The coming of age of mass spectrometry in peptide and protein ...

f r o r e h Scierlcc. (1995). 4:1920-1927. Cambridgc Univcrsity Press. Printed i n the USA. Copyright : 1995 The Protein Society

RECOLLECTIONS

The coming of age of mass spectrometry in peptide and protein chemistry

KLAUS BIEMANN ~)cparrmcnl of Chcmi~try. Sla\Wchu\c[[\ In\titu[c of Technology, Cambridge, &fxs\ac.hu\c[[\ 02139

Glancing through a recent issue (October 1994) of Protein Sci- Efforts to use mass spectrometry for the structur a I c -h aracter- ence, one runs across publications that reveal how routinely ization and sequencing of peptides and proteins go back to the mass spectrometry is now used and accepted by protein chemists late 1950s. Shortly before, Sanger had developed the dinitro- and biologists. One ofthe papers mentions (p . 1787) a 51-amino phenyl-labeling method of N-terminal amino acids of peptides acid peptide “synthesized by standard solid-state peptide syn- and successfully used i t for the determination of the primary thesis methods” (which) “was purified . . . and the purity and structure of insulin. Also during the 1950s. Edman carried out molecular weight were confirmed by mass spectrometry.’’ An- his pioneering studies that later led to the automated sequential other one reports (p . 1830) the expression of ful ly “N-labeled sequencing method, which carries his name and for so long was polypeptides 16-58 amino acids in length and simply states that the only way to determine the primary structure of a protein. “The molecular mass of each gene product was confirmed by As outlined in more detail elsewhere (Biemann, 1994), i t was laser desorption mass spectrometry.” I indeed the work of Sanger that got me interested in devising a

Only a few years ago. such statements would have required chemical conversion that would specifically mark the C-terminal an extensive description of the methodology and to be backed amino acid o f a small peptide. This idea formed the basis of an up by experimental data. Furthermore, u n t i l the early 1980s, NIH proposal submitted i n 1957, which was approved at $IOK/ such measurements of accurate molccular weishts would not year for 3 years. The grant is still active (fortunately at slowly have been possible at all. For the “N-labeled peptides, one increasing higher funding over the years) and has supported could, of course, have determined the “ N content by the iso- much of the work in the author’s laboratory discussed i n this tope ratio measurements pioneered by A.O.C. Nier and intro- paper. duced into biochemistry by Rittenberg in the 1940s. However, Fate would have it that, at about the same time, I became in- this required combustion of milligrams of sample, sophisticated terested in the use of mass spectrometry for the determination instrumentation, and an experienced operator.

Kcprint rcque\t\ to: Klaus I3icmann. Ikp t . ofChemi\try, Km. 18-587. Sla\\achu\ctts Instituteof Tcchnolopy, 77 Sla\\achu\ctt\ Avenue. Cam- bridge, Sla\\achusett\ 02139-4307: e-mail: h;hicmann~.rnit.cdu.

Klaus Riemann rcceivcd his 1’h.D. (organic chcmktry) from the Uni- vcrsity of Inn\bruck (Amtria) in 1951. He came t o the Massachusett\ In\titutc of Technolopy on a Fulbright I~cllo~vship i n 1954 antl rcturncd again as a Kescarch As\ociarc ( w i t h Ci. I3iichi) i n 1955. wa\ appoinrctl Itntructor (1957). Assktant Prol’c\\or (l959), A\sociate Professor (1962). and Profc\\or of Chemistry (1963).

Dr. Hicmann is an honorary mcmbcr of thc Iklgian Chemical Soci- ety (since 1962) and of the Japanese Society for Medical Mass Spcctroni- err) (1981). a Fellow of the American Academy of Arts antl Scicncc\ (1966). and a Mcmhcr of thc National Academy of Scienccs (19%). For hi\ work in mass \pectromctry hc ha\ rcccivcd nunlcrous awards, among them the Outstanding Spectroscopist Award of the New York Scction o f the Society for Applied Spcctroscopy (1974), thc Fritz Prcgl Medal of the Austrian Microchemical Society (1977). the Esceptional Scicn- tific Achievement Medal of NASA (1977). the Field and Franklin Award i n Mass Spectrometry of the American Chemical Society (1986). the Maurice I-. Hasler Award of the Spcctroxopy Society of Pittsburgh (1989). the Thomson Medal (1991). and the Pchr Edman Award (1992).

Dr. Riemann is also a member of the editorial board of sevcral pres- tigious journal\ and has published over 3 3 0 scicntil‘ic paper\.

’ Hccausc this i \ a pcrwnal rccollecrion of cvcnts rather than a rc- view. rcl‘erences to the work discusscd arc not included. Instead (with the esccption of the first two cntric\) a l i \ t of reviews of the field puh- lished by the author periodically since 1962 is appendcd. Thcse may be K l a u \ Bicmann i n the earlier clay5 o f hi\ career (photograph taken by consulted for reference\ to all the w o r k of thc author and that of oth- J o h n M . H a y \ i n 1965, then a graduntc \tutlcnt, non Dirtinguishetl Pro- er\ mentioned i n this article. fc\wr a t Indiana Univcr\ity. I3loominpton. Indiana).

1920

Mass spectrometry in peptide and protein chemistry 1921

of the structure of natural products. This was in 1958, a time when this type of instrumentation was chiefly used in the petro- leum industry for the quantitative analysis of complex hydrocarbon mixtures (gasoline, jet fuel, and crude oils) and the major chemical companies started to explore its utility for small non- hydrocarbon compounds, work that was reviewed by E McLaf- ferty in 1956.

Our work on the determination of the structure of a large number of indole alkaloids quickly convinced the organic chemists of the power of mass spectrometry. To prove its utility for peptide sequencing was a much harder row to hoe and required two decades to overcome all the skepticism. Fortunately, NIH’s policy of permitting the Principal Investigator to change the approach to the proposed overall goal allowed me to switch the chemical method described in the grant application mentioned earlier to mass spectrometric peptide sequencing.

