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October 2015
www.nature.com/milestones/mass-spec
MILESTONES
Mass Spectrometry
Produced with support from:
Produced by: Nature Methods, Nature,
Nature Biotechnology, Nature Chemical
Biology and Nature Protocols
MILESTONES
Mass Spectrometry
M I L E S TO N E S CO L L E C T I O N
4 Timeline
5 Discovering the power of mass-to-charge (1910) NATURE METHODS: COMMENTARY
23 Mass spectrometry in high-throughput
6 Development of ionization methods (1929) proteomics: ready for the big time
7 Isotopes and ancient environments (1939) Tommy Nilsson, Matthias Mann, Ruedi Aebersold,
John R Yates III, Amos Bairoch & John J M Bergeron
8 When a velocitron meets a reflectron (1946)
8 Spinning ion trajectories (1949)
NATURE: REVIEW
9 Fly out of the traps (1953)
28 The biological impact of mass-spectrometry-
10 Breaking down problems (1956) based proteomics
10 Amicable separations (1959) Benjamin F. Cravatt, Gabriel M. Simon &
John R. Yates III
11 Solving the primary structure of peptides (1959)
12 A technique to carry a torch for (1961) NATURE: REVIEW
12 The pixelation of mass spectrometry (1962) 38 Metabolic phenotyping in clinical and surgical
environments
13 Conquering carbohydrate complexity (1963)
Jeremy K. Nicholson, Elaine Holmes,
14 Forming fragments (1966) James M. Kinross, Ara W. Darzi, Zoltan Takats &
14 Seeing the full picture of metabolism (1966) John C. Lindon
15 Electrospray makes molecular elephants fly (1968)
16 Signatures of disease (1975)
16 Reduce complexity by choosing your reactions (1978)
17 Enter the matrix (1985)
18 Dynamic protein structures (1991)
19 Protein discovery goes global (1993)
20 In pursuit of PTMs (1995)
21 Putting the pieces together (1999)
CITING THE MILESTONES CONTRIBUTING JOURNALS UK/Europe/ROW (excluding Japan):
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NATURE MILESTONES | MASS SPECTROMETRY O CTOBER 2015 | 1
N
ature Milestones are special supplements that aim to highlight the outstanding
technological developments and scientific discoveries that have helped to define a
particular field. Nature Milestones in Mass Spectrometry, a collaboration between five
Nature Publishing Group journals, presents a historical look back at the key technical
developments in mass spectrometry and the chemical and biological applications that stemmed
from these advances. Each short Milestone article, written by a Nature Publishing Group editor,
covers one breakthrough, highlighting the main papers that contributed to the advance and
discussing both their value at the time and their lasting influence on mass spectrometry today.
The Milestone topics and papers were selected with the help of expert advisers, but the
ultimate decisions on what to include were made by the editors. Nature Milestones in Mass
Spectrometry is not meant to be a comprehensive overview of this field, and despite our and
the advisers’ best efforts, omissions of important literature are inevitable. Our intent is to give
readers a taste of the key advances in this technique, with a special focus on biological and
biomedical applications, areas in which much of the research using mass spectrometry is
currently concentrated.
▶ cover: Design by Erin Dewalt
Original mass spectrum taken from Käll, L. et al., The seeds of mass spectrometry were planted just over a century ago with the pioneering
Nat. Methods 4, 923–925 (2007). work of physicist J.J. Thomson (see Milestone 1). The development of ionization methods
EDITORIAL OFFICES (Milestone 2) and instrumentation (see Milestones 4–6) was fueled in part by the Manhattan
NEW YORK Project during the Second World War. The first applications of mass spectrometry in the field of
Springer Nature
One New York Plaza, Suite 4500, chemistry were reported soon after, and to this day, mass spectrometry serves as a workhorse
New York, NY 10004-1562
T: (212) 726 9200
technique for molecular and elemental analysis in laboratories worldwide (see Milestones 3, 7,
Coordinating editors: Allison Doerr, 10 and 12).
Joshua Finkelstein, Irene Jarchum, Catherine Goodman
and Bronwen Dekker
The development of the soft ionization techniques of electrospray ionization (Milestone 15)
production editor: Jennifer Gustavson and matrix-assisted laser desorption/ionization (MALDI; Milestone 18), and also of tandem
Copy editorS: Rebecca Barr and Ashley Stevenson
Editorial Assistant: Tanyeli Taze mass spectrometry (Milestone 13) and of the combination of chromatographic separation
web production editorS: Jayce Childs and with mass spectrometry (Milestone 8), further revolutionized the field, allowing mass
James McSweeney
web Design: Sam Rios and Luke Stavenhagen spectrometry to become an essential tool not just in chemical research but also in the biological
Manufacturing Production: Susan Gray arena. Today, mass spectrometry is the central technology employed in the field of proteomics
marketing: Hannah Phipps
Head of Publishing Services: Ruth Wilson (Milestone 20), enabling the analysis of post-translational modifications (Milestone 21) and
editor-in-chief, Nature Publications:
Philip Campbell
protein interactions (Milestone 22), and it is also as an important tool in structural biology
Sponsorship: David Bagshaw and Yvette Smith (Milestone 19).
Copyright © 2015 Nature America, Inc. The supplement includes a Timeline that lists the key developments (by the year in which the
first milestone paper pertinent to each breakthrough was published), a reprinted Commentary
from Nature Methods and two reprinted Reviews from Nature (these articles will be made freely
available online until March 2016). The Milestones website also includes an extensive Library
listing of mass spectrometry–related papers published in Nature Publishing Group journals.
We would like to sincerely thank our advisers and acknowledge support from SCIEX, Thermo
Fisher Scientific Inc. and Waters Corporation. As always, Nature Publishing Group takes
complete responsibility for the editorial content.
