Medal in Chemistry Presented to Klaus Biemann, Ph.D., of The Massachusetts Institute of Technology Cambridge, Massachusetts
Journal of the Franklin Institute 349 (2012) 3203–3210 www.elsevier.com/locate/jfranklin
The 2007 Benjamin Franklin Medal in Chemistry Presented to Klaus Biemann, Ph.D., of The Massachusetts Institute of Technology Cambridge, Massachusetts George Preti Monell Chemical Senses Center, 3500 Market Street, Philadelphia, PA 19104, United States Received 27 February 2007; accepted 21 December 2007 Available online 16 May 2010
Abstract Mass spectrometry is an analytical technique used to measure mass and the structure of complex organic compounds. Mass spectrometry had its origins in J.J. Thomson’s vacuum tube, with which, in the early part of the 20th century, the existence of electrons and ’’positive rays’’ was demonstrated. Thomson, the physicist, observed in his book ’’Rays of Positive Electricity and Their Application to Chemical Analysis’’ that the new technique could be used profitably by chemists to analyze chemicals. Despite this far-sighted observation, the primary application of mass spectrometry remained in the realm of physics; the technique was used to discover a number of isotopes, to determine the relative abundance of the isotopes, and to measure their ’’exact masses’’, i.e. atomic masses, with high precision. America’s entry into WWII moved mass spectrometry out of the physics laboratory into industrial settings. War-time needs included using mass spectrometry for the analyses of petroleum distillates: determination of high octane fuels; analyses of synthetic rubbers for vehicles, planes and other uses. & 2010 Published by Elsevier Ltd. on behalf of The Franklin Institute.
1. Introduction The intense pace of research and development during the war lead to greater understanding of ion formation and molecular decomposition within the mass spectrometer and instrument development. By the end of World War II, the mass spectrometer E-mail address: [email protected] 0016-0032/$32.00 & 2010 Published by Elsevier Ltd. on behalf of The Franklin Institute. doi:10.1016/j.jfranklin.2007.12.006
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had evolved into an invaluable analytical tool which was used primarily by the petroleum industry and nuclear chemists. Klaus Biemann, trained in synthetic organic chemistry, was appointed a junior faculty member in the department of chemistry at MIT in 1957. He learned about mass spectrometry shortly after coming to MIT, but it had not yet entered the realm of the organic chemist. Biemann decided to use this technique for the determination of the structure of complex natural product molecules produced by plants and other organisms. Mass spectra of organic molecules measure the mass of the fragments formed upon ionization with an electron beam. From these fragments, Biemann learned to deduce the way a molecule is put together, i.e. its chemical structure. His early successes included the major component of the fragrance of roses (in collaboration with a group of Swiss scientists) and the structures of alkaloids—the first anti-cancer drugs. Isolated by Eli Lilly & Co. from a tropical plant, these alkaloids are still used today as a chemotherapeutic. In 1962, Biemann authored the seminal book, ‘‘Mass Spectrometry: Organic Chemical Applications.’’ Throughout his career at MIT, Biemann maintained a keen interest in applying mass spectrometry to determine protein structure. His mass spectrometric approach enabled him to solve many problems in protein structure not amenable to classical methods. Among the first were the structures of monellin and bacteriorhodopsin. The former protein was the first isolated protein with an intense sweet taste; the latter is the light-capturing protein used by certain bacteria. Structure determination of numerous complex biomolecules such as these allowed Biemann and the numerous graduate and postdoctoral students he trained to establish the groundwork for a new field in biochemistry—proteomics. Proteomics makes it possible to study the cascade of proteins and the modifications that occur in the cell which determine its function from formation to death. Understanding these processes is crucial to designing drugs that prevent aberrations (disease). Mass spectrometry is now indispensable in the synthesis and manufacture of such pharmaceuticals. 2. Background In addition to his pioneering efforts in proteomics, Biemann was an innovator in the field of mass spectrometry and mentor to a large number of graduate and postdoctoral students. Together, they created and influenced many changes in mass spectrometers and mass spectrometry. His laboratory was among the pioneers who interfaced gas chromatographs with mass spectrometers as well as coupling computers with the GC/MS combination to create one of the most powerful analytical combinations in use today. This methodology, which allows for fast, efficient measurement of the mass spectra of the components within complex mixtures, is now widely used for clinical, forensic, environmental and other analyses. Consequently, Biemann has made and influenced major contributions to medicine and pharmacology through his studies. In 1976, Biemann, as the leader of the Viking Molecular Analysis Team, even helped send a miniaturized instrument to Mars as part of the Viking Mission to look for organic compounds on the surface of the Red Planet. 3. History Modern organic mass spectrometry had its origins in J.J. Thomson’s vacuum tube with which, in the early part of the 20th century, the existence of electrons and ’’positive rays’’