The origin of this work was the realization that peptides produced by enzymatic digestion or partial acid hydrolysis are linear molecules with a repeating backbone specifically substituted by side chains of limited structure. Furthermore, all of these are of different masses, with the exception of the isomeric pair leucine/isoleucine and, less problematic, the isobaric pair lysine and glutamine. Even from the limited published mass spectrometric behavior of simpler linear molecules, one could predict that fragmentation along the backbone would result in ions, the mass of which should reveal the amino acid sequence of a peptide. The major stumbling block was, of course, the highly polar, zwitterionic character of peptides, which makes it impossible to vaporize such molecules into the sample reservoir of a mass spectrometer without thermal decomposition. This requirement was the major limitation of mass spectrometry at that time, when bombardment of a vaporized molecule with an electron beam was the only practical ionization method available.

Thus, it became imperative to develop a microchemical procedure to reduce the polarity of a peptide and to produce a derivative, which not only retained the sequence-related structural features but also gave a simple mass spectrum dominated by fragment ions due to unique cleavage of what had been the orig- inal peptide bond. Here, training in synthetic organic chemistry, rather than mass spectrometry, became very useful. Acetylation of all the basic amino groups, including the N-terminus, and esterification of all free carboxyl groups (including the C-terminus), destroyed the zwitterionic character and greatly reduced the polarity of these groups. At the same time, the acetylation of the e-amino group of lysine eliminated the mass equivalence of its side chain and that of glutamine.

The key reaction toward the conversion of a peptide to a rather volatile derivative particularly suited for mass spectrometry was the reduction of the N-acetyl-peptide methyl ester to a polyamino alcohol with lithium aluminum hydride. This reagent re- duces amido groups to amines and esters to alcohols. Strictly speaking, the resulting compounds are substituted polyethylene- diamines terminating in an aminoethanol group of the general structure H(NH-CHR-CH,),-OH, in which R is the side chain (or its reduction product) of each of the consecutive amino acids. More importantly, the C-C bond flanked by the basic nitrogens is prone to facile fragmentation in the mass spectrometer, thus leading to ions whose mass (or more exactly the mass-to-charge ratio, m/z, where z generally is unity) reveals the consecutive amino acids. This methodology was described in my very first paper on mass spectrometry (Biemann et al., 1959). In order to

prevent that the side chains of threonine and aspartic acid become isomeric (and thus indistinguishable) LiAID, was later used in the reduction step.

Excellent interpretable mass spectra of models up to tetrapep- tides could be obtained, as long as the amino acids did not contain hydroxyl groups, or carboxylic acids reduced to hydroxyl groups, which substantially reduce the volatility of the polyamino alcohols. Their replacement with hydrogen (or actually deuterium to avoid that the side chains of two different amino acids, such as serine and alanine, become identical in mass) solved this problem. However, it was a tour-de-force on a mi- croscale, because the entire conversion sequence now required five steps: acetylation, esterification, reduction, conversion of -CH-OH to -CH-CI, and reduction.

Because the ultimate goal was a method useful for the determination of the primary structure of a protein, it had to be applicable to sequencing the individual components of the complex mixtures of peptides produced upon enzymatic or chemical cleavage of a protein. Separation could be carried out either at the peptide level or after their conversion to the polyamino alcohols. However, isolating the peptides first would require carrying out their individual chemical conversion to the polyamino alcohols, an unsurmountable - or at least boring and time-consuming - task. Furthermore, the much less polar conversion products lent themselves to a separation technique that had been invented just a few years earlier: gas-liquid chromatography (GC).

It so happened that E.R.H. Jones, Professor of Chemistry at Oxford University, spent a few weeks at M.I.T. as a Guest Lec- turer in the summer of 1955. He told us that GC was used extensively at IC1 in England by organic chemists for micropre- parative isolation of natural and synthetic products, including the details of how to build one of these contraptions for that use. The version constructed at M.I.T. was quite simple: Bend a 2-m/5-mm glass tube into a U shape, fill it with finely ground fire-brick coated with a thin layer of a readily available stopcock grease (Apiezon), connect the tube to a cylindrical piece of stain- less steel containing a gas inlet, a thermistor (control), a rubber septum for sample injection, connections to the two ends of the U-tube, a second thermistor (for measurement), and a gas out- let (in that order). The entire system was placed into an aluminum tube wrapped with heating tape. The two thermistors were connected in a Wheatstone bridge circuit that contained the most expensive part, a pen-and-ink recorder to follow the changes in the difference of the thermal conductivity between the pure gas (at the inlet) and the gas carrying the eluant (at the exit). The total cost of parts and labor (at M.I.T.3 machine shop) was about $1,800, a sum, which even at that time, each research group could afford.

Upon injection of the reduction product of a mixture of peptides into such a system, and watching the pen deflection of the recorder, we collected the individual fractions in melting-point capillaries (cooled with a chip of dry ice) that were inserted into the exit port when the pen started to rise. Each capillary was then individually placed into the heated-inlet system of the mass spectrometer, the sample vaporized, and the spectrum recorded.

Although we had thus demonstrated the feasibility of mass spectrometric sequencing of small peptides, even when present as a mixture, the method turned out to be too complex, and also too instrument- and labor-intensive to be used routinely for protein sequencing. This the more so because, at that time, P. Ed- man had devised the ingenious “spinning cup” version of his

1922 K . Biernann

sequential sequencing methodology. This “protein sequenator” soon became commercially available.

Nevertheless, our work had been sufficiently noticed by the community of peptide chemists that I was invited to present a paper at the European Peptide Symposium held in Basel, Swit- zerland, in September 1960. Although the conference was limited to European research groups, in the invitation to attend, Professor M. Brenner (the chief organizer) considered me, for this purpose, still as an Austrian and thus a legitimate partici- pant. I don’t know whether the same argument needed to be made for the two other participants from the US, Miklos Bodansky (then at Squibb) and Klaus Hofmann (Univ. of Pitts- burgh), already well-established peptide chemists at that time. On the other hand, there was an abundance of East Europeans and Russians (including the late M.M. Schemjakin), which was quite natural in those “pre-Berlin Wall” times. This Symposium was an opportunity for me to meet many of the “card-carrying” peptide chemists of the Continent for the first time. A good number of the attendees participated in the Third American Pep- tide Symposium held in Boston 12 years later, which gave me an opportunity to gather them at a “reunion” party at my home in Cambridge.