Allison Doerr, Senior Editor, Nature Methods
Joshua Finkelstein, Senior Editor, Nature
Irene Jarchum, Associate Editor, Nature Biotechnology
Catherine Goodman, Senior Editor, Nature Chemical Biology
Bronwen Dekker, Senior Editor, Nature Protocols
MILESTONES ADVISORS
*Ruedi Aebersold, ETH Zürich, Switzerland *Fred W. McLafferty, Cornell University, USA
*Peter Armentrout, University of Utah, USA Howard R. Morris, Imperial College London, UK
Daniel Armstrong, University of Texas, USA David C. Muddiman, North Carolina State University, USA
*H. Alex Brown, Vanderbilt University, USA Francis Pullen, University of Greenwich, UK
*Richard Caprioli, Vanderbilt University, USA *Joshua Rabinowitz, Princeton University, USA
Steven Carr, Broad Institute of MIT and Harvard, USA *Paula J. Reimer, Queen’s University Belfast, UK
*Brian Chait, The Rockefeller University, USA *Carol Robinson, University of Oxford, UK
David Clemmer, Indiana University, USA David H. Russell, Texas A&M University, USA
*Anne Dell, Imperial College London, UK *Uwe Sauer, ETH Zürich, Switzerland
*Rob Ellam, University of Glasgow, UK *Antonio Simonetti, University of Notre Dame, USA
Michael H. Gelb, University of Washington, USA *Gary Siuzdak, The Scripps Research Institute, USA
*Gary Glish, University of North Carolina at Chapel Hill, USA Luke Skinner, University of Cambridge, UK
*Michael A. Grayson, American Society for Mass Spectrometry, USA Richard Smith, Pacific Northwest National Laboratory, USA
*Jürgen H. Gross, University of Heidelberg, Germany *Giulio Superti-Furga, Research Center for Molecular Medicine of the Austrian Academy of Sciences,
*Steven Gygi, Harvard Medical School, USA Austria
Donald F. Hunt, University of Virginia, USA *Jonathan Sweedler, University of Illinois at Urbana-Champaign, USA
*Akihiko Kameyama, National Institute of Advanced Industrial John Todd, University of Kent, UK
Science and Technology, Japan *John Yates III, The Scripps Research Institute, USA
Neil Kelleher, Northwestern University, USA *Richard Yost, University of Florida, USA
*Bernhard Küster, Technische Universität München, Germany *Joseph Zaia, Boston University, USA
*Joseph A. Loo, University of California, Los Angeles, USA *Renato Zenobi, ETH Zürich, Switzerland
*Matthias Mann, Max Planck Institute of Biochemistry, Germany
Raymond March, Trent University, Canada *indicates advisers who assisted with multiple stages of the project
NATURE MILESTONES | MASS SPECTROMETRY O CTOBER 2015 | 3
M I L E S TO N E S T I M E L I N E 1910 The beginnings (Milestone 1) 1929 Development of ionization methods (Milestone 2) 1939 Environmental analysis (Milestone 3) 1946 Time of flight (Milestone 4) 1949 Trapping mass analyzers (Milestone 5) 1953 Quadrupole and triple-stage quadrupole mass filters (Milestone 6) 1956 Small-molecule analysis (Milestone 7) 1959 Separations (Milestone 8) 1959 Peptide sequencing (Milestone 9) 1961 Inductively coupled plasma mass spectrometry (Milestone 10) 1962 Imaging mass spectrometry (Milestone 11) 1963 Carbohydrate analysis (Milestone 12) 1966 Tandem mass spectrometry (Milestone 13) 1966 Metabolomics (Milestone 14) 1968 Electrospray ionization (Milestone 15) 1975 Medical applications (Milestone 16) 1978 Selected reaction monitoring (Milestone 17) 1985 Matrix-assisted laser desorption/ionization (Milestone 18) 1991 Structural biology applications (Milestone 19) 1993 Proteomics (Milestone 20) 1995 Post-translational modification analysis (Milestone 21) 1999 Interactome analysis (Milestone 22) 4 | O CTOBER 2015 www.nature.com/milestones/mass-spec
MILESTONES
© Proceedings of the Royal Society
MILESTONE 1
Discovering the power of mass-to-charge
The twenty-first century is an exciting time for Cambridge, made another crucial observation: time using an electrometer, doing away with
mass spectrometrists. But things were quite he found that in the purest preparations of the cumbersome photographic plates.
exhilarating even at the technique’s birth, more neon gas there were two parabolas, one Dempster also introduced electron
than a hundred years ago. The discovery of the corresponding to an atomic weight of 20 and bombardment as a method to generate positive
electron and of the isotopes of neon, and another one to 22. Although he could not ions. Both of these discoveries made ripples in
year-to-year leaps in the degree of accuracy explain it at the time, this discovery would later the field, and Dempster’s ‘mass spectrometer’,
and resolution of the data, were just some of be recognized as the first indication that stable as it came to be called, became the basis of later
the reasons scientists were motivated to push elements can have isotopes. commercially developed instruments.
ahead with the nascent technology. Though regarded as a major advance, Dempster and Aston also carried out critical
It was perhaps Wilhelm Wien’s discovery Thomson’s technique had limitations, as he work toward determining the isotopic
showing that rays of positively charged himself recognized. In particular, some of the abundance and mass of the elements. Among
particles could be deflected with very powerful rays hit the walls of the tube as they traveled, these was uranium. Others had shown that
magnetic fields that gave mass spectrometry its filling the tubes with ‘metallic dust’ and splitting the uranium atom released a large
start. Wien measured the deflection of these requiring frequent cleanings, and the intensities amount of energy, and on the brink of the
positive particles and was able to calculate of the parabolas on the photographic plate were Second World War, the idea that the fission of
their mass. sometimes insufficient for accurate high-purity uranium could be used as a
Following up on these discoveries, measurements. powerful weapon was born. In 1940, Alfred
Joseph John (J.J.) Thomson showed that Francis Aston, also at the University of Nier (see Milestone 2) provided the missing
positive rays traveling along an axis x and Cambridge, shortly thereafter took on these piece: he was able to make pure preparations of
striking a plane at right angles could be challenges with the aim of increasing the 235
U and 238U, which were then used to identify
deflected by parallel electric and magnetic intensity of the signal. He did this by designing 235
U as responsible for slow neutron fission.
forces on axis y. This caused the rays to be an instrument that would focus the rays in the Efforts to isolate 235U were named the
deflected and strike the plane at a different form of a line hitting the plate at a specific point ‘Manhattan Project’ and occupied leading
place depending on their charge-to-mass on a focal plane. Aston’s device incorporated physicists during the war.
ratio. The rays hit the plane on a parabolic two parallel slits and used two electromagneti- Investments toward a nuclear bomb led to
arc, so to capture this information, Thomson cally charged plates to focus the rays, the development of techniques that advanced
allowed the particles to fall on a photo- mimicking the focusing effect of an optical the field of mass spectrometry in the postwar
graphic plate. He then measured the lens. This first mass spectrograph had not only years. As we now know, and as is described in
parabolas on the photograph and calculated greater measurement intensity and accuracy the following milestones, there would be many
the charge-to-mass ratio of the particles but also better resolution than Thomson’s more critical developments to follow.
using mathematical equations. instrument. Aston used his spectrograph to Irene Jarchum, Associate Editor,
Thomson, resolve the puzzle of neon, demonstrating for Nature Biotechnology
working at the the first time that stable elements can be
ORIGINAL RESEARCH PAPERS Thomson, J.J. Rays of positive
University of isotopic.
© Proceedings of the Royal Society
electricity. Philos. Mag. Ser. 6, 20, 752–767 (1910) |
Another important technological Dempster, A.J. A new method of positive ray analysis. Phys.
Rev. 11, 316–324 (1918) | Aston, F.W. A positive ray
J.J. Thomson captured development came from Arthur Dempster at spectrograph. Philos. Mag. 38, 707–715 (1919)
the parabolas of
deflected rays on a the University of Chicago. Dempster’s FURTHER READING Wien, W. Untersuchungen über die
photographic plate. spectrograph, referred to as a magnetic sector elektrische Entladung in verdünnten Gasen. Ann. Phys. 313,
Reproduced with 244–266 (1902) | Lawrence, E.O. Method and apparatus for
permission from Proc.
analyzer, deflected the rays by 180° by applying the acceleration of ions. US patent 1,948,384 (1934) |
Roy. Soc. A 89, 1–20 a strong magnetic field. This focused the rays of Washburn, H.W., Wiley, H.F. & Rock, S.M. The mass
(1913), J.J. Thomson, spectrometer as an analytical tool. Ind. Eng. Chem. Anal. Ed. 15,
‘Bakerian Lecture: rays
a specific mass-to-charge ratio through a
541–547 (1943)
of positive electricity’. narrow slit. These were then detected in real
NATURE MILESTONES | M A SS S PECTROMETRY O CTOBER 2015 | 5
M I L E S TO N E S
MILESTONE 2 chromatography (HPLC) instrument
(Milestone 8). Modern versions of these
instruments are compact and inexpensive.