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was demonstrated. Thomson is ‘‘linked’’ to Professor Biemann in the lineage of Franklin Institute medal recipients since he was awarded three Franklin Institute medals—1910, 1922 and 1923. These were the only Franklin Institute medals awarded for research stemming from mass spectrometry until the present medal to Klaus Biemann. Thomson observed that mass spectrometry could be used profitably by chemists to analyze chemicals. Despite this far-sighted observation, the primary application of mass spectrometry remained in the realm of physics up to the start of WWII. After America’s entry into WWII, mass spectrometry moved out of the physics and academic laboratories into industrial settings. War-time needs included using mass spectrometry for the analyses of petroleum distillates: determination of high octane fuels; analyses of synthetic rubbers for vehicles, planes and other uses. The intense pace of research and development during the war led to greater understanding of ion formation and molecular decomposition within the mass spectrometer and instrument development. Klaus Biemann received his Ph.D. in organic synthetic chemistry from the University of Innsbruck, Austria in 1951. He came to MIT as a postdoctoral fellow with the renowned natural products chemist, George Buchi. He and the vast majority of organic chemists from this era were unaware of mass spectrometry, but as luck would have it, Firmenich & Cie (currently Firmenich, SA, Geneva, Switzerland) who supported Biemann’s studies in Buchi’s lab, asked him to attend and report on talks given at the Conference on Food Flavors. One of the talks at the Chicago conference described the use of a mass spectrometer (‘‘a fancy instrument he had never heard of’’) to identify fruit flavor components by comparing their mass spectra to spectra published in the collection of the American Petroleum Institute. It occurred to Biemann that mass spectrometry could be used to identify synthetic or natural organic compounds in the same way that organic chemists were using infrared and ultraviolet spectra. 3.1. What is a mass spectrometer? In its simplest form, the mass spectrometer consists of a sample inlet (e.g. heated inlet, gas chromatograph) where energy is put into a molecule or molecules to be analyzed to put them into the vapor phase; a source where the molecules are bombarded with energy (e.g. high energy electron beam, laser energy) and ions are formed; the resulting ions are sorted by their mass-to-charge ratio in a mass analyzer (e.g. electromagnet, quadrapole); ions are detected using one of several methods (e.g. electron or photomultipliers) and the resulting mass spectrum or spectra are stored and manipulated by a data system with its attendant software. When the vaporized organic sample passes into the ionization chamber of a mass spectrometer, it is bombarded by a stream of electrons. These electrons impart enough energy to knock an electron off an organic molecule to form a positive ion. This ion is called the molecular ion—or sometimes the parent ion. This is a very valuable piece of information since it gives you the molecular weight of the compound being measured (elemental composition too, if measured accurately enough). The molecular ions are energetically unstable, and some of them will break up into smaller pieces. The simplest case is that a molecular ion breaks into two parts—one of which is another positive ion, and the other is an uncharged free radical: M þ -X þ þ Y
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The ion, Xþ, will travel through the mass spectrometer just like any other positive ion—and will produce a signal in the detector (a line on the stick diagram). Many fragmentations of the original molecular ion are possible, yielding a host of lines (ions) in the mass spectrum. For example, the mass spectra of pentane and pentanone look like this:
The tallest (most abundant) ions in the spectra (m/z=43; m/z=57) are called the base peak. This is usually given an arbitrary height of 100, and the height of everything else is measured relative to this. The base peak is the tallest peak because it represents the commonest fragment ion to be formed—either because there are several ways in which it could be produced during fragmentation of the parent ion, or because it is a particularly stable ion. 3.2. Organic mass spectrometry and Klaus Biemann In the 1950s a vigorously pursued area of research for organic chemists was the study of the structure and chemistry of natural products that could serve as pharmaceuticals. The ‘‘rational drug design’’ of the times utilized a global search for pharmacologically useful plant products, particularly from the tropics. Many of these initially isolated products were alkaloids which could be easily isolated by aqueous acid extraction from complex plant mixtures. ‘‘Reserpine’’ was the first antihypertensive drug isolated from these sources; its huge financial success spurred further discovery. At the time Biemann acquired his first mass spectrometer (a Consolidated Electrodynamics Corporation model 23–101), complex molecules had not been subjected to analysis by mass spectrometry and spectra were recorded on photographic paper which had to be developed. Fast oscillographic recorders with light-sensitive paper had not yet been invented and computers were room-sized. Consequently, structural determination of complex molecules was time and material consuming. Biemann carried out the conversion of a proposed unknown to a known or analogous structure with subsequent comparison of the mass spectra. The structure proof of several new alkaloids ensued, but more importantly, these studies showed how mass spectrometry could be used in structural determinations of complex molecules. This spurred contemporaries to follow suit and in 1960 Professor Carl Djerassi invited Biemann to Stanford to help with the installation of a mass spectrometer in his lab. The impact was noted by Djerassi 32 years later, when he recounted, ‘‘yit was the elegant rationalization by Biemann of the mass spectral fragmentation behavior of alkaloids of the
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aspidospermine class that stimulated a serious effort at Stanford on organic chemical applications of mass spectrometry.’’ Concomitant with this activity was Biemann’s research aimed at obtaining mass spectra of amino acids, peptides and years later, proteins. The known (and sometimes unusual) amino acids combine to form long repeating structures known as peptides and proteins. A peptide chain will therefore look like the general structure shown below.