Although my laboratory had concentrated on the reduction of the polarity of the amide groups to amines, others had attempted less extensive modifications to obtain sufficiently volatile peptide derivatives. As early as 1958, the N-trifluoroacetyl (TFA) methyl ester of Ala-Phe was shown by C. -0 . Anderson (University of Uppsala, Sweden) to be sufficiently volatile to provide a mass spectrum exhibiting a molecular ion that could be rationalized in terms of their sequence. A few years later, E. Stenhagen (Univ. Goteborg, Sweden) published a paper show- ing mass spectra of TFA di- and tri-peptide esters (the largest Val-Gly-Ala). Beyond these observations the Swedish group did not pursue this topic, but during the 196Os, a number of other laboratories explored the effects of a wide variety of acyl groups attached to the N-terminus in efforts to either increase volatility or to simplify the fragmentation of the derivative upon electron ionization. None of these efforts led anywhere until in 1965, E. Lederer (CNRS, Gif-sur-Ivette, France) in collaboration with M. Barber (at AEI, a mass spectrometer manufacturer in Man- chester, UK), attempted to use mass spectrometry to verify the structure of a peptidolipid, fortuitin, isolated from Mycobac- terium fortuifum. Its methyl ester turned out to give a respect- able mass spectrum, even though it indicated a molecular weight of 1,359! The interpretation of the spectrum was facilitated by the fact that it exhibited a series of pairs of peaks spaced 28 Da apart, eventually recognized as due to the presence of equimolar Cz0 and Czz acyl groups at the N-terminus. Secondly, three of the nine amino acids lacked an amide nitrogen (2 N-MeLeu, 1 Pro) and the two threonines were @acetylated. The remain- ing three amino acids were small aliphatic ones, three valines and one alanine. Thus, this large nonapeptide had only six hydro- gens that could become involved in intermolecular hydrogen bonds. It was the latter feature that pointed to another approach for increasing the volatility of a peptide, and Lederer’s group began to explore the permethylation of N-acetylated peptides as a means of reducing their polarity. Starting with the reaction developed by Hakomori for carbohydrates, which turned out to be too harsh for some of the functionalized amino acids, conditions were gradually modified to provide relatively clean N,O-methylation of N-acetylated peptides. A11 these studies

were carried out with small, single, and pure peptides of known structure.

The mass spectra of these derivatives were complicated by the fact that the carbonyl group of the peptide bonds could trigger a rearrangement that produced, in effect, an apparently new peptide ion that then fragmented on its own. These fragmenta- tions were elucidated chiefly in D.H. William’s laboratory in Cambridge (UK), where H.D. Morris then developed a strategy for the sequencing of acetyl-N,O-permethylated peptides present in simple mixtures. It involved the enzymatic cleavage of the protein followed by gel filtration. Fractions in the range of M, < 1,000 were derivatized directly, whereas those of higher molecular weight were pooled and recycled using enzymes of other specificity, until a large set of fractions, each containing a few peptides, had been produced. These were then N,O-methylated and vaporized slowly into the ion source of the mass spectrometer. Based on the difference in volatility of the components of each mixture, consecutive mass spectra were obtained exhibiting sets of peaks rising and falling in unison and thus representing fragments from the same peptide.

This method was tested on a number of small proteins of known sequence, for which it provided a partial confirmation. One example is the identification of Met- and Leu-encephalin in a mixture isolated from brain where the dansyl-Edman method failed to determine the C-terminal amino acid. Finally, the permethylation technique was used by Morris et al. for a protein of unknown sequence (a dihydrofolate reductase). Partial sequences were reported in a series of three papers during the period of 1974-1977 and the final mass spectrometrically determined sequence was announced at a conference but apparently was never published. It became quite clear that this procedure was too laborious to compete with the Edman method, which by that time had been considerably automated. It was only ham- pered by the inability of dealing with N-terminally acylated peptides and proteins, as well as the difficulties of quickly and reliably identifying the phenylthiohydantoin (PTH) derivatives of the amino acid liberated in each step. Even this problem was soon overcome by the emergence of HPLC as a routine method.

Convinced of the notion that any mass spectrometric method for peptide sequencing with the ultimate goal of determining the primary structure of a protein of reasonable size must be able to deal efficiently and relatively fast with rather complex mixtures of peptides, we continued to pursue the “reductive” method to polyaminoalcohols separable by gas chromatography and amenable to mass spectrometry. Progress along these lines was made in spurts.

First, we applied the on-line combination of the gas chro- matograph with the mass spectrometer (GCIMS), originally developed for our studies of alkaloid structure, to the problem. Fortunately, the widespread efforts of others to extend the ap- plicability of gas chromatography to larger and polar molecules considerably aided our work: the development of the trimethyl- silylation of hydroxyl groups eliminated the previously mentioned cumbersome need for their chemical elimination; the very sensitive flame ionization detector allowed efficient stream split- ting; precise temperature programming of the column became routine; this, combined with silicone-based liquid phases, ex- tended the temperature range at which the columns could be reliably operated and increased the size of molecules amenable. All these developments were taken advantage of, while we improved the mass spectrometric side of the instrumentation;


chiefly in the area of data acquisition, processing and interpretation, which finally allowed the analysis of mixtures of O-tri- methylsilyl-polyaminoalcohols derived from very complex mixtures of peptides up to six amino acids in length. In parallel, chemical steps were modified and improved, such as triflu- oroacetylation instead of acetylation that enhanced the volatility of the products and the interpretability of their mass spectra, as well as the use of BrD, instead of LAID4, a change that eliminated the need of extracting the amino alcohols from the sometimes jelly-like aluminum hydroxide.