Development of ionization methods During this time period, other scientists
were trying to obtain mass spectra of solid
In 1929, following the pioneering work of electric field led to the gentle ionization of the samples. In 1949, Richard Herzog and Franz
Arthur Dempster and Francis Aston analyte at the microscope’s tip. Whereas an EI Viehböck showed that an ion source that
(Milestone 1), Walker Bleakney described a spectrum of acetone, for example, contains 19 produces a beam of positive ions can be used
new method of positive ray analysis that low-molecular-weight peaks, its FI spectrum to bombard the surface of a solid; the impact
involved heating a tungsten wire filament to contains only a single peak that corresponds to of these positively charged particles with the
generate a stream of electrons and then using a the molecular ion. sample results in the ionization and ejection of
uniform magnetic field to focus those In 1969, Hans Beckey showed that some of the atoms, as ‘secondary ions’, from
electrons into a narrow beam. Bleakney used adsorbing a sample onto a tungsten wire the surface. Less than a decade later, Richard
this technique to measure the first four containing a dense array of ‘micro needles’ Honig reported a mass spectrometer that uses
ionization energies of mercury. Five years later, increased the sensitivity of the FI method and a similar approach—termed ‘sputtering’—to
John Tate and Philip Smith showed that this reduced the amount of analyte needed to identify a broad range of neutral, positively
ionization method—called ‘electron impact’ acquire a high-quality mass spectrum. Beckey charged and negatively charged species on the
(EI) at the time, but now known as ‘electron used this field desorption (FD) method to surfaces of silver, germanium and
ionization’—could be used to measure the obtain mass spectra of monosaccharides, germanium-silicon alloy samples. Other
ionization energies of several other elements. It which represented a challenge for other scientists showed that this method, eventually
was even possible to generate highly ionized ionization methods because of their thermal named secondary ion mass spectrometry
species, such as Cs7+. lability. The EI spectrum of d-glucose (SIMS), could be used to determine the
Nearly 20 years later, Alfred Nier, whom exclusively contains low-molecular-weight chemical composition of rocks from the Moon
many would call the ‘father of modern mass fragment ions, and its FI spectrum contains a and to produce images of cells and other
spectrometry’, published a detailed description peak from the protonated monosaccharide, as biological samples (Milestone 11).
of an EI mass spectrometer that was able to well as others resulting from the successive Although mass spectra of small organic
measure the relative isotopic abundances of dehydration of this species. The FD spectrum molecules were, at this point, relatively easy to
carbon, nitrogen and oxygen in a sample. Nier of this sugar, in contrast, consists obtain using EI, CI, FI and FD, larger
had reported a simpler version of the predominantly of a large peak that biomolecules were still difficult to characterize
instrument in 1940, but his work on the corresponds to the protonated sugar and a using mass spectrometry. In 1976, Ronald
Manhattan Project (Milestone 1) prevented smaller peak that corresponds to the molecular Macfarlane and David Torgerson showed that
him from publishing his improvements until ion. Other researchers reported soon after that nuclear fission of the radioactive element 252Cf,
after the Second World War had ended. FD could be used to obtain spectra of other which generates high-energy particles, could
EI became the ‘gold standard’ ionization heat-sensitive organic molecules, including be used to ionize a biological molecule that
method for many years. However, the glycosides, nucleotides and short peptides. had been deposited on a nickel foil. Using this
conditions required for EI proved too harsh for The harsh conditions of EI inspired technique, known as plasma desorption mass
many organic molecules; the molecular ion scientists to develop new ionization methods spectrometry (PDMS), they obtained mass
usually decomposed into smaller ions. In the that would not result in the decomposition of spectra of several molecules, including the
mid-1950s, Mark Inghram and Robert Gomer the molecular ion. In 1966, Burnaby Munson thermally labile neurotoxin tetrodotoxin,
published two papers that described a ‘softer’ and Frank Field found that when a small vitamin B12 and the antibiotic gramicidin A.
ionization method—field ionization (FI)—in amount of an analyte is mixed with methane Other researchers soon used PDMS to obtain
which an analyte was ionized in close gas, electrons that pass through the mixture mass spectra of oligonucleotides and small
proximity to the tungsten tip of a field almost exclusively ionize the methane. The proteins.
emission microscope. Application of a high resulting methane ions can then react with the In 1981, Michael Barber and his colleagues
analyte, and these ‘chemical ionization’ (CI) published two papers that described a new
a events will produce ions of the analyte. ionization method called fast atom
EI
Munson and Field obtained CI and EI spectra bombardment (FAB), in which a beam of
of several organic molecules and found that neutral argon atoms is aimed at an analyte on
Rel. int (%)
“[g]enerally, the ions in the chemical a copper sample stage containing a
ionization mass spectrum are predominantly low-volatility organic matrix, such as glycerol.
in the high molecular weight end of the The analyte is ionized via the same sputtering
m/z
b spectrum, whereas the converse is true for the mechanism as SIMS. Barber and colleagues
CI4Cl
CH
electron impact mass spectrum.” used FAB to obtain high-quality spectra of
In the mid-1970s, E.C. Horning and oligosaccharides, nucleotides, organometallic
colleagues and D.I. Carroll and colleagues complexes and small proteins, and the
described modifications to Munson’s and technique became the first ionization
Springer
Field’s device: they replaced the ionization method able to sequence longer peptides
m/z source with a 63Ni foil or a corona discharge. (Milestone 9). The popularity of FAB waned
The EI and CI spectra of methionine. Adapted from Gross, J.H., Because the analyte was ionized in a stream of quickly, however, as the arrival of electrospray
Mass Spectrometry—A Textbook (Springer, Berlin, Germany,
2011), with kind permission from Springer Science and
flowing gas at ambient pressure, it could be ionization (ESI; Milestone 15) and
Business Media. coupled to a high-performance liquid matrix-assisted laser desorption/ionization
6 | O CTOBER 2015 www.nature.com/milestones/mass-spec
M I L E S TO N E S
(MALDI; Milestone 18) a few years later
MILESTONE 3
meant that FAB would only be needed when
other ionization methods failed to produce
PHOTOALTO
high-quality spectra.
With such an assortment of ionization Isotopes and ancient environments
methods available, one might expect modern
scientists to cease their efforts to develop new In the early twentieth century, the relatively new Glacial landforms can be dated by mass spectrometry, using
radioactive isotopes such as 14C and 10Be.
ones. But that has not been the case. For technique of mass spectrometry provided an
example, two recently reported ionization opportunity to assess whether there were mass-to-charge ratios. Muller estimated that
methods—desorption electrospray ionization systematic variations in the ratio of the isotopes similar techniques should allow 14C and 10Be to
(DESI) and direct analysis in real time of matter. In 1939, Alfred Nier and Earl be measured in far smaller samples than had
(DART)—have generated quite a bit of Gulbransen showed that this was the case for been possible at the time. Indeed, the cyclotron
excitement because they can directly ionize an carbon. They found that the isotopic proved useful in measuring 10Be, a powerful
analyte from a solid surface at ambient composition of carbon varied depending on method for determining the age of glacial
pressure without any sample preparation. how and when the various types of rock and landforms such as moraines. In a slight variation
DESI was able to detect the presence of an organic matter formed. For example, they found on the cyclotron technique, Charles Bennett
antihistamine on a human fingertip subtle but consistent differences between and colleagues showed that a linear accelerator
40 minutes after oral administration, and carbon minerals formed by volcanic processes coupled with a negative ion source could detect
DART could detect trace amounts of and those derived from seawater. The isotope very small amounts of radiocarbon.
