Plasma Desorption Mass Spectrometry: Coming of Age Robert J. Cotter Middle Atlantic Mass Spectrometry Facility Department of Pharmacology and Molecular Sciences The Johns Hopkins University Baltimore. MD 21205 Plasma desorption mass spectrometry (PDMS) uses the high-energy fragments emitted from the spontaneous fission of ii'Cf to desorb and ionize large and nonvolatile molecules for mass analysis. The method is also known as fission fragment induced desorption (FFID). Much of the development of PDMS has occurred in nuclear laboratories, where accelerators have been used to generate heavy particles with energies (about 1 MeVInucleou) similar to those of fission fragments. Thus the term heavy ion induced desorption (HIID) is often used to distinguish PDMS from the lighter and less energetic primary particle methods used in secondary ion mass spectrometry (SIMS) and fast atom bombardment mass spectrometry (FAB-MS). Since plasma desorption was introduced by Macfarlane and Torgersou in 1974 ( I ) , the size of molecules successfully mass analyzed by the technique has steadily increased and now includes a large number of peptide hormones, growth factors, and other small proteins (2) in the mass range of 5,00025,000 m u . Particularly impressive is the recent recording of the PDMS spectrum of porcine pepsin (Figure 1) with a molecular weight close to 35 kDa 0003-2700/88/0360-781A/$Ot Sol0 @ 1988 American Chemical Society
lNS7RUMEN7A7lON
1 Figure 1. Plasma desorption mass spectrum of porcine pepsin obtained on a BIOION 20 (Uppsala, Sweden). The sample is a mixture ot residues 1-42 and 1-44 (34,504and 34,686 m u . respectively,calculated
from the sequence). Which are unresolved in the spectrum.(Adapted with permission horn Reference 3.)
ANALYTICAL CHEMISTRY, VOL. BO, NO. 13, JULY 1, 1988 * 781 A
portant complement to the structural information obtained by Edman degradation, gas-phase sequencing, or the sequence analysis of enzymatic digests by FAB-MS (5).
Flgure 2. Rising cost and capabi
ers as a function of the
The point labeled "POMS" has been added to the wlglnal flgve tram the Plmente repat (4and r e p sen18 the 1985 pwchase price 01 the BIO-ION ( U p p h , Sweden) BIN 10K mass speclrOmB18r. (Adapted wim ~ 1 6 8 i O hom n Reference 4:copyright 1985 by the National Academy of Sciences.)
(3).Such achievements by PDMS have challenged our assumptions about the real mass limits of MS. Because of its high mass range, high ion transmission, and ability to record ions of different mass simultaneously, the time-of-flight (TOF) mass analyzer is most commonly used with PDMS. A disadvantage of the TOF analyzer is its low mass resolution. Thus in PDMS spectra the isotopic contributions to the molecular ion signal are not resolved,and measurements are made for the average rather than the monoisotopic mars. At the same time, advances in magnet technology have greatly extended the mass range of double-focusing sector instruments that can make both unit and high-resolution measurements. However, as shown in Figure 2 (41, the increasing cost of such highperformance instruments has been of some concern. PDMS offers a lower cwt alternative, and instruments that are used with this technique are available commercially. Following more than a decade of fundamental research, interest in the technique now focuses primarily on its application to structural problems in biochemistry and biophysics. This is not to ignore the early contributions of the instrument to structural problems or the continued interest in the energetics of macromolecules bound to, and desorbed from, surfaces; but rather to
-
recognize the availability of the technique to the nonphysicist as an inexpensive, reliable tool for relatively accurate assessment of molecular weights of small proteins and other polymers. Such measurements can provide an im-
Instrumentation The general features of plasma desorption mass spectrometers are shown in Figure 3. The ionization source is (typically) a IO-FCi sample of t$f sandwiched between two thin sheets of nickel foil and held a t the same electrical potential (-20 kV) as the sample to be analyzed. g2Cf decays with a halflife of 2.65 years: 97%as alpha particles and 3% as two simultaneous, multiply charged fission fragments emitted in opposite directions. A typical decay would involve the simultaneous emission of i T c and :fBa with energies of 104 and 79 MeV, respectively (6). At the beginning of each timing cycle, one of the fission fragments strikes the start detector, a grounded foil that emits secondary electrons collected by a dual channel plate detector. The output pulse from this detector is amplified, passed through a constant fraction discriminator, and recorded as the start pulse by a time-to-digital converter. The detector foil and discriminator are also designed to distinguish fission fragments from lower energy (6.1 MeV) alpha particles. At the same time, the second fission fragment penetrates the foil (aluminum or aluminized mylar) on which sample has been deposited on the reverse side. From 1to 10 secondary (sample) ions are desorbed from the foil and accelerated toward a grid held at ground potential, where thev
I
stop
IFigure 3. Block diagram of a F
7 a 2 ~ ANALYTICAL CHEMISTRY, VOL. BO, NO. 13. JULY 1, 1988
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783A
enter a long (15 cm to 3 m) drift region with velocities inversely proportional to the square root of their masses. The amount of time needed to traverse the drift region, recorded when the secondary particles reach the stop detector, becomes a measure of their mass. A complete mass spectrum is assembled by accumulating the signal from many such timing cycles.