One requirement for efficiency was to generate mainly peptides that were sufficiently small (from up to four polar amino acids to six chiefly nonpolar ones). This was necessary to cover the entire protein with sufficient overlap to enable the alignment of the short peptide sequences to one unique primary structure of the protein. Partial acid hydrolysis seemed to be the simplest way to generate such a peptide mixture and most of our model experiments were either mimicking such a mixture or producing it from larger peptides of known structure.

By the late 1960s, our work had attracted sufficient attention that I was invited to present it at the Biochemical Conference on Protein Structure and Function, held at the Alpine Inn, Ste. Marguerite, Quebec, in early March 1969 (scheduled then and there to provide an additional attraction for skiing enthusiasts). It so happened that at that time, I was busy at NASA’s Lunar Receiving Laboratory, at the Johnson Spacecraft Center in Houston, Texas, preparing for the organic analysis of the ma- terial to be returned by the Apollo 1 1 mission. This was a side- line of my research career that had begun in the early 1960s, when a P.I. scout from the Jet Propulsion Laboratory in Pasa- dena, CA (Dr. Gerald Soffen, later Project Scientist of NASA’s 1976 Viking mission to Mars, in which 1 played a role similar to that in the Apollo Project) visited my laboratory. He found out that we had “real data!!” because, as part of his Ph.D. thesis, Jim McCloskey (now a distinguished professor at the Uni- versity of Utah) had produced and interpreted the mass spectra of free amino acids. Nevertheless, I felt i t important for my long- range research interests to fly to Quebec to participate in the protein conference.

After 1 had given my talk outlining our strategy and results, I fielded some of the questions during the discussion period, which preceded the coffee break. While walking into the corri- dor, I bumped into Stanford Moore, who was wearing an im- peccable dark suit and tie up there in ski country! After some congratulatory comments about my talk, he softly patted me on the shoulder. Shaking his white mane, he commented that “it will never work” because of the wide differences in the rate of hydrolysis of various peptide bonds: some will hydrolyze so fast that we would never find them (no overlap) and others would never cleave. Of course, this statement was perfectly true, based on Moore’s extensive work on total hydrolysis of proteins to single amino acids for the purpose of their quantitative analysis. I could only reply that I would not be worried about the stable bonds, like those between sterically hindered amino acids, such as valine and isoleucine, because encountering more than six of them in a row would be highly unlikely. The very labile ones were of more concern in the correct reassembly of a large number of peptides, but at least we knew, thanks in great part to the extensive work of Stein and Moore, which bonds would cleave very fast. At this point, we had to leave it to the experiments that followed to assess the magnitude of this potential problem.

After a period of refinements and model studies, the stage was finally set to start tackling some “real” problems, namely, the determination of the hitherto entirely unknown primary sequence of small proteins. The first example worth mentioning was that of monellin, a sweet-tasting protein isolated from “ser- endipity berries,” a fruit of Dioscoreophyllum comminsii. It was known to consist of two subunits, I and 11, of a total of 94 amino acids. We were able to separate these by liquid chromatography (on Biorex-70) and began work on subunit I. Upon partial acid hydrolysis, followed by our chemical conversion procedure, the resulting mixture gave a gas chromatogram exhibiting about 30 distinct peaks or discernable shoulders. The mass spectral data obtained on-line in about 20 min revealed the presence and sequence of 5 5 peptides ranging from di- to hexapeptides. The mass spectrum of the peak eluting [ast indicated that it must be the trimethylsilylated reduction product of Gly-Pro-Val-Pro- Pro-Pro.

While we were in the process of assembling these pieces to one single sequence, there appeared a paper by Bohak and Li on the primary structure of both subunits of monellin. These authors had completely sequenced subunit I1 by stepwise Edman degradation but were unable to complete the sequence of subunit 1 because of a rapid decline in the yields of PTH-amino acids after step 29, resulting in an uncertain sequence 28-37 and no information on the C-terminal region beyond. Our data already in hand immediately allowed us to establish the correct and complete sequence, which turned out to be 44 amino acids long. It was evident that the very hydrophobic hexapeptide mentioned above (spectrum #55) caused the loss of the C-terminal portion of the protein by “wash out.” We also had sufficient information to characterize the few amino acids that Bohak and Li were not able to identify.

This work was, perhaps, the first demonstration of the com- plementarity, rather than competition, of the Edman method and our mass spectrometric strategy. Although we could complete the sequence, we could also use the published Edman results to assign all Leu versus Ile positions, a differentiation by mass spectrometry that had to wait for another decade and an entirely different methodology.

Recalling the skepticism expressed by Sanford Moore years earlier, it should be noted that there was only one single peptide bond that was not represented in the partial acid hydrolyzate of monellin-I. Thus, there would have been only two ways to assemble all the small peptide sequences. Once the partial structure appeared in the literature, the missing bond turned out to be between Lys(4) and Gly(5). Even without that information, it would have been relatively easy to rule out the other possibil- ity, Gly(1) to Lys(44), because one would expect to find peptides extending the Pro-Pro-Pro C-terminus.

Another example of the value of combining the mutual ad- vantages of the Edman degradation (using the spinning-cup se- quencer, as well as Laursen’s solid-phase technique) with our GC/MS method was the determination of the primary structure of bacteriorhodopsin isolated from Halobacterium halobium. This protein contains seven transmembrane segments and 70% of its amino acids are hydrophobic. This is the reason why this protein was not amenable to the well-established protocols for water-soluble proteins. On the other hand, hydrophobic peptides are particularly well suited for our mass spectrometric strategy. It was, in part, for this reason that my colleague, H. Gobind Khorana, asked us to collaborate with his group (which was

1924 K. Biemann

using the Edman methodology) in the determination of the primary structure of bacteriorhodopsin.