nitroglycerin on a man’s necktie eight hours composition even varied between the flesh and The issue of sample size for stable isotope
after he walked past a construction site where the shell of clams. analysis was solved with a modification to the
demolition work was taking place. Mass It later became clear that carbon isotope gas inlet system of the mass spectrometer
spectrometry ionization methods have made a ratios are even more variable than initially found developed by Nicholas Shackleton. In his setup,
tremendous journey—from EI to ESI and in this early work: they also reflect changes in the molecular leak system by which carbon
MALDI in fewer than 60 years—affecting carbon cycling in the terrestrial biosphere and dioxide produced from carbonate samples
nearly every scientific discipline along the way. throughout the oceans . These discoveries entered the mass spectrometer was applicable
Joshua Finkelstein, Senior Editor, Nature opened up the possibility of tracing the to small sample sizes, but this system also
evolution of life and the carbon cycle through caused the sample to undergo further
ORIGINAL RESEARCH PAPERS
Bleakney, W. A new method of positive ray analysis and its time. fractionation. Automation of the inlet valves
application to the measurement of ionization potentials in Stable oxygen isotopes, particularly 18O and enabled the amount of time the sample and a
mercury vapor. Phys. Rev. 34, 157–160 (1929) | Tate, J.T. & 16
O, also proved to be important tracers. The reference standard flowed through the leak to
Smith, P.T. Ionization potentials and probabilities for the
formation of multiply charged ions in the alkali vapors and in work of Willi Dansgaard demonstrated that the be equalized, thus allowing for correction of the
krypton and xenon. Phys. Rev. 46, 773–776 (1934) | Nier, A.O. oxygen isotope composition of precipitation fractionation. With this system, samples as
Mass spectrometer for isotope and gas analysis. Rev. Sci. Instrum.
18, 398–411 (1947) | Herzog, R.F.K. & Viehböck, F.P. Ion source
could be used to trace both the temperature at small as 100 µl could be analyzed.
for mass spectrography. Phys. Rev. 76, 855–856 (1949) | which the water droplets formed and the Collectively, these developments meant that
Inghram, M.G. & Gomer, R. Mass spectrometric analysis of ions history of the air mass the water originated for fossil material such as tooth or bone, only
from the field microscope. J. Chem. Phys. 22, 1279–1280 (1954) |
Gomer, R. & Inghram, M.G. Applications of field ionization to from as it moved away from the initial vapor small parts of the fossil needed to be sacrificed.
mass spectrometry. J. Am. Chem. Soc. 77, 500 (1955) | source. Oxygen isotopes became the primary This also allowed measurements of fossil
Honig, R.E. Sputtering of surfaces by positive ion beams of low
means to interpret ice cores collected from the carbonate within marine sediments to be made
energy. J. Appl. Phys. 29, 549–555 (1958) | Munson, M.S.B. &
Field, F.H. Chemical ionization mass spectrometry. I. General Greenland and Antarctic ice sheets, and are at a much higher resolution, resolving the
introduction. J. Am. Chem. Soc. 88, 2621–2630 (1966) | Beckey, also key to interpreting biogenic carbonates in patterns of glacial-interglacial temperature
H.D. Field desorption mass spectrometry: a technique for the
study of thermally unstable substances of low volatility. Int. J.
marine sediment cores. change as well as the timing of more recent
Mass Spectrom. Ion Phys. 2, 500–503 (1969) | Horning, E.C., Mass spectrometry–based analyses of ice fluctuations. Such high-resolution
Horning, M.G., Carroll, D.I., Dzidic, I. & Stillwell, R.N. New and sediment cores soon showed that dramatic measurements ultimately revealed that swings
picogram detection system based on a mass spectrometer with
an external ionization source at atmospheric pressure. Anal. and repeated periods of climate upheaval had from glacial to interglacial states over the past
Chem. 45, 936–943 (1973) | Carroll, D.I., Dzidic, I., Stillwell, R.N., occurred for the past few million years. But half million years were paced by changes in the
Haegele, K.D. & Horning, E.C. Atmospheric pressure ionization
there were impediments to fully realizing the Earth’s orbit around the sun.
mass spectrometry. Corona discharge ion source for use in a
liquid chromatograph-mass spectrometer-computer analytical potential of these environmental archives, Alicia Newton, Senior Editor, Nature Geoscience
system. Anal. Chem. 47, 2369–2373 (1975) | Macfarlane, R.D. & including the large sample size required for
Torgerson, D.F. Californium-252 plasma desorption mass ORIGINAL RESEARCH PAPERS Nier, A.G. & Gulbransen, E.A.
spectroscopy. Science 191, 920–925 (1976) | Barber, M.,
isotopic analyses, particularly with Variations in the relative abundance of the carbon isotopes. J. Am.
Bordoli, R.S., Sedgwick, R.D. & Tyler, A.N. Fast atom radioisotopes. The decay counting techniques Chem. Soc. 61, 697–698 (1939) | Muller, R.A. Radioisotope dating
with a cyclotron. Science 196, 489–494 (1977)
bombardment of solids (F.A.B.): a new ion source for mass of the early 1970s required large amounts of
spectrometry. J. Chem. Soc. Chem. Commun. 7, 325–327 (1981) | FURTHER READING Dansgaard, W. The abundance of O18 in
Barber, M., Bordoli, R.S., Sedgwick, R.D. & Tyler, A.N. Fast atom often irreplaceable samples to be destroyed. atmospheric water and water vapour. Tellus 5, 461–469 (1953) |
bombardment of solids as an ion source in mass spectrometry. Richard Muller’s report of the use of a Shackleton, N.J. The high precision isotopic analysis of oxygen and
Nature 293, 270–275 (1981) carbon in carbon dioxide. J. Scient. Instrum. 42, 689–692 (1965) |
cyclotron to measure tritium in water samples Hays, J.D., Imbrie J. & Shackleton, N.J. Variations in the Earth’s
FURTHER READING Takats, Z., Wiseman, J.M., Gologan, B. &
Cooks, R.G. Mass spectrometry sampling under ambient in 1977 was therefore a welcome development. orbit: pacemaker of the ice ages. Science 194, 1121–1132 (1976) |
conditions with desorption electrospray ionization. Science 306, In conventional mass spectrometry, stable Nelson D.E., Korteling, R.G. & Stott, W.R. C-14—Direct detection at
471–473 (2004) | Cody, R.B., Laramée, J.A. & Durst, H.D. natural concentrations. Science 198, 507–508 (1977) | Bennett, C.L.