The ’’R’’ groups come from the 20 amino acids which generally occur in proteins. The peptide chain is known as the backbone, and the ’’R’’ groups are known as side chains. These complex, non-volatile structures were incompatible with the mass spectrometer inlets of the time. However, Biemann used his knowledge of organic chemical reactions (acylation of amino groups and esterification of carboxyl groups and reduction of the resulting peptide with lithium–aluminum hydride) to diminish the peptide’s polarity and increase the structure’s volatility to obtain mass spectra. In addition to demonstrating the peptide’s volatility, he was able to demonstrate how the peptides were joined (‘‘sequenced’’) using its fragmentation pattern. The first paper to examine the mass spectrometry of amino acids and peptides appeared in 1959. The first proteins of unknown structure sequenced by this technique were not reported until 1976. The two seminal examples are listed here: G. Hudson, K. Biemann, Mass spectrometric sequencing of proteins. The structure of subunit I of monellin. Biochemical & Biophysical Research Communications 71(1) (1976) 212–20. G.E. Gerber, R.J. Anderegg, W.C. Herlihy, C.P. Gray, K. Biemann, H.G. Khorana, Partial primary structure of bacteriorhodopsin: sequencing methods for membrane proteins, Proceedings of the National Academy of Sciences of the United States of America 76(1) (1979) 227–31. Concomitant with the effort to determine the structure of complex biomolecules was the desire to couple the separating power of the gas chromatograph (GC) with the mass spectrometer (MS). The combined system, GC/MS, was first demonstrated by scientists at Philip-Morris and Dow Chemical and published in 1959. However, the technique used in this initial report did not resemble what we use today. These early experiments used a fraction of the packed column eluant (3–5%) to pass by the molecular leak of the mass spectrometer to give mass spectra. Direct recording of total gas chromatographic eluants was demonstrated almost simultaneously in 1964 by Ryhage (Anal Chem. 36 (1964) 759–764) as well as Watson and Biemann (Anal. Chem. 36 (1964) 1135–1137). Widespread use of Ryhage’s technique was hampered by the patent restricting use of the molecular jet separator to the LKB Corporation, but the Watson–Biemann all-glass molecular separator was not restricted by patent protection and was available for all to use. From this point on, the Biemann group also pioneered the application of computerized GC/MS data acquisition, providing the template for today’s modern systems. Further to this point, Biemann’s group also developed many of the initial algorithms for searching computerized
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data files for ‘‘mass chromatograms,’’ for library searching and for automated data processing. The latter applications were published in several articles in the 1970s. Professor Biemann’s initial, seminal accomplishments established mass spectrometry as a technique with broad utility in solving problems for organic and biological chemistry. In the following three decades, under his direction, his laboratory made numerous significant contributions in a variety of areas involving mass spectrometry, including instrumental development, computer interfacing and software, identification of drugs and their metabolites, organic-geochemistry and extraterrestrial biology as well as ever more diverse and complex biomolecules. One of the letters of support commented upon the lasting effect of these early developments upon the field today: His laboratory developed the widely used ‘‘Watson–Biemann’’ jet separator that allowed GC to be interfaced directly to the mass spectrometer obviating the need to hand collect fractions from the GC. This advance led to the development of computer based methods to collect data on-line, a huge advance over the current method of the time using photographic plates or UV sensitive paper. Again these pioneering efforts paved the way for the incorporation of computers into mass spectrometers and some of the data display concepts developed in the Biemann laboratory are still used today. Additional letters of support from contemporaries of Klaus Biemann as well as younger mass spectrometrists attest to his impact on the field, particularly in the area of recognition, proteomics. Two of the letters of support highlight this; the first letter states, ‘‘He was the founder of the field of biological mass spectrometry at a time when nobody was anticipating its principal feasibility. He (Biemann) described the potential of mass spectrometry for peptide sequencing already in a 1959 paper, long before desorption ionization techniques or even chemical ionization was invented. The development of the field (protein sequencing by mass spectrometry) starting from the 1990s finally made him a successful visionary.’’ In addition, the second supporter states, ‘‘Biemann was one of the first to apply mass spectrometry to the structural characterization of natural product molecules. His classic work on indole alkaloids was among the first research to attract the attention of practicing organic chemists to the possibilities of MS, and his pioneering research on the sequencing of peptides has blossomed into the new rapidly expanding field of proteomics mass spectrometry.’’ Although one of Professor Biemann’s most notable contributions to science was enabling proteomics, the latter is a relatively new concept. Proteomics is a recently coined term that was not ‘‘invented’’ until the last decade of the 20th Century. It, in turn, stems from the term ’’proteome’’ which was coined in 1994 to mean the total protein complement encoded by a genome. Consequently, the proteome of an organism includes all the proteins contained in the organism. The study of these proteomes and specific individual proteins is known as ‘‘Proteomics.’’ Proteomics, as a field, is growing in importance in this new millennium. A large number of initiatives have been launched worldwide to conduct research in this field. Proteomics usually pertains to four broad categories: identification of all the proteins; quantification of all the proteins; study of protein–protein interactions that affect the various complex pathways and networks; and structural characterization. The identification and quantification of proteins in complex pathways and networks may be achieved using mass spectrometry techniques.