One of the difficulties with this protein was its resistance to proteases, except that chymotrypsin cleaved a single peptide bond (later shown to be Phe(71)-Gly(72), at an accessible part of an extra-membrane loop). This made it possible to elaborate the structure in two steps. The experimental problems encountered and their solution were, however, the same for both. Either of the two parts was only soluble in 88% formic acid but allowed dilution to 70% for CNBr cleavage at the total of nine methio- nines. The major contributions of the GC/MS methodology were as follows: ( 1 ) direct sequencing of a tri- and a tetrapeptide too small for the Edman method; (2) determination of hydrophobic C-terminal sequences lost by “wash out” during the Edman sequencing of the longer CNBr-peptides; (3) confirmation of the obtainable Edman information; (4) identification of the blocked N-terminus; and, perhaps most importantly, ( 5 ) “fish- ing out” methionine-containing sequences (using a computer search for methionine-specific fragment ions) from the very complex mixture produced by partial acid hydrolysis of either of the two chymotrypsin-generated parts of bacteriorhodopsin (finally found to encompass amino acids 1-71 and 72-248, re- spectively). Determination of the amino acid sequences sur- rounding Met was, of course, necessary for the proper alignment of the CNBr-peptides. The N-terminal blocking group was established by high-resolution mass spectrometry to be pyroglutamic acid followed by alanine.

The value of the simultaneous utilization of two entirely different methodologies for the reliable sequencing of a protein exhibiting uncommon physical properties is best demonstrated by the fact that Yu. A. Ovchinnikov’s group in Moscow published, slightly ahead of us, a primary structure of bacteriorhodopsin, which proved to be incorrect. I t was missing a tryptophan and five amino acids were misassigned.

This was the period during which the methodologies for DNA sequencing (first, Maxam and Gilbert and then Sanger et al.) became sufficiently reliable and established for the translation to protein sequences. Soon an article appeared in the “News and Views” section of Nature (Sept. 14, 1978) entitled “The decline and fall of protein chemistry?” The thrust of the paper was that the zenith of direct protein sequencing had just been reached by Fowler, Zabin, and their colleagues, who, in a herculean effort, had determined the primary structure of the 1,021-amino acid long &galactosidase from Escherichia coli. The prediction was made that, in the future, primary protein structure would be de- duced from DNA sequences, thus moving protein chemistry backstage or worse. History has proven this view to be too pes- simistic, but a discussion of the reasons and the actual increase in the importance of studies on the protein level are beyond the scope of this article.

In fact, Paul Schimmel, whose laboratory was located a few floors above mine, was at that time interested in the structure of alanyl-tRNA synthetase, a protein of M , - 100,000. For this reason, he chose to sequence the genomic DNA coding for the protein. However, he was aware of the pitfalls when working with such a long DNA, even when sequencing both strands. At the time, the generally accepted criteria for determining the correct reading frame had to satisfy two requirements: The N-terminal and C-terminal amino acid sequences of the gene product must fit the same reading frame and that there should not be a stop codon (i.e., i t must be an “open reading frame”). Con-

sidering the high accuracy of DNA sequencing even then, for short stretches of a few hundred bases these criteria may suf- fice. However, for a 100,000-Da protein, about 3,000 bases must be accounted for and correctly identified (unless a misidentification simply leads to a base triplet coding for the same amino acid). Any erroneous deletion or insertion of a base followed later by an insertion or deletion of another one, switches to another reading frame in between and generates a stretch of an entirely incorrect amino acid sequence. Such a pair of errors would have no effect on the N- and C-termini and thus escape detec- tion, unless it happened to create a stop codon, which is unlikely if the switched portion of the reading frame is relatively short. Furthermore, it has been shown that, in certain cases, a stop codon (e.g., UGA) is read as Trp.

When discussing this problem, i t occurred to us that i t would be very useful to have a random check across the entire protein, not just at the two termini. Again, this was an ideal case to be dealt with by our GC/MS method: the sequence of short peptides generated by partial acid and/or enzymatic degradation of the gene product must all fit the base sequence of one- and only one- reading frame of the experimentally determined DNA sequence. Any one of the above-mentioned back-and- forth switches between two reading frames would be detected immediately because, although the majority of the peptides would f i t one reading frame (the correct one), others would match a stretch of an apparently incorrect frame. Furthermore, the region and direction of the switch delineate the type (deletion or insertion of a base) and region of the error.

Using two large proteins of then known sequence, human se- rum albumin and &galactosidase from E. co/j, we were able to calculate that the minimum size of practically unique sequences are tetra- and pentapeptides (in contrast, dipeptides would f i t at many places). Thus, we developed the following strategy: digest the protein with thermolysin, a protease that cleaves at relatively many different amino acids (thus maximizing the generation of small peptides); subject the digest to our reductive derivatiza- tion chemistry; inject the entire product into the GC/MS system and continuously record the mass spectra of the GC effluent. Because we could predict the retention time of the fast- est moving tetrapeptide-reaction product (that from Ala,), one could concentrate attention on the mass spectra recorded from then onward. Even though we could not, at that time, differentiate Leu from Ile, and because the esterification step resulted in the same products from Asn and Asp or Gln and Clu , these shortcomings would only leave those base misidentifications undetected, which merely convert the codon of one member of such a pair to that of the other one.

The generation of these sequence data was so fast that this part of our collaboration effort ran ahead of the DNA sequencing. This allowed us to match each new set of DNA sequence data produced by Schimmel’s group with our previously accu- mulated peptide sequences. Any error in the former that led to a frame shift or misidentified base that changed a codon to that of another amino acid in a position covered by any of the peptides detected became immediately obvious. In most cases, re- inspection of the corresponding sequencing gel corrected the error. On the other hand, those regions of the DNA sequence that fit well a number of peptides did not need to be redundantly reanalyzed, thus resulting in great savings of time.

Now, more than 15 years later, during which period the DNA- sequencing technology has vastly increased in speed and relia-


bility, it is hardly ever necessary to carry out such parallel checks using the protein. But, at that time, the approach described above greatly decreased the time required to confidently establish a correct cDNA sequence and, thus, ensure that the derived primary protein structure was also correct. Nevertheless, the DNA sequence dictates only the primary structure of the gene product, but gives n o clue about the multitude of posttranslational modifications that convert it to the ultimate biologically active protein. Thus, the structure of the latter has to be inves- tigated in detail and mass spectrometry has become an important technique in this field.