History The PDMS technique introduced by the Texas A&M group (1, 7) was an outgrowth of earlier TOF methods developed for the identification of alpha and beta radioactive nuclei (8), but it was intended at the outset as a new method for structural analysis of large biomolecules. In 1980 Macfarlane and co-workers used their new instrument to determine the molecular weight (2681 amu) of the marine toxin, palytoxin. McNeal et al. (10, 11)investigated the advantages of negative ion PDMS for the sequencing of fully protected synthetic oligonucleotides. In 1981 a plasma desorption mass spectrometer was constructed a t Rockefeller University. With this instrument Chait and Field focused their attention on the abundant (but generally unfocused) ion signal that arises from the fragmentation of large molecules (12, 13). Their investigations of both prompt and metastable fragmentation have not only improved our understanding of the factors that affect mass resolution in TOF analyzers, but have fostered further interest (both theoretical and experimental) in the applicability of quasi-equilibrium theory and other fragmentation models to very large molecules (14-16). As noted, much of the development of PDMS occurred at several nuclear laboratories, where the availability of heavy ions from an accelerator made possible detailed studies of heavy ion induced desorption of nonvolatile molecules as a function of the mass, energy, and charge state of the primary ion. These included the nuclear laboratories at Darmstadt, West Germany ( 1 7 ) , Orsay, France (18,19),Erlangen, West Germany ( 2 0 ) , Pasadena, CA ( 2 1 ) , Marburg, West Germany ( 2 2 ) , and Uppsala, Sweden (23). In addition to their more fundamental studies, the group at Orsay (and the Curie Institute) used the PDMS technique for the analysis of nucleic acids modified by carcinogenic polycyclic hydrocarbons (24).An LC/PDMS system was developed at Marburg (25), where PDMS was also used in conjunction with thinlayer chromatography to investigate the pharmacokinetics of the antitumor drugs etoposide and teniposide (26). The Uppsala group reported the first PDMS mass spectrum of bovine insulin using 1271+20 ions from a tandem ac784A
celerator (27). Researchers from that group also reported the mass spectrum of the neufotoxin from the Naja naja siamensis cobra snake venom (28)and continued to advance the mass range of PDMS with their molecular weight measurements ( 2 ) for cytochrome-C (12,361 amu), bovine ribonuclease (13,682 amu), and porcine phospholipase A2 (13,980 amu). PDMS instruments were constructed in other laboratories as well. An instrument built by the Texas group was installed in the laboratory of Henry Fales at the National Institutes of Health in Bethesda, MD. He reported the combined use of high-speed countercurrent chromatography and PDMS for the separation and identification of methyl violet 2B (29),commercial digitonin (30),and the 252Cfmass spectra of skin lipids (31).An instrument was constructed at the Curie Institute for the study of unprotected oligonucleotides (32).An instrument was also constructed a t Argonne (33),and the use of 252Cf as an ion source for Fourier transform mass spectrometry (FT-MS) has been reported (34). In 1984 the first commercial PDMS instruments became available from BIO-ION Nordic (Uppsala) and in the next two years were delivered to laboratories a t Odense University (Denmark), The Johns Hopkins University (Baltimore, MD), and the Mayo Clinic (Rochester, MN). The instrument has a remarkably short (15 cm) drift region, which improves the secondary ion transmission and shortens the analysis time. When using the PDMS instrument it is not unusual to accumulate spectra to a preset ion count of lo6 counts, which results in a mass spectrum in about 11 min for a source for which approximately 1500 fission fragments per second are recorded (35). Mass resolution is low, about 1/500, so that isotopic contributions to molecular and fragment ion peaks are not resolved. At the same time, mass accuracy ranges from 0.01% (below 5000 amu) to 0.1% a t higher masses. The instrument is equipped with automatic valves for loading up to eight samples and to prevent any possibility for exposure of the californium source to the outside world. The complete data system provides simultaneous acquisition and processing of the same or different spectra, with background subtraction, smoothing, and mass Calibration. The introduction of this instrument, and its initial achievements during the past two years, coincides well with increasing reports by a number of laboratories on the applications of PDMS to the solution of structural problems in biochemistry. Its availability to the general scientific community can be expected to result in an increased use of the technique for the