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During the 1980s, three techniques were invented for ionizing and vaporizing large, polar molecules such as proteins. They were fast atom bombardment (FAB, which appeared in 1981), matrix-assisted laser desorption ionization (MALDI, which appeared in 1988) and electrospray ionization (ESI, which appeared in 1989). Although Biemann’s group was sequencing proteins long before these techniques for ionization and vaporization of large molecules were ‘‘invented,’’ he adapted two of these for use in his lab shortly after they appeared. These adaptations were commented upon in his support letters. For example: He was the first one to demonstrate that fast atom bombardment (FAB was the forerunner of MALDI) in conjunction with 4-sector mass spectrometers could be used to sequence peptides. Peptide sequencing by mass spectrometry is now commonplace, but at the time he started it, he had to face a lot of opposition not only from biochemists who had adopted Edman sequencing as their principal tool, but also from mass spectrometrists who did not believe that this could ever be a useful application. It is this vision and his perseverance in the pursuit of this goal which makes him an outstanding scientist and a worthy candidate for an award as prestigious as the Benjamin Franklin Medal. In addition, ‘‘Biemann was one of the first to apply mass spectrometry to the structural characterization of natural product molecules. His classic work on indole alkaloids was among the first research to attract the attention of practicing organic chemists to the possibilities of MS, and his pioneering research on the sequencing of peptides has blossomed into the new rapidly expanding field of proteomics mass spectrometry (emphasis added).’’ In addition to Professor Biemann’s direct impact upon the field of organic mass spectrometry and its application to proteomics, a number of the responders commented upon his continuing legacy through the students and postdoctoral fellows trained in his lab. Some examples are quoted here: ‘‘yNearly every prominent mass spectrometrist in the United States and even some in Europe have gone through the lab of Dr. Biemann, today the third generation is growing and there will be hardly any leading mass spectrometrist in the US who is not the grandson/daughter of Klaus Biemann.’’ yHe also trained many of the most prominent scientists in mass spectrometry and in this way had enormous influence on the development of the subject. Finally, we hear ‘‘y.Professor Biemann’s vision for biological mass spectrometry has brought us to where we are today. His research inspired and challenged many research groups around the world and set important standards of excellence. Furthermore, his commitment to training young scientists has been honored by his former students and postdoctoral fellows by endowing an award in his name through the American Society for Mass Spectrometry to recognize significant achievements in basic and applied mass spectrometry made by an individual early in his or her career.’’ Professor Klaus Biemann has been the recipient of numerous honors and awards, most are listed here: Guggenheim Fellow American Chemical Society: Field and Franklin Award in Mass Spectrometry International Mass Spectrometry Society: Thomson Medal
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American Chemical Society: Analytical Chemistry Award Beckman-ABRF Award Pehr Edman Award NASA Principle Investigator; leader of molecular analysis team for analyses of lunar and Martian soil samples NIH National Research Facilities grant: 1964–1996 Biemann was accorded membership in the National Academy of Sciences in 1993. He is the author of over 350 peer-reviewed articles, chapters and reviews focused upon mass spectrometry and its application to a variety of problems. He authored the book ‘‘Mass Spectrometry: Organic Chemical Applications’’ 1962 (McGraw-Hill Book Company, New York) which was reprinted in 1998 as vol. 1 of the American Society of Mass Spectrometry Publications of ‘‘Classical works in mass spectrometry.’’ Finally, Professor Biemann’s enduring legacy has been his role as mentor to more than 150 graduate and postdoctoral students currently using and advancing the area of mass spectrometry in a myriad of scientific directions. 4. 2007 Benjamin Franklin Medal in Chemistry Citation: For his pioneering work in the development and application of mass spectrometry instrumentation and techniques for the structure determination of complex molecules of great biological and medicinal interest, particularly in peptide and protein sequencing and its contributions to proteomics. The Benjamin Franklin Medal in Chemistry Medal Legacy Previous laureates in chemistry who share a common intellectual thread with Klaus Biemann include: 1913 1923 1923 1945 1947 1954 1958 1959
Emil Fischer, Cresson Medal Francis W. Aston, Scott Medal Joseph John Thomson, Scott Medal Robert B. Woodward, Scott Medal Robert Robinson, Franklin Medal Vincent de Vigneaud, Scott Medal A.J.P. Martin, Scott Medal A.J.P. Martin, A.T. James, and R.L.M. Synge, Wetherill Medal
The 2007 Benjamin Franklin Medal in Chemistry Presented to Klaus Biemann, Ph.D., of The Massachusetts Institute of Technology Cambridge, Massachusetts George Preti Monell Chemical Senses Center, 3500 Market Street, Philadelphia, PA 19104, United States Received 27 February 2007; accepted 21 December 2007 Available online 16 May 2010