At this point it should be noted that, since about the mid- 1970s, mass spectrometry had begun to play a significant role in the determination of structural features of proteins that could not be addressed by either the manual or the automated Edman degradation, then widely and successfully employed. These cases involved mainly blocked N-termini or amino acids that were not stable under the Edman conditions.

N-terminal pyroglutamic acid was a typical problem that could easily be addressed by mass spectrometry. Upon N-methylation, it is converted to N-methyl pyroglutamic acid that added 14 Da to its mass, whereas reduction with LiAlD, or BzD6 reduced the residue mass by 12 Da producing dideuteroproline. The former approach, for example, was used by H. Morris (by then at Imperial College, London) in the case of fibronectin, whereas we employed the reductive method in a collaboration with H. Neurath’s group on the carboxypeptidase inhibitor from potatoes. A third approach was high-resolution mass spectrometry, which allows one to determine the elemental composition of the molecular ion and each fragment thereof, an approach by which we determined that N-terminal amino acid of bacteriorhodopsin is pyroglutamic acid.

Another notorious problem in amino acid sequencing was the identification of labile modified amino acids. A prime example is y-carboxyglutamic acid (Gla), which being a substituted ma- lonic acid easily decarboxylates to glutamic acid, from which it is then undistinguishable. Taking advantage of this reaction, we developed a procedure of decarboxylation in the solid phase by DCI gas, which cleanly introduced two atoms of deuterium at the y-carbon of glutamic acid and increased its residue mass by 2 Da. This strategy was used in the determination of the amino acid sequences of chicken and monkey osteocalcin (in collaboration with P. Hauschka at Boston Univ.), both of which turned out to contain three Glas. As an aside, it should be noted that, at that time, we had already found a way to differentiate leucine from isoleucine, if we could find a dipeptide in which either of these amino acids represented the C-terminal position. Such dipeptides were formed upon harsher partial acid hydrolysis, again pointing out that one could manipulate S. Moore’s concerns, mentioned earlier, to one’s advantage.

Other applications of the G U M S method, in combination with the Edman degradation and/or the soon discovered fast atom bombardment (FAB) ionization (see below), involved the correction of previously published amino acid sequences. For example, once we had determined the primary structure of mac- romomycin (where the G U M S method provided crucial overlap information), that of the related neocarcinostatin became suspect. The sequences of two corresponding tryptic peptides appeared to be considerably different. Most of the molecular weights of the peptides obtained by digestion of this tryptic peptide did not match anything compatible with the published struc-

ture of neocarcinostatin. The reductive GC/MS method clearly established the correct sequence of this crucial tryptic peptide.

Another important contribution of mass spectrometry to the recognition of N-terminal blocking groups was the first identification (in collaboration with K . Walsh’s group, Seattle, WA) of a myristyl group at the N-terminus of the catalytic subunit of cyclic AMP-dependent protein kinase from bovine cardiac muscle. Both direct chemical ionization on an N-terminal tri- peptide and FAB ionization of a heptapeptide indicated the presence of a saturated C,,-acyl group, which was identified by GC/MS (after total hydrolysis and esterification) as myristic acid,

All the peptide sequencing using the GC/MS method had been carried out at the 50-100 nmol level. Realizing that there is often not that much protein available, we set out to modify the methodology so that it would be applicable to much smaller amounts. The major problem in simply miniaturizing the procedure was that there is a limit by which solvent volumina and reagents can be scaled down. The accumulating impurities from these sources and the handling and transfer of reaction products soon became the major components and obliterated the spectra of the peptide derivatives. A similar problem had been encountered during improvements of the Edman chemistry and was solved by automation of the procedure. A further reduction in sample requirements was achieved by Hood, Hunkapiller et al. by carrying out the reactions in the “gas phase.” This approach seemed to be the most logical direction to take in our de- rivatization procedure. One of my postdocs, Marcus Zollinger, undertook the project. With typical Swiss attention to detail, he designed and constructed a system capable of carrying out all the chemical steps, starting with the peptide mixture, using gas- eous reagents, and ending with automatic injection of the final products into the CC/MS system.

Halfway through the successful testing of the apparatus, an event occurred that drastically changed the field of mass spectrometry, particularly with respect to its application to peptide and protein chemistry. Michael Barber at UMIST (Manchester, UK) invented a novel method for the direct ionization of large and polar molecules: FAB. While attempting to use secondary ion mass spectrometry (SIMS, a method long established for inorganic analysis) to organic molecules, he noticed that pure crystalline compounds produced only a short burst of intact molecular ions upon bombardment with argon ions of a few kilo- volts of kinetic energy. However, much more lasting signals were obtained using argon atoms as the primary beam and when the sample was in a less pure noncrystalline state. Concluding that the sample must be present in a liquid medium, he irradiated glycerol solutions of the analyte. Surprisingly, signals were easily obtained that lasted long enough (a few minutes) to record an entire mass spectrum. Probably remembering the instant attention that the electron ionization spectrum of fortuitine had received 15 years earlier, Barber tried his new ionization method on underivatized peptides and it worked beautifully! In the first publication, he used Met-Lys-bradykinin as an example. It gave a pronounced signal at m/z 1,319 for the protonated molecule, [M + HI+, and some fragment ions that could be correlated with the sequence of the peptide. In retrospect, the careful reader of this first paper (in early 1981) describing the technique will notice that no mention of the addition of glycerol, nor of any other “matrix,” was made. However, the secret soon leaked out and as the FAB ionization method was easily adaptable to ex-

1926 E(. Biemann

isting mass spectrometers, including the type we had, FAB-MS was immediately used for the determination of molecular weights of peptides ranging up to 2,000 Da and soon beyond. A few nanomoles of smaller peptides gave sufficient fragment ions to be interpreted in terms of their known amino acid sequence.

These developments sounded the death knell for our G U M S method with its elaborate chemical conversion and thus to M. Zollinger’s research project, into which considerable time and effort had been invested. 1 still feel uneasy about the fact that this work was never published, but we made it obsolete ourselves by developing tandem mass spectrometry of FAB-generated protonated peptide ions for their fast and reliable sequencing at high sensitivity (about 1 nmol and soon even less).