Versatile new ion source for the analysis of materials in open air
isotopes overwhelm any radioisotope signal, et al. Radiocarbon dating using electrostatic accelerators—
under ambient conditions. Anal. Chem. 77, 2297–2302 (2005) | but at the high energy reached in the cyclotron, negative ions provide key. Science 198, 508–510 (1977) | Raisbeck,
G.M., Yiou, F., Fruneau, M. & Loiseaux, J.M. Be-10 mass-
Gross, J.H. Mass Spectrometry—A Textbook (Springer, Berlin, it was possible to distinguish the radioisotopes
Germany, 2011) spectrometry with a cyclotron. Science 202, 215–217 (1978)
from other isotopes with similar
NATURE MILESTONES | M A SS S PECTROMETRY O CTOBER 2015 | 7
M I L E S TO N E S
MILESTONE 4 velocitron’. As Stephens had proposed, the veloc-
itron did not need a magnetic field and used ion
When a velocitron meets a reflectron
pulses of 5 microseconds. Cameron and Eggers
showed that in this first TOF spectrometer, mercu-
ry ions with different charge could be resolved, but
A drawback of sector mass spectrometers acquired in just hundreds of microseconds, or their isotopes could not. The poor mass resolution
(Milestone 1) was the narrow range of mass-to- as long as it takes the heaviest ions to reach the was the result of initial distributions in both energy
charge ratio (m/z) that could be analyzed at any detector. Stephens pinpointed the advantages of and spatial location of the ions, which led to dis-
given time. These analyzers could be thought of his method: “The response time should be limited persions in the measured times not related to m/z.
as ‘mass filters’, so that acquiring a complete m/z only by the repetition rate (milliseconds). The However, in contrast to previous technologies, the
spectrum required ‘tuning’ the filter across all the indication would be continuous and visual and TOF spectrometer did allow mass spectra to be
relevant ranges of interest. This took time, and it easily photographed. Magnets and stabilization readily visualized on an oscilloscope and updated
was not apt at capturing the spectrum of a sample equipment would be eliminated. Resolution would every millisecond or so.
that either was short-lived or had a composition not be limited by smallness of slits or alignment.” The mass resolution of TOF was improved
that changed quickly. Moreover, there were upper The seed of time-of-flight mass spectrometry by W.C. Wiley and I.H. McLaren in 1955. They
limits to the detectable mass that were inherent to (TOF-MS) was thus planted. devised an improved ion source with two acceler-
the spectrometer. It took two years before a proof-of-principle ating regions that could correct for the initial ion
In 1946, W.E. Stephens from the University TOF spectrometer was developed by A.E. spatial distribution that is generated by the finite
of Pennsylvania proposed a new technology to Cameron and D.F. Eggers, then working at Clinton width of the ionization electron beam. A mass
circumvent these limitations. He called it “A pulsed Engineer Works–Tennessee Eastman Corpora- resolving power of up to 300 could be achieved in
mass spectrometer with time dispersion.” The key- tion. They gave it a more concise name, the ‘ion this type of spectrometer, which was significant
words ‘pulsed’ and ‘time dispersion’ give away the enough to open the path to commercialization.
two main features of this mass analyzer: the use of
© 1948, AIP Publishing LLC
In 1973, B.A. Mamyrin and colleagues solved
microsecond pulses of ions, and the fact that ions the issue of the initial ion energy distribution. They
with different m/z reach the detector at different proposed the use of an electrostatic reflector to
times, allowing ion species to be distinguished detour ions with the same mass but higher veloci-
by their ‘time of flight’ (TOF) in the analyzer. The ties, in what they termed a ‘reflectron’ TOF-MS.
ion pulses are accelerated by an electric field to Such ions penetrate the electric field to a greater
the same energy and travel down a vacuum tube. depth, increasing the length of their path toward
Because ions with different m/z have differ- the detector and thus compensating for the initial
ent initial velocities, they will hit the detector at The velocitron developed by Cameron and Eggers could differences in energy. The introduction of the re-
slightly different times, with lighter and/or more resolve mercury ions with one, two or three positive
charges, but not their isotopes. Reprinted with permission flectron enabled a further increase in mass resolv-
charged ions arriving first. In this way, the entire from Cameron, A.E. & Eggers, D.F. Rev. Sci. Instrum. 19, ing power, by an order of magnitude, as compared
m/z spectrum of the sample under study can be 605–607 (1948). to TOF-MS in which ions propagate linearly.
MILESTONE 5 is known as ion cyclotron resonance (ICR) John Hipple in 1943. Image
reproduced from Encyclopedia of
mass analysis and powers the highest-
Spinning ion trajectories
Mass Spectrometry: Vol. 9:
performance mass spectrometers. Historical Perspectives, Part A: The
Development of Mass Spectrometry
By 1951, Hipple’s team was already (Keith A. Nier, Alfred L. Yergey &
In 1932, Ernest Lawrence invented the envisioning their omegatron as a powerful P. Jane Gale), Newnes, 2015,
p. 112, with permission from
Elsevier
cyclotron—a particle accelerator using a static mass analyzer and discussing ways to improve Elsevier.
magnetic field in which charged particles its resolution using higher magnetic fields,
follow an outward spiral, accelerated by a better trapping and enhanced detection. magnets, these fields can
rapidly varying radiofrequency (RF) field; for Although trapping was improved in the go ever higher: the National High Magnetic
this work, he was awarded the Nobel Prize in subsequent development of magnetic ion traps, Field Laboratory’s FT-ICR mass spectrometers
Physics in 1939. At around that time, John substantial improvements in detection had to hold the current world record of 21 tesla, an
Hipple was working for Westinghouse Electric wait until the 1974 work of Melvin Comisarow impressive but very expensive achievement.
Co. on the design of 90° magnetic sector mass and Alan Marshall. Instead of detecting the But were magnetic fields actually needed?
spectrometers (see Milestone 1). A few years charged particles directly, Comisarow and Back in 1923, Kenneth Kingdon had described
later, after joining the US Bureau of Standards, Marshall measured the image current the trapping of charged particles in a simple
Hipple combined his knowledge of magnetic generated by the charges in the detector plates. electrostatic device—the Kingdon trap—
sector mass spectrometers with the principles Specifically, turning off the RF excitation causes consisting of a cylinder with a wire along its
of cyclotron acceleration in a new device he bunches of ions to rotate at the cyclotron axis, with a voltage difference between the two.
called the omegatron. frequency. As the ions repeatedly pass the As Kingdon saw it, an ion would be imprisoned
In the first prototype, Hipple and two detector plates, they produce a free induction in the tube, forced to orbit to and fro around
colleagues trapped hydrogen ions using a static current that can be detected and subsequently the axis until it lost its transverse velocity and
electric potential and a magnetic field. Tuning converted to a frequency spectrum using the collapsed into the wire.
the frequency of an additional RF field to Fourier transform—hence the name ‘Fourier In 2000, Alexander Makarov revisited this
resonance with the cyclotron frequency transform ICR’ (FT-ICR) given to the new concept to create an equally simple and elegant
ensured that only ions with a desired technique. design known as the orbitrap. Makarov
charge-to-mass ratio would be accelerated. With trapping and detection taken care of, replaced the wire with a spindle-shaped
The ions would then be pushed along the exact the most obvious route to even higher electrode and the cylinder with a barrel-like
outward-spiraling trajectory necessary for resolution and mass accuracy was to increase electrode. The ions would follow intricate
them to hit the detector. Today, this technique the magnetic field. Thanks to superconducting spiraling trajectories around the spindle, much
8 | O CTOBER 2015 www.nature.com/milestones/mass-spec
M I L E S TO N E S
MILESTONE 6 beyond what was possible with conventional
Introduced commercially in the early 1960s, techniques. One person who saw such an
TOF-MS has seen alternating fortunes, experi-
encing a renaissance in popularity in the early Fly out of the traps opportunity was Robert Finnigan.