Abstract Mass spectrometry is an analytical technique used to measure mass and the structure of complex organic compounds. Mass spectrometry had its origins in J.J. Thomson’s vacuum tube, with which, in the early part of the 20th century, the existence of electrons and ’’positive rays’’ was demonstrated. Thomson, the physicist, observed in his book ’’Rays of Positive Electricity and Their Application to Chemical Analysis’’ that the new technique could be used profitably by chemists to analyze chemicals. Despite this far-sighted observation, the primary application of mass spectrometry remained in the realm of physics; the technique was used to discover a number of isotopes, to determine the relative abundance of the isotopes, and to measure their ’’exact masses’’, i.e. atomic masses, with high precision. America’s entry into WWII moved mass spectrometry out of the physics laboratory into industrial settings. War-time needs included using mass spectrometry for the analyses of petroleum distillates: determination of high octane fuels; analyses of synthetic rubbers for vehicles, planes and other uses. & 2010 Published by Elsevier Ltd. on behalf of The Franklin Institute.
1. Introduction The intense pace of research and development during the war lead to greater understanding of ion formation and molecular decomposition within the mass spectrometer and instrument development. By the end of World War II, the mass spectrometer E-mail address: [email protected] 0016-0032/$32.00 & 2010 Published by Elsevier Ltd. on behalf of The Franklin Institute. doi:10.1016/j.jfranklin.2007.12.006
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had evolved into an invaluable analytical tool which was used primarily by the petroleum industry and nuclear chemists. Klaus Biemann, trained in synthetic organic chemistry, was appointed a junior faculty member in the department of chemistry at MIT in 1957. He learned about mass spectrometry shortly after coming to MIT, but it had not yet entered the realm of the organic chemist. Biemann decided to use this technique for the determination of the structure of complex natural product molecules produced by plants and other organisms. Mass spectra of organic molecules measure the mass of the fragments formed upon ionization with an electron beam. From these fragments, Biemann learned to deduce the way a molecule is put together, i.e. its chemical structure. His early successes included the major component of the fragrance of roses (in collaboration with a group of Swiss scientists) and the structures of alkaloids—the first anti-cancer drugs. Isolated by Eli Lilly & Co. from a tropical plant, these alkaloids are still used today as a chemotherapeutic. In 1962, Biemann authored the seminal book, ‘‘Mass Spectrometry: Organic Chemical Applications.’’ Throughout his career at MIT, Biemann maintained a keen interest in applying mass spectrometry to determine protein structure. His mass spectrometric approach enabled him to solve many problems in protein structure not amenable to classical methods. Among the first were the structures of monellin and bacteriorhodopsin. The former protein was the first isolated protein with an intense sweet taste; the latter is the light-capturing protein used by certain bacteria. Structure determination of numerous complex biomolecules such as these allowed Biemann and the numerous graduate and postdoctoral students he trained to establish the groundwork for a new field in biochemistry—proteomics. Proteomics makes it possible to study the cascade of proteins and the modifications that occur in the cell which determine its function from formation to death. Understanding these processes is crucial to designing drugs that prevent aberrations (disease). Mass spectrometry is now indispensable in the synthesis and manufacture of such pharmaceuticals. 2. Background In addition to his pioneering efforts in proteomics, Biemann was an innovator in the field of mass spectrometry and mentor to a large number of graduate and postdoctoral students. Together, they created and influenced many changes in mass spectrometers and mass spectrometry. His laboratory was among the pioneers who interfaced gas chromatographs with mass spectrometers as well as coupling computers with the GC/MS combination to create one of the most powerful analytical combinations in use today. This methodology, which allows for fast, efficient measurement of the mass spectra of the components within complex mixtures, is now widely used for clinical, forensic, environmental and other analyses. Consequently, Biemann has made and influenced major contributions to medicine and pharmacology through his studies. In 1976, Biemann, as the leader of the Viking Molecular Analysis Team, even helped send a miniaturized instrument to Mars as part of the Viking Mission to look for organic compounds on the surface of the Red Planet. 3. History Modern organic mass spectrometry had its origins in J.J. Thomson’s vacuum tube with which, in the early part of the 20th century, the existence of electrons and ’’positive rays’’
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was demonstrated. Thomson is ‘‘linked’’ to Professor Biemann in the lineage of Franklin Institute medal recipients since he was awarded three Franklin Institute medals—1910, 1922 and 1923. These were the only Franklin Institute medals awarded for research stemming from mass spectrometry until the present medal to Klaus Biemann. Thomson observed that mass spectrometry could be used profitably by chemists to analyze chemicals. Despite this far-sighted observation, the primary application of mass spectrometry remained in the realm of physics up to the start of WWII. After America’s entry into WWII, mass spectrometry moved out of the physics and academic laboratories into industrial settings. War-time needs included using mass spectrometry for the analyses of petroleum distillates: determination of high octane fuels; analyses of synthetic rubbers for vehicles, planes and