The success of the phase check of DNA sequences quickly led to other collaborations. An artist’s conception of the sequence of the alanyl-tRNA synthetase, with the mass spectrometrically derived information highlighted, even made the cover of Science (Sept. 25, 1981). At that time, Dieter So11 at Yale was in the process of sequencing the cDNA coding for glutaminyl-tRNA synthetase when he learned from P. Schirnmel about our work and asked us to collaborate with him too. We had just completed one experiment using the GC/MS method when we learned about Barber’s FAB ionization, which we immediately implemented on one of our mass spectrometers. Because this method produces mainly protonated peptide molecules (i.e., [M + H]+ ions) with little fragmentation, it was ideally suited as the basis of a different strategy for checking the correctness of a DNA sequence. Rather than utilizing the actual sequence of small (tri- penta)peptides, the molecular weights determined by FAB of the peptides of a tryptic digest were compared with those predicted from the still incomplete DNA sequence. Because larger peptides span a longer sequence, they are more useful and revealing. When testing this approach on the Glu-tRNA synthetase, we caught the misidentification of two bases (Cs originally read as Ts) just in time to correct amino acid 7 from serine to proline, which differ by 10 Da. Because the G U M S data did not cover this portion, the reading error had remained undetected, but the molecular weights of two overlapping N-terminal peptides were 10 Da heavier than expected. Unfortunately, errors in preliminary data usually are not discussed in final papers and the efficiency of such checking procedures was not apparent and appreciated by the general audience.

In addition to the two cases discussed above, we collaborated in a number of similar situations and other mass spectrometry laboratories adopted the same approach. Nowadays, checking new or suspect-published protein sequences by measuring the molecular weights of proteolytic peptides is common practice. I t is particularly useful for the identification of posttranslational processing, natural mutations, or biosynthetic site-specific modifications, etc., all of which change the molecular weight of the protein and that of certain proteolytic peptides.

As had been mentioned earlier, FAB-generated [M + H]+ ions have little tendency to fragment. This is because these ions are very stable and contain little excess energy to cause the rup- ture of covalent bonds. Thus, the FAB spectrum of a peptide shows abundant [M + HI+ ions, but the peaks due to fragments are very small and are sometimes interfered with by the matrix (glycerol or other related polar liquids). It had been known for a number of years from the work of McLafferty, Beynon, Hunt, etc., that small peptide molecules or derivatives

thereof, once ionized, can be fragmented by collision with a neu- tral gas. In 1985, we placed an order with JEOL (Tokyo, Ja- pan) to design and build for us a tandem mass spectrometer that consisted of two double-focusing sector instruments. Between these two, there was a region where the [M + H]+ ions produced by FAB ionization in the first mass spectrometer with a kinetic energy of a few kV collided with He or Xe atoms, which caused fragmentation (collision-induced decomposition, CID). The fragment ions were then mass analyzed in the second mass spectrometer. As pointed out above, the principle was not new, but this instrument was the first one to be built to high- performance specifications, as far as mass range (up to 15 kDa), resolution, and collision energy (up to 10 kV) were concerned. Within a few months of its delivery in July of 1986, we had established a method for the sequencing of peptides up to 25 amino acids in length. The next 2 years were spent applying the technique to protein sequencing, working out the details of the experimental strategy, elucidating the fragmentation processes, and assigning the previously unknown amino acid sequence of proteolytic peptides. This method also proved to be able to un- ambiguously distinguish the two isomeric amino acids leucine and isoleucine.

Having thus demonstrated the practicality of high energy CID mass spectrometry, a number of other laboratories, led by those in the biotechnology industry such as Genentech (South San Francisco, California) and Genetics Institute (Andover, Mas- sachusetts), acquired the same type of instrument (named JEOL HXI 1O/HX1 IO), in spite of the high cost (in excess of 1 million dollars). A single, similar instrument from another source (VG, Manchester, UK) had already been ordered by SmithKline Beecham (King of Prussia, Pennsylvania).

The next events in the development of mass spectrometric techniques useful in biology, particularly in protein chemistry, were the inventions of matrix-assisted laser desorption ionization (MALDI), by F. Hillenkamp and M. Karas, and electro- spray ionization (ESI), by J. Fenn and coworkers. Both made it possible, after some refinements, to determine the molecular weight of a protein in the 104-105 Da range with an accuracy of _+O. 1-0.01 To and requiring only a few picomoles (or less for smaller molecules). In either case, it was the application to proteins in 1988 that caught the attention of the broader scientific world. John Fenn had, a few years earlier, successfully ionized and analyzed large molecules, such as polyethylene glycols and other synthetic polymers, but it was not until the Annual Con- ference of the American Society for Mass Spectrometry held in June 1988, in San Francisco, where he showed ESI mass spectra of proteins, ranging from insulin t o alcohol dehydrogenase, that the method became instantly accepted. ESI produces highly multiply charged (protonated) molecules and their mass-to- charge ratio (m/z ) is, therefore, low because z is relatively large (in proteins relatively proportional to M,) . Thus, a simple quadrupole mass spectrometer of limited mass range (m/z 1,000-2,OOO) suffices. The new ionization method could be easily adapted to this then widely available type of spectrometer and ESI-MS is now a well-established method for the determination of the molecular weight of proteins, even in mixtures.

An even simpler technique is MALDI, which involves pulse (ns) irradiation with a UV or IR laser of a co-crystallized mixture of about a picomole of peptide or protein and a large excess of an organic substance absorbing light at the laser’s wavelength. In this case, mainly singly and doubly protonated


protein molecules are produced, the mass of which is then measured in a time-of-flight (TOF) mass spectrometer. Because these instruments were not readily available, the widespread use of MALDI-MS took a little bit longer to materialize. Fortunately, the next 5-year renewal proposal of my NIH Mass Spectrom- etry Resource grant (it had been active since 1966) was just coming up, which provided a good opportunity to apply for funds for such an instrument. At that time (1988), there was no MALDI instrument particularly suitable for organic molecules that was commercially available (Hillenkamp and Karas had to modify a spectrometer designed for imaging inorganic elements). Fortunately, our proposal was funded at a level sufficient to in- terest the Vestec Corporation (Houston, Texas) to build us a MALDI spectrometer, using a design developed by Chait and Beavis at the Rockefeller University’s NIH MS Resource.