In 1967, Finnigan co-founded Finnigan
1990s due to new methods to produce pulsed
ion sources. In particular, the gentle ioniza-
Perhaps fittingly for a machine that works by Instruments, which aimed to combine these
tion of soft biological macromolecules, whose sending beams of charged particles on mass filters with gas chromatography
importance was underlined by the awarding of undulating paths, the journey of the quadrupole (Milestone 8) to achieve a single, computerized
part of the 2002 Nobel Prize in Chemistry to mass filter from conception to laboratory bench system that could separate and identify the
John Fenn and Koichi Tanaka, for “their devel- was not particularly straightforward. Wolfgang constituents of a mixture. Importantly,
opment of soft desorption ionisation methods Paul, at the University of Bonn, first published quadrupole mass filters were able to generate
for mass spectrometric analyses of biological
the concept behind the mass filter—or mass spectra at unparalleled sampling speeds.
macromolecules,” spread the application of
TOF-MS in the biological sciences. Nowadays, spectrometer—in 1953. Today, he is regarded as This gave chemists, biochemists and countless
TOF-MS is one of the main mass analyzer the technology’s father. Less known, however, is others a simple system that could analyze
technologies available, alongside quadrupoles that a researcher at the University of California samples within hours.
(Milestone 6), ion traps (Milestone 5) and (later Lawrence) Radiation Laboratory, Richard In the late 1970s, as quadrupole
Fourier transform ion cyclotron resonance Post, came up with a similar idea around the technologies became more widely adopted, Jim
(Milestone 5), and is distinguished by a same time. But he never published his work: his Morrison discovered an innovative use for
relatively high mass resolving power of up to
ideas made it only into his personal notebooks them. With a line of three quadrupoles, a
60,000 at fast scan speeds.
and a Lawrence Radiation Laboratory report. specific ion can be isolated in the first filter,
Elisa De Ranieri, Senior Editor,
Nature Energy Paul is known also for his development of broken into fragments in the second and then
quadrupole ion traps, for which he shared the analyzed and detected in the third, providing a
ORIGINAL RESEARCH PAPERS Stephens, W.E. A pulsed Nobel Prize in Physics in 1989. Quadrupole ion method for probing chemical structure or
mass spectrometer with time dispersion. Phys. Rev. 69, 674– traps and mass filters both use electric selecting and monitoring specific chemical
792 (1946) | Cameron, A.E. & Eggers, D.F. An ion
quadrupole fields to manipulate ionized atoms reactions (Milestone 17). Morrison used light
“velocitron”. Rev. Sci. Instrum 19, 605–607 (1948) | Wiley,
W.C. & McLaren, I.H. Time-of-flight mass spectrometer with or charged particles. In ion traps, the ions are to break up ions, but this was not a practical
improved resolution. Rev. Sci. Instrum. 26, 1150–1157 (1955) | confined to a small region in which they can be approach for analytical studies.
Mamyrin, B.A., Karataev, V.I., Shmikk, D.V. & Zagulin, V.A.
The mass-reflectron, a new nonmagnetic time-of-flight mass
laser cooled and used for spectroscopy, Along with his student Richard Yost, Christie
spectrometer with high resolution. Sov. Phys.-JETP 37, 45–48 ultracold chemistry or quantum information Enke showed that energetic gas-phase
(1973) [Russian original: Zh. Eksp. Teor. Fiz. 64, 82, 1973] processing, whereas quadrupole mass filters collisions in the second quadrupole could be
FURTHER READING The 2002 Nobel Prize in Chemistry—
advanced information (Nobel Media AB, 2014) guide the ions to a detector. The filters consist used to break apart the ions, without the need
of four metal rods that have both direct-current for light. This collision-induced fragmentation
and oscillating radiofrequency voltages applied was achieved by adding an inert gas, such as
like a thread spun from a yarn. At the same in a constant ratio between the opposing pairs. argon, to increase the pressure. Similar to what
time, they would swing back and forth along The exact nature of the trajectories of the ions happens in particle accelerators, energetic ions,
the axis of the spindle, trapped in an depends on their mass-to-charge ratios. This when accelerated by electric fields applied
electrostatic harmonic potential. The ratio can be determined relatively easily, as between the first and second quadrupoles,
charge-to-mass ratio of the ions can be derived different electric-field strengths and oscillating collide with these gas molecules and separate
from these harmonic axial oscillations. frequencies are required to transmit different into smaller pieces.
Combined with image current detection, as in species of ions through the electrode structure Enke, Yost and Morrison realized that this
FT-ICR, the orbitrap provides a high-accuracy, and onto the detector. triple-stage system could be extended to the
high-resolution, simple and compact mass By the early 1960s, several companies were analysis of more complex organic ions (see
analyzer that is now routinely used in producing quadrupole mass filters, but Milestone 7), as well as to ions formed from
proteomics research (Milestone 20). widespread adoption of the technology was proteins (see Milestone 20). And although
Whether achieved by using magnetic fields surprisingly slow. Compared with early the initial adoption of quadrupole mass filters
or electrostatic potentials, the idea of spinning magnetic sector analyzers, the mass filters was not as fast as one might expect for such a
ions on spiraling trajectories that betray their were smaller, cheaper, tolerant of more revolutionary technology, triple-stage
charge-to-mass ratio by the frequencies extreme conditions and generally easier to quadrupole mass spectrometers rose quickly
measured is a surprisingly simple yet very automate—so why such resistance to them? to popularity and are firmly established as
powerful concept. Magnetic-based devices were trusted, and invaluable tools for a range of disciplines in
Iulia Georgescu, Senior Editor, Nature Physics researchers knew both how they worked and laboratories around the world.
how to use them. The quadrupole mass filters Luke Fleet, Associate Editor, Nature Physics
ORIGINAL RESEARCH PAPERS Hipple, J.A., Sommer, H. & therefore needed to do something spectacular,
Thomas, H.A. A precise method of determining the Faraday by
magnetic resonance. Phys. Rev. 76, 1877–1878 (1949) | ORIGINAL RESEARCH PAPERS Paul, W. & Steinwedel, H. Ein
Sommer, H., Thomas, H.A. & Hipple, J.A. The measurement of neues Massenspektrometer ohne Magnetfeld. Zeitschrift für
eM by cyclotron resonance. Phys. Rev. 82, 697–702 (1951) | Naturforschung A. 8, 448–450 (1953) | Paul, W. Apparatus for
Comisarow, M.B. & Marshall, A.G. Fourier transform ion separating charged particles of different specific charges. US
cyclotron resonance spectroscopy. Chem. Phys. Lett. 25, 282– patent 2,939,952 A (1953) | Yost, R.A., Enke, C.G., McGilvery,
283 (1974) | Makarov, A. Electrostatic axially harmonic orbital D.C., Smith, D. & Morrison, J.D. High-efficiency collision-
trapping: a high-performance technique of mass analysis. Anal. induced dissociation in an RF-only quadrupole. Int. J. Mass
Chem. 72, 1156–1162 (2000) Spectrom. Ion Phys. 30, 127–136 (1979)
FURTHER READING Kingdon, K.H. A method for the FURTHER READING Finnigan, R.E. Quadrupole mass
neutralization of electron space charge by positive ionization at spectrometers: from development to commercialisation. Anal.
very low gas pressures. Phys. Rev. 21, 408–418 (1923) Diagram of Wolfgang Paul’s patent for a quadrupole mass Chem. 66, 969A–975A (1994)
filter. Image adapted from US patent 2,939,952 A (1953).
NATURE MILESTONES | M A SS S PECTROMETRY O CTOBER 2015 | 9
©1956, American Chemical Society
M I L E S TO N E S
MILESTONE 7 The mass spectrum of styrene chlorohydrin,
as reported by McLafferty. Reprinted with
Breaking down problems
permission from McLafferty, F.W., Mass
spectrometric analysis broad applicability to
chemical research, Anal. Chem. 28, 306–316 (1956).