other uses. The intense pace of research and development during the war led to greater understanding of ion formation and molecular decomposition within the mass spectrometer and instrument development. Klaus Biemann received his Ph.D. in organic synthetic chemistry from the University of Innsbruck, Austria in 1951. He came to MIT as a postdoctoral fellow with the renowned natural products chemist, George Buchi. He and the vast majority of organic chemists from this era were unaware of mass spectrometry, but as luck would have it, Firmenich & Cie (currently Firmenich, SA, Geneva, Switzerland) who supported Biemann’s studies in Buchi’s lab, asked him to attend and report on talks given at the Conference on Food Flavors. One of the talks at the Chicago conference described the use of a mass spectrometer (‘‘a fancy instrument he had never heard of’’) to identify fruit flavor components by comparing their mass spectra to spectra published in the collection of the American Petroleum Institute. It occurred to Biemann that mass spectrometry could be used to identify synthetic or natural organic compounds in the same way that organic chemists were using infrared and ultraviolet spectra. 3.1. What is a mass spectrometer? In its simplest form, the mass spectrometer consists of a sample inlet (e.g. heated inlet, gas chromatograph) where energy is put into a molecule or molecules to be analyzed to put them into the vapor phase; a source where the molecules are bombarded with energy (e.g. high energy electron beam, laser energy) and ions are formed; the resulting ions are sorted by their mass-to-charge ratio in a mass analyzer (e.g. electromagnet, quadrapole); ions are detected using one of several methods (e.g. electron or photomultipliers) and the resulting mass spectrum or spectra are stored and manipulated by a data system with its attendant software. When the vaporized organic sample passes into the ionization chamber of a mass spectrometer, it is bombarded by a stream of electrons. These electrons impart enough energy to knock an electron off an organic molecule to form a positive ion. This ion is called the molecular ion—or sometimes the parent ion. This is a very valuable piece of information since it gives you the molecular weight of the compound being measured (elemental composition too, if measured accurately enough). The molecular ions are energetically unstable, and some of them will break up into smaller pieces. The simplest case is that a molecular ion breaks into two parts—one of which is another positive ion, and the other is an uncharged free radical: M þ -X þ þ Y
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The ion, Xþ, will travel through the mass spectrometer just like any other positive ion—and will produce a signal in the detector (a line on the stick diagram). Many fragmentations of the original molecular ion are possible, yielding a host of lines (ions) in the mass spectrum. For example, the mass spectra of pentane and pentanone look like this:
The tallest (most abundant) ions in the spectra (m/z=43; m/z=57) are called the base peak. This is usually given an arbitrary height of 100, and the height of everything else is measured relative to this. The base peak is the tallest peak because it represents the commonest fragment ion to be formed—either because there are several ways in which it could be produced during fragmentation of the parent ion, or because it is a particularly stable ion. 3.2. Organic mass spectrometry and Klaus Biemann In the 1950s a vigorously pursued area of research for organic chemists was the study of the structure and chemistry of natural products that could serve as pharmaceuticals. The ‘‘rational drug design’’ of the times utilized a global search for pharmacologically useful plant products, particularly from the tropics. Many of these initially isolated products were alkaloids which could be easily isolated by aqueous acid extraction from complex plant mixtures. ‘‘Reserpine’’ was the first antihypertensive drug isolated from these sources; its huge financial success spurred further discovery. At the time Biemann acquired his first mass spectrometer (a Consolidated Electrodynamics Corporation model 23–101), complex molecules had not been subjected to analysis by mass spectrometry and spectra were recorded on photographic paper which had to be developed. Fast oscillographic recorders with light-sensitive paper had not yet been invented and computers were room-sized. Consequently, structural determination of complex molecules was time and material consuming. Biemann carried out the conversion of a proposed unknown to a known or analogous structure with subsequent comparison of the mass spectra. The structure proof of several new alkaloids ensued, but more importantly, these studies showed how mass spectrometry could be used in structural determinations of complex molecules. This spurred contemporaries to follow suit and in 1960 Professor Carl Djerassi invited Biemann to Stanford to help with the installation of a mass spectrometer in his lab. The impact was noted by Djerassi 32 years later, when he recounted, ‘‘yit was the elegant rationalization by Biemann of the mass spectral fragmentation behavior of alkaloids of the
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aspidospermine class that stimulated a serious effort at Stanford on organic chemical applications of mass spectrometry.’’ Concomitant with this activity was Biemann’s research aimed at obtaining mass spectra of amino acids, peptides and years later, proteins. The known (and sometimes unusual) amino acids combine to form long repeating structures known as peptides and proteins. A peptide chain will therefore look like the general structure shown below.