This instrument was delivered in October 1990, and immediately provided us with the capability of measuring the molecular weights of the proteins that we were working on at the time. Needless to say, it is comforting to know that a final sequence agrees with the accurately measured molecular weight, eliminat- ing redundant experiments or digests to ensure that nothing had been missed. We just regretted that this capability was not available to us many years earlier.

Furthermore, because the high sensitivity, mass accuracy, and mass range of both MALDI and ESI permit the fast and facile measurement of the molecular weight of a protein, these methods are now widely used to confirm the primary structure of ma- terials produced by overexpression of a natural gene or one that has been modified in the laboratory. It is surprising how often we find it to differ from that assumed by the investigator for whom we carry out such a measurement. In these cases, the proteins are generally smaller than expected, either because the translation begins further downstream than assumed or posttranslational events have shortened the protein at either the N-terminus or the C-terminus, sometimes at both. A further powerful aspect of mass spectrometry is its capability of ana- lyzing mixtures without prior separation. Depending on the re- solving power of the instrument and the molecular weight of the protein, differences by a single amino acid can be easily detected at the picomole level in less than 15 min.

Not yet mentioned was a method for the direct ionization of large, polar molecules, developed as early as 1976 by Macfarlane and Torgerson: 252Cf plasma desorption. Although not applied to proteins until about 1982 and eclipsed by MALDI in 1988, it showed for the first time that large molecules can be ionized intact and mass analyzed in a time-of-flight mass spectrometer.

The rapid developments occurring during the 1980s led to a broadening of the use of mass spectrometry in protein chemistry and structure analysis. It is impossible to mention the many laboratories that employed the technique for studies on known systems in efforts (some real, some imaginary) to improve the methodology. However, a number of groups beyond our own applied it more and more to the solution of real problems of biochemical significance. Most noteworthy (without attempting to generate a comprehensive list) are the groups in biotechnology companies led, for example, by S. Carr (SmithKline), J. Stults (Genentech), H. Scoble and S. Martin (Genetics Institute); at medical research facilities, T. Lee at the Beckman Research In- stitute of the City of Hope; and at academic institutions, A. Bur- lingame and B. Gibson (both at UC, San Francisco), B. Chait (Rockefeller University), C. Fenselau (Univ. of Maryland, Bal-

timore County), P. Roepstorff (Odense University, Denmark), Y. Shimonishi (Osaka University), etc. Quite a few of these in- dividuals had been trained in my laboratory (Biemann, 1994).

Particularly important contributions have been made by D.F. Hunt’s research group at the University of Virginia. He pioneered the use of the triple quadrupole tandem mass spectrometer system for the sequencing of peptides ionized by various techniques, starting with positive and negative chemical ionization, followed by FAB and then ESI. Various collaborations with members of the biological community, both in Virginia and elsewhere, greatly increased their awareness of the utility of mass spectrometry in biology. One of his most significant contributions is the miniaturization of LC-ESI-MS interfaces, designed by his able associate, J. Shabanowitz, for the analysis (at the femtomole level) of the extremely complex mixtures of peptides associated with Class I and Class I1 MHC molecules. This work has great impact on immunology. Hunt, now a University Pro- fessor (Chemistry and Pathology) at the University of Virginia, obtained his initial training in mass spectrometry in my laboratory in 1967 and then went his own way. We converged again at the 1992 Conference on Methods in Protein Sequence Anal- ysis held in Otsu, Japan, where we both independently were the recipients of the Pehr Edman Award, a gratifying demonstration that the peptide and protein science community has come to recognize the important contributions that mass spectrometry did make to this field and surely will continue to do so.

Acknowledgments

lt is a particular privilege to have had the enthusiastic cooperation of many graduate students, postdoctoral fellows, visiting scientists, and support staff (see Biemann, 1994), who carried out a good part of the work described here. I also acknowledge the mutual learning experience and frequent encouragement from biochemists and biologists from other laboratories with whom we collaborated. Equally important is the long- standing support of federal funding agencies, particularly from the Na- tional Institutes of Health through grants GM05472 (since 1958) and RR00317 (since 1966), as well as a training grant (during the crucial period from 1966 to the mid-1970s).

References

Biemann K. 1994. The Massachusetts Institute of Technology mass spectrometry school. J A m Soc Mass Spectrom 5:332-338.

Biemann K, Gapp F, Seibl J. 1959. Applications of mass spectrometry to structure problems. 1. Amino acid sequence in peptides. J A m Chem Soc 81:2274.

Biemann K . 1962. Mass spectrometry: Organic chemical applications. New York: McGraw-Hill Book Company, Inc. Chapter 7.

Biemann K. 1963. Mass spectrometry. Annu Rev Biochem 32:755-780. Biemann K. 1972. Amino acid sequence in oligopeptides. In: Waller GR, ed.

Biochemical applications of mass spectrometry. New York: John Wiley and Sons. pp 405-428.

Biemann K. 1980. Amino acid sequence in oligopeptides and proteins. In: Waller GR, Dermer OC, eds. Biochemical applications of massspectrom- etry. 1st suppl. vol. New York: John Wiley and Sons. pp 469-525.

Carr SA, Biemann K. 1984. The identification of posttranslationally modified amino acids in proteins by mass spectrometry. Methods Enzymol 106~29-58.

Martin SA, Biemann K. 1987. Mass spectrometric determination of the amino acid sequence of peptides and proteins. Mass Spectrom Rev 6:l-76.

Biemann K. 1990. Peptides and proteins: Overview and strategy. Methods Enzymol 193:351-360.

Biemann K. 1990. Sequencing of peptides by tandem mass spectrometry upon

479. high-energy collision-induced dissociation. Methods Enzymol 193:455-

Biemann K. 1992. Mass spectrometry of peptides and proteins. Annu Rev Biochem 61:977-1010.

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