At its simplest, mass spectrometry gives onto IBM punched cards. These could be
information about the atomic or molecular compared rapidly with the significant peaks of general rule that major bond breakage occurs
weight of an element or compound. Initial an unknown sample, thus allowing automated β to a benzene ring, he could predict the main
applications of mass spectrometry focused on matches to be made in many cases. characteristics of the molecules’ expected
confirming the weight of a known compound Even when reference spectra were not spectra: 2-chloro-2-phenylethanol should
or analyzing elemental samples. The next step available, scientists learned how to extract generate large peaks corresponding to
was its use in determining molecular increasingly greater amounts of information PhCHCH2OH+ (loss of chloro) and PhCHCl+
structures. from spectral peaks, through a combination (loss of CH2OH). 2-Chloro-1-phenylethanol,
In the 1950s, there was a pressing need for of empirical evidence and chemical intuition. in contrast, should result in peaks for
such techniques. NMR spectroscopy was in its Fred McLafferty, John Beynon and others PhCHCH2Cl+ and PhCHOH+, also from
infancy, with commercial machines only examined many spectra, which led to a β-bond cleavage. McLafferty observed the
beginning to emerge. Elemental analysis could number of general observations. For latter scenario and was able to assign the
confirm the composition of a sample, but it example, they determined that when structural isomer confidently.
did not help with bond connectivity. carbon-carbon double bonds are present, Mass spectra offer insight beyond
Crystallography only worked for molecules including in aromatic rings, cleavage occurs structural assignments and have been
that form single crystals. typically at the β-position. Molecules with employed to monitor gas-phase reactions of
It was in this climate that scientists began carbonyls fragment at the position α to the ions. Ionization techniques produce reactive
to use mass spectra as a means to reveal all, or double bond. Saturated rings fragment gas-phase species within a mass
part, of a molecule’s structure. Some methods adjacent to the ring. spectrometer, and the products that arise
for small-molecule structure determination Applied together, these rules could from collisions can subsequently be
were straightforward; the simplest was to distinguish between closely related isomers. monitored by the instrument’s detector.
compare the molecule’s spectrum with known As an example, McLafferty examined styrene F.H. Field, J.L Franklin and F.W. Lampe were
reference spectra. By this time, laboratories at chlorohydrin, which has two possible isomers instrumental in developing this field, starting
Dow Chemical, for example, had encoded depending on the location of the hydroxyl in the mid-1950s. They studied the
reference spectra for thousands of compounds and chlorine substituents. Employing the secondary ions formed through gas-phase
MILESTONE 8 Patrick Arpino brings the incompatibility of LC and MS to life. carrier liquid. Other ionization
© 1982, Elsevier
Reprinted from Arpino, P.J., On-line liquid chromatography/mass techniques were trialed with mixed
Amicable separations
spectrometry? An odd couple!, Trends Analyt. Chem. 1, 154–158
(1982), with permission from Elsevier.
results—electrospray ionization
(Milestone 15) was a particular
chromatography (LC)—used for the triumph—and the most successful
By the 1950s, mass spectrometry was a well- separation of non-volatile and thermally were soon incorporated by all
established technology for the analysis of volatile unstable compounds—with major manufacturers into the new generation
compounds in the petroleum, pharmaceutical and mass spectrometry proved of commercial instruments. These technologies
chemical industries. However, the deconvolution of more difficult. gave LC–mass spectrometry (LC-MS) a new
spectra comprising multiple analytes was proving Initially, V.L. Tal’roze level of usability in terms of compatible sol-
problematic—there was a growing desire for a and G.V. Karpov tried vents and analytes. This flexibility, along with
rapid, online separation method. direct liquid injection. the improved speed and precision of modern
In fact, the chromatographic techniques neces- By leaking a minute LC–MS, makes it an invaluable method for the
sary for such separation were themselves just volume of LC effluent unequivocal detection of trace molecules—for
coming to the market. Although gas chromatogra- into the high-vacuum example, testing for banned drugs in athletes’
phy (GC) was achieving previously unimaginable conditions of the ioniza- blood or urine.
separation performances, the detection methods tion chamber, they could The softness of the new ionization methods
then available gave limited chemical insight. The vaporize the sample and then meant that even quite large molecular ions were
answer lay in coupling the powerful separation ionize it through electron impact (now called elec- detected intact, simplifying interpretation of the
ability of chromatography with the specificity and tron ionization; see Milestone 2). Michael Baldwin data considerably and, importantly, widening the
precision of mass spectrometry. and McLafferty improved this approach by switch- scope for potential biological applications. Indeed,
This solution was first explored in 1955 by ing to a chemical ionization technique (later devel- when coupled with capillary zone electrophoresis,
Roland Gohlke and Fred McLafferty of Dow Chemi- oped and sold commercially by Hewlett-Packard). another liquid-based separation method whereby
cal Company, who hooked up a homemade gas Meanwhile, others experimented with belt-drying charged species move under an applied potential,
chromatograph to a time-of-flight (TOF) instru- to remove solvent before ionization (later result- these ionization techniques proved to be useful for
ment. This TOF instrument had been developed ing in a commercial instrument from Finnigan) the identification of peptides and proteins.
only recently (Milestone 4) and generated spectra or concentrating the analytes using membrane In a similar way to GC, ion-mobility separation
much faster than did magnetic sector instruments separation. A real game-changer was the develop- (IMS) lent itself well to a partnership with mass
(see Milestone 1). Soon, the team could separate ment of charged droplet evaporation techniques. In spectrometry, because both handle ions in the
mixtures of organic species and identify them—in 1978, Calvin Blakley, Mary McAdams and Marvin gaseous phase. Pairing IMS with a magnetic sector
real time. Vestal reported a method for forcing liquid through or a TOF instrument allowed the analysis of gas-
Of course, the marriage of GC and mass a heated capillary at increased pressure to effect phase reactions, such as the formation of H3+ after
spectrometry was always going to be harmoni- nebulization, a process that they termed thermo- ionization of hydrogen. Later, experiments showed
ous; the gaseous exhaust of the GC was primed, spray. They found that ionization could be achieved that IMS could separate different conformations
ready for ionization. In contrast, pairing liquid chemically by adding ammonium acetate to the of intact proteins that have identical m/z values,
10 | O CTOBER 2015 www.nature.com/milestones/mass-specM I L E S TO N E S
collisions, allowing reaction rates and rate MILESTONE 9
constants to be estimated.
The structural information that could be
deduced from mass spectra continued to
Solving the primary structure of peptides
increase. Carl Djerassi was instrumental in By the late 1950s, chemists had realized that as Edman sequencing—which was streamlined
applying mass spectrometry analysis to mass spectrometry could be used to decipher by that point—as well as DNA sequencing,
natural products. In 1963, he and his the structures of molecules (Milestone 7). which yielded gene sequences that could be
co-workers performed a systematic study of Scientists were also just beginning to identify translated into the protein sequence. Ultimately,
fragmentation patterns in pentacyclic the primary structure, or sequence, of peptides mass spectrometry proved complementary to
triterpenes, describing fragmentation and proteins using chemical approaches. In these techniques, allowing researchers to
behaviors and enabling unknown substances 1953, Fred Sanger used N-terminal labeling of determine the C termini of proteins that were
to be assigned to a particular subclass. peptide fragments, followed by hydrolysis and too long for Edman sequencing and to confirm
Key ion fragments and the reactions that analysis via paper chromatography, to translated sequences.
gave rise to them could be deduced. Even sequence insulin (a feat that earned him the The early 1980s brought further innovations.
stereochemistry at carbon bridges could be Nobel Prize in Chemistry in 1958). Around the In 1981, Donald Hunt and co-workers carried
determined—a powerful demonstration of same time, Pehr Edman devised a method for out the first sequencing of peptides by tandem
how much these techniques had progressed, sequencing proteins by stepwise degradation mass spectrometry (Milestone 13). They
and foreshadowing their continued use today. starting from their N termini. analyzed permethylated peptides on a triple
Enda Bergin, Senior Editor, The application of mass spectrometry to quadrupole (Milestone 6) mass spectrometer
Nature Communications peptide sequencing came shortly thereafter, in following chemical ionization; this allowed
1959, when Klaus Biemann and colleagues direct analysis of a complex mixture of
ORIGINAL RESEARCH PAPERS McLafferty, F.W. Mass described an innovative way to elucidate peptides, generated by protease cleavage of a
spectrometric analysis broad applicability to chemical research.