The ’’R’’ groups come from the 20 amino acids which generally occur in proteins. The peptide chain is known as the backbone, and the ’’R’’ groups are known as side chains. These complex, non-volatile structures were incompatible with the mass spectrometer inlets of the time. However, Biemann used his knowledge of organic chemical reactions (acylation of amino groups and esterification of carboxyl groups and reduction of the resulting peptide with lithium–aluminum hydride) to diminish the peptide’s polarity and increase the structure’s volatility to obtain mass spectra. In addition to demonstrating the peptide’s volatility, he was able to demonstrate how the peptides were joined (‘‘sequenced’’) using its fragmentation pattern. The first paper to examine the mass spectrometry of amino acids and peptides appeared in 1959. The first proteins of unknown structure sequenced by this technique were not reported until 1976. The two seminal examples are listed here: G. Hudson, K. Biemann, Mass spectrometric sequencing of proteins. The structure of subunit I of monellin. Biochemical & Biophysical Research Communications 71(1) (1976) 212–20. G.E. Gerber, R.J. Anderegg, W.C. Herlihy, C.P. Gray, K. Biemann, H.G. Khorana, Partial primary structure of bacteriorhodopsin: sequencing methods for membrane proteins, Proceedings of the National Academy of Sciences of the United States of America 76(1) (1979) 227–31. Concomitant with the effort to determine the structure of complex biomolecules was the desire to couple the separating power of the gas chromatograph (GC) with the mass spectrometer (MS). The combined system, GC/MS, was first demonstrated by scientists at Philip-Morris and Dow Chemical and published in 1959. However, the technique used in this initial report did not resemble what we use today. These early experiments used a fraction of the packed column eluant (3–5%) to pass by the molecular leak of the mass spectrometer to give mass spectra. Direct recording of total gas chromatographic eluants was demonstrated almost simultaneously in 1964 by Ryhage (Anal Chem. 36 (1964) 759–764) as well as Watson and Biemann (Anal. Chem. 36 (1964) 1135–1137). Widespread use of Ryhage’s technique was hampered by the patent restricting use of the molecular jet separator to the LKB Corporation, but the Watson–Biemann all-glass molecular separator was not restricted by patent protection and was available for all to use. From this point on, the Biemann group also pioneered the application of computerized GC/MS data acquisition, providing the template for today’s modern systems. Further to this point, Biemann’s group also developed many of the initial algorithms for searching computerized
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data files for ‘‘mass chromatograms,’’ for library searching and for automated data processing. The latter applications were published in several articles in the 1970s. Professor Biemann’s initial, seminal accomplishments established mass spectrometry as a technique with broad utility in solving problems for organic and biological chemistry. In the following three decades, under his direction, his laboratory made numerous significant contributions in a variety of areas involving mass spectrometry, including instrumental development, computer interfacing and software, identification of drugs and their metabolites, organic-geochemistry and extraterrestrial biology as well as ever more diverse and complex biomolecules. One of the letters of support commented upon the lasting effect of these early developments upon the field today: His laboratory developed the widely used ‘‘Watson–Biemann’’ jet separator that allowed GC to be interfaced directly to the mass spectrometer obviating the need to hand collect fractions from the GC. This advance led to the development of computer based methods to collect data on-line, a huge advance over the current method of the time using photographic plates or UV sensitive paper. Again these pioneering efforts paved the way for the incorporation of computers into mass spectrometers and some of the data display concepts developed in the Biemann laboratory are still used today. Additional letters of support from contemporaries of Klaus Biemann as well as younger mass spectrometrists attest to his impact on the field, particularly in the area of recognition, proteomics. Two of the letters of support highlight this; the first letter states, ‘‘He was the founder of the field of biological mass spectrometry at a time when nobody was anticipating its principal feasibility. He (Biemann) described the potential of mass spectrometry for peptide sequencing already in a 1959 paper, long before desorption ionization techniques or even chemical ionization was invented. The development of the field (protein sequencing by mass spectrometry) starting from the 1990s finally made him a successful visionary.’’ In addition, the second supporter states, ‘‘Biemann was one of the first to apply mass spectrometry to the structural characterization of natural product molecules. His classic work on indole alkaloids was among the first research to attract the attention of practicing organic chemists to the possibilities of MS, and his pioneering research on the sequencing of peptides has blossomed into the new rapidly expanding field of proteomics mass spectrometry.’’ Although one of Professor Biemann’s most notable contributions to science was enabling proteomics, the latter is a relatively new concept. Proteomics is a recently coined term that was not ‘‘invented’’ until the last decade of the 20th Century. It, in turn, stems from the term ’’proteome’’ which was coined in 1994 to mean the total protein complement encoded by a genome. Consequently, the proteome of an organism includes all the proteins contained in the organism. The study of these proteomes and specific individual proteins is known as ‘‘Proteomics.’’ Proteomics, as a field, is growing in importance in this new millennium. A large number of initiatives have been launched worldwide to conduct research in this field. Proteomics usually pertains to four broad categories: identification of all the proteins; quantification of all the proteins; study of protein–protein interactions that affect the various complex pathways and networks; and structural characterization. The identification and quantification of proteins in complex pathways and networks may be achieved using mass spectrometry techniques.