ANALYTICAL CHEMISTRY, VOL. 60, NO. 13, JULY 1, 1988
types of problems presented in this article.
Sulfates, matrices, and the desorption energy barrier The successful application of any of the so-called desorption techniques appears to be highly dependent on the way that samples are prepared, purified, and presented to the mass spectrometer. The presence or absence of cations in the sample, the use of liquid matrices or clean surfaces to support the sample, and presentation as a monolayer or bulk sample all appear to be crucial factors for the different techniques. PDMS is no exception, and as with the other techniques, there has been some attempt to explore the relationship between our observation of successful sample presentation and our understanding of how the technique works. In the plasma desorption mass spectrometer, the thin foil allows penetration of the sample by the fission fragment from the opposite side. This is important only when this geometry is used for obtaining a start signal from the fission fragment moving in the opposite direction. Indeed, techniques that employ accelerators use a geometry in which the heavy ions strike a much thicker surface on the same side on which the sample is deposited. For many reasons it appears desirable to make the sample layer deposited on the foil as thin as possible, and this has been achieved by the use of electrospraying techniques (36). In addition to reducing interactions between sample molecules, desorption close to the equipotential surface in the presence of a high ion extracting electrical field ensures that all ions are accelerated to the same kinetic energy-a critical factor for mass focusing in the TOF analyzer. As heavy ions penetrate the solid surface, they produce a microscopic fission track, a region of intensive damage reflecting the deposition of primary particle energy in the solid lattice. The major charged species sputtered from this region include atomic ions (H+, Na+, etc.) and low-mass fragment ions (CH3+, CN-, etc.) that do not provide specific structural information for the sample deposited on the surface. It was suggested early on (37) that the desorption of intact macromolecular species takes place a t some distance from the fission track, and this notion has been generalized (38,Figure 4) for other desorption techniques. Isomerization of the deposited energy results in rapid expansion of the interaction area from which energy can be transferred to adsorbed molecules. Ultimately this interaction area includes regions for which the energy density a t the surface is low. Energy transferred to molecules adsorbed in this region is sufficient to
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overcome the weak interactions (van der Waals forces and hydrogen bonds) binding molecules to surfaces and to each other, hut it does not result in the cleavage of covalent bonds. Generally, molecular ion yields from MeV primary particles are higher than those from KeV particles. Macfarlane has suggested that the effective interaction areagenerated by the passage of 100 KeV ions through a material extends much farther than that of a few KeV particles (39) and that this becomes crucial for a peptide whose contactareawiththesurfacemaybeon the order of 20 A. In any case, an appreciation of the sensitivity of desorption to weak forces (espoused by this model) may in part explain some of the recent successes in approaches designed to improve desorption efficiency. One approach to improving desorption has been to change the composition of the surface. One of the first changes was to reverse the aluminized mylar sample foil, desorbing ions directly from the mylar surface. An ionexchange surface, Nafion, has also been used (40). Most successful, however, has been the use of a proteinbinding surface, nitrocellulose, electrosprayed onto the aluminum foil (41). The sample is deposited and evaporated on the surface and then washed with a mild acid solution to remove alkali ions that do not bind to the surface hut can interfere with sample desorption. Another approach, developed in our laboratory (42), includes in the electrosprayed sample solution reagents that are known to preserve or promote