Anal. Chem. 28, 306–316 (1956) | Beynon, J.H. The use of the
peptide structures using the reduction of small large protein, without prior fractionation.
mass spectrometer for the identification of organic compounds. peptides to generate polyamino alcohols with Tandem mass spectrometry soon became the
Microchimica Acta 44, 437–453 (1956) | Field, F.H., Franklin, J.L. characteristic spectra. Their key advance was standard method for peptide sequencing.
& Lampe, F.W. Reactions of gaseous ions. I. Methane and
ethylene. J. Am. Chem. Soc. 79, 2419–2429 (1957) | identifying chemistry to reduce the highly polar, Another revolution was the introduction of
Budzikiewicz, H., Wilson, J.M. & Djerassi, C. Mass spectrometry zwitterionic character of peptides to allow them ‘soft’ ionization methods, which can be used on
in structural and stereochemical problems. XXXII. Pentacyclic
to be vaporized for ionization. In the years that polar, thermally labile compounds and yield
triterpenes. J. Am. Chem. Soc. 85, 3688–3699 (1963)
followed, numerous groups pioneered methods ions that are not highly fragmented (see
for mass spectrometry–based peptide Milestone 2). In 1981, Michael Barber and
and, in 1998, David Clemmer and colleagues sequencing, developing ways to chemically colleagues developed one of the first of these
developed an instrument that could record modify peptides to be compatible with techniques: fast atom bombardment (FAB),
mass-resolved ion mobilities for all analyte ions
technical innovations that allowed direct which involves mixing samples in solution with
simultaneously. This approach has since become
a powerful tool in the characterization of confor- introduction of samples into the ion source for a matrix and bombarding them with
mational dynamics of large biomolecules. mass spectrometry. high-energy atoms. FAB allowed the group to
Thomas Faust, Associate Editor, In parallel, Biemann and colleagues built on sequence unmodified peptides. Although
Nature Communications their previous work to develop a sequencing important, FAB was ultimately surpassed by
ORIGINAL RESEARCH PAPERS Gohlke, R.S. Time-of-flight strategy that was both fast and generally soft ionization methods such as matrix-assisted
mass spectrometry and gas-liquid partition chromatography. applicable for use on short peptides. However, laser-desorption/ionization (MALDI;
Anal. Chem. 31, 535–541 (1959) | Tal’roze, V.L., Karpov,
they soon found that the mass spectra were Milestone 18), which are used widely today.
G.V., Gordetski, I.C. & Skurat, V.E. Russ. J. Phys. Chem. 42,
1658–1664 (1968) | McFadden, W.H., Schwartz, H.L. & complicated by factors such as side-chain The past few decades have been fruitful, and
Evans, S. Direct analysis of liquid chromatographic fragmentation and variable ion abundance. To the use of mass spectrometry for peptide and
effluents. J. Chromatogr. A 122, 389–396 (1976) | Blakley, C.R.,
McAdams, M.J. & Vestal, M.L. Crossed-beam liquid
sort these out, they used a computational protein analysis has become commonplace. It is
chromatograph–mass spectrometer combination. approach to interpret the mass spectra. The clear that these and other seminal works have
J. Chromatogr. A 158, 261–276 (1978) | Hoaglund, C.S., technique relied on using the exact masses of had a lasting impact on the analysis of protein
Valentine, S.J., Sporleder, C.R., Reilly, J.P. & Clemmer, D.E. Three-
dimensional ion mobility/TOFMS analysis of electrosprayed ion fragments to compute all possible peptide primary structures.
biomolecules. Anal. Chem. 70, 2236–2242 (1998) sequences, from which they could select the Rita Strack, Assistant Editor, Nature Methods
FURTHER READING McDaniel, E.W., Martin, D.W. &
most probable sequence on the basis of the
Barnes, W.S. Drift tube-mass spectrometer for studies of low-
energy ion-molecule reactions. Rev. Sci. Instrum. 33, 2–7 most abundant ions. They confirmed their ORIGINAL RESEARCH PAPERS Biemann, K., Gapp, G. & Seibl, J.
Application of mass spectrometry to structure problems. I.
(1962) | McAfee, K.B. Jr. & Edelson, D. Identification and choice by looking for other ions that should be Amino acid sequence in peptides. J. Am. Chem. Soc. 81, 2274–
mobility of ions in a Townsend discharge by time-resolved
mass spectrometry. Proc. Phys. Soc. 81, 382–384 (1963)|
present were the selected structure correct. 2275 (1959) | Biemann, K., Cone, C., Webster, B.R. & Arsenault,
Baldwin, M.A. & McLafferty, F.W. Liquid chromatography- This work was among the first to use computers G.P. Determination of the amino acid sequence in oligopeptides
by computer interpretation of their high-resolution mass spectra.
mass spectrometry interface—I: the direct introduction of to analyze mass spectra, setting an important J. Am. Chem. Soc. 88, 5598–1606 (1966) | Hunt, D.F., Buko, A.M.,
liquid solutions into a chemical ionization mass spectrometer.
Org. Mass Spectrom. 7, 1111–1112 (1973) | Smith, R.D., Olivares, precedent for the field. Ballard, J.M., Shabanowitz, J. & Giordani, A.B. Sequence analysis
J.A., Nguyen, N.T. & Udseth, H.R. Capillary zone The late 1960s and 1970s saw many of polypeptides by collision activated dissociation on a triple
electrophoresis–mass spectrometry using an electrospray quadrupole mass spectrometer. Biomed. Mass Spectrom. 8, 397–
advances in peptide sequencing by mass 408 (1981) | Barber, M., Bordoli, R.S., Sedgwick, R.D. & Tyler,
ionization. Anal. Chem. 60, 436–441 (1988) | Gohlke, R.S. &
McLafferty, F.W. Early gas chromatography/mass spectrometry, including a permethylation A.N. Fast atom bombardment of solids as an ion source in mass
spectrometry. Nature 293, 270–275 (1981)
spectrometry. J. Am. Soc. Mass Spectrom. 4, 367–371 (1993) | technique that Howard Morris and colleagues FURTHER READING Morris, H.R., Williams, D.H. & Ambler, R.P.
Pullen, F. The fascinating history of the development of
LC-MS; a personal perspective. Chromatography Today
used for partial sequencing of proteins. Determination of the sequences of protein-derived peptides and
February/March, 4–6 (2010) However, mass spectrometry methods had peptide mixtures by mass spectrometry. Biochem J. 125, 189–201
(1971)
competition from alternative approaches, such
NATURE MILESTONES | M A SS S PECTROMETRY O CTOBER 2015 | 11You can also read