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During the 1980s, three techniques were invented for ionizing and vaporizing large, polar molecules such as proteins. They were fast atom bombardment (FAB, which appeared in 1981), matrix-assisted laser desorption ionization (MALDI, which appeared in 1988) and electrospray ionization (ESI, which appeared in 1989). Although Biemann’s group was sequencing proteins long before these techniques for ionization and vaporization of large molecules were ‘‘invented,’’ he adapted two of these for use in his lab shortly after they appeared. These adaptations were commented upon in his support letters. For example: He was the first one to demonstrate that fast atom bombardment (FAB was the forerunner of MALDI) in conjunction with 4-sector mass spectrometers could be used to sequence peptides. Peptide sequencing by mass spectrometry is now commonplace, but at the time he started it, he had to face a lot of opposition not only from biochemists who had adopted Edman sequencing as their principal tool, but also from mass spectrometrists who did not believe that this could ever be a useful application. It is this vision and his perseverance in the pursuit of this goal which makes him an outstanding scientist and a worthy candidate for an award as prestigious as the Benjamin Franklin Medal. In addition, ‘‘Biemann was one of the first to apply mass spectrometry to the structural characterization of natural product molecules. His classic work on indole alkaloids was among the first research to attract the attention of practicing organic chemists to the possibilities of MS, and his pioneering research on the sequencing of peptides has blossomed into the new rapidly expanding field of proteomics mass spectrometry (emphasis added).’’ In addition to Professor Biemann’s direct impact upon the field of organic mass spectrometry and its application to proteomics, a number of the responders commented upon his continuing legacy through the students and postdoctoral fellows trained in his lab. Some examples are quoted here: ‘‘yNearly every prominent mass spectrometrist in the United States and even some in Europe have gone through the lab of Dr. Biemann, today the third generation is growing and there will be hardly any leading mass spectrometrist in the US who is not the grandson/daughter of Klaus Biemann.’’ yHe also trained many of the most prominent scientists in mass spectrometry and in this way had enormous influence on the development of the subject. Finally, we hear ‘‘y.Professor Biemann’s vision for biological mass spectrometry has brought us to where we are today. His research inspired and challenged many research groups around the world and set important standards of excellence. Furthermore, his commitment to training young scientists has been honored by his former students and postdoctoral fellows by endowing an award in his name through the American Society for Mass Spectrometry to recognize significant achievements in basic and applied mass spectrometry made by an individual early in his or her career.’’ Professor Klaus Biemann has been the recipient of numerous honors and awards, most are listed here: Guggenheim Fellow American Chemical Society: Field and Franklin Award in Mass Spectrometry International Mass Spectrometry Society: Thomson Medal
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American Chemical Society: Analytical Chemistry Award Beckman-ABRF Award Pehr Edman Award NASA Principle Investigator; leader of molecular analysis team for analyses of lunar and Martian soil samples NIH National Research Facilities grant: 1964–1996 Biemann was accorded membership in the National Academy of Sciences in 1993. He is the author of over 350 peer-reviewed articles, chapters and reviews focused upon mass spectrometry and its application to a variety of problems. He authored the book ‘‘Mass Spectrometry: Organic Chemical Applications’’ 1962 (McGraw-Hill Book Company, New York) which was reprinted in 1998 as vol. 1 of the American Society of Mass Spectrometry Publications of ‘‘Classical works in mass spectrometry.’’ Finally, Professor Biemann’s enduring legacy has been his role as mentor to more than 150 graduate and postdoctoral students currently using and advancing the area of mass spectrometry in a myriad of scientific directions. 4. 2007 Benjamin Franklin Medal in Chemistry Citation: For his pioneering work in the development and application of mass spectrometry instrumentation and techniques for the structure determination of complex molecules of great biological and medicinal interest, particularly in peptide and protein sequencing and its contributions to proteomics. The Benjamin Franklin Medal in Chemistry Medal Legacy Previous laureates in chemistry who share a common intellectual thread with Klaus Biemann include: 1913 1923 1923 1945 1947 1954 1958 1959
Emil Fischer, Cresson Medal Francis W. Aston, Scott Medal Joseph John Thomson, Scott Medal Robert B. Woodward, Scott Medal Robert Robinson, Franklin Medal Vincent de Vigneaud, Scott Medal A.J.P. Martin, Scott Medal A.J.P. Martin, A.T. James, and R.L.M. Synge, Wetherill Medal