protein folding. The most successful of these has been the tripeptide glutathione (y-L-glutamyl-L-cysteinylglycine). Interestingly, Saxena and Wetlaufer (43) observed that a t a 1 0 1 mixture of reduced (GSH) to oxidized (GS-SG) glutathione formed the optimal conditions for the refolding of reduced and denatured hen egg white lysozyme. This finding was consistent with our observations that the reduced form was far more effective than the oxidized form in promoting desorption of intact molecular ions. Most interesting is the fact that the use of the nitrocellulose surface or glutathione matrix has similar effects on the appearance of the spectra (35,42). These effects include an increase in molecular ion signal (yield) to hackground, an increase in the intensity of multiply charged ions (see Figure 0 , and a decrease in the peak widths (42). Narrow peak widths can be associated with a reduction in initial kinetic energy distributions. Chait (16) has noted that ions desorbed from nitrocellulose have less internal energy as well; they fragment less. One possible explanation for the observed changes in the spectra is that the energy required to release a molecular ion from the surface has been reduced. Ions are then desorbed from a broader interaction area, where the energy available to feed both translational and internal degrees of freedom is also reduced. Desorption from a broader interaction area is also consistent with the large increase in molecular ion signal. However, it is not clear why the nitrocellulose surface or glutathione ma-
nergy aeposio
I
Desorption energy
decreases with distance Tom impam
\r /
lodmdecule reactions
/
,/'
Dissxiatmn Desdvation
Uniixdeculardissxiations
Figure 4 tralizei ram for desorption techniques that shows the decreaa energy density on the surface as a function of the distance from the point of particle or photon impact and processes occurring both on and above the surface. (Adapted wilh pwmlsslon lrm Reference 38.)
788A
ANALYTICAL CHEMISTRY, VOL. BO. NO. 13. JULY 1, 1988
trix should enhance the ease of desorption of molecular ions. An important feature of the nitrocellulose technique is the ability to remove alkali ions, by washing with de-ionized water or mild acid solutions, while adsorbed peptides remain hound to the surface. Sodium ions, which are ubiquitous in biological samples and solvents, appear to be detrimental in many techniques, including field desorption, FAB, and plasma desorption. In addition, the use of nitrocellulose permits the adsorption of a thinner and more even sample layer (effectively, a monolayer) than electrospraying. This may bring about a reduction in intramolecular interactions, thereby reducing the energy needed for desorption. Peptide samples generally are prepared for electrospraying as solutions of glacial acetic acid or concentrated trifluoroacetic acid. Such solutions usually can be electrosprayed more easily and form more uniform layers than solutions in water or dilute acid. In addition, the low pH of the solution appears to improve the formation of protonated species. Strongly acidic solutions can, of course, denature peptides, and we have observed that chromatographically pure insulin samples reveal an abundance of A and B chains when rechromatographed following electrospray from their acid solutions. When peptides are dissolved in a solution of glutathione and acetic acid before electrospraying, the enhanced effects on the spectra noted above are observed. When glutathione is added after dissolution of the peptide in glacial acetic acid, or when glutathione is electrosprayed on the foil before deposition of the peptide solution, the enhanced molecular ion yield is not ohserved. We conclude that an important role for the glutathione matrix may be simply to stabilize the peptide sample against the damaging effects of acidic solutions used in the electrospray technique. Although we havenoted that the reduced form of glutathione has been used to promote the refolding of peptides containing disulfide bridges (42), mass spectra of peptides without disulfide bridges also are enhanced. I t is quite possible that peptide folding is important to desorption of peptides as ions in the gas phase, if in fact this reduces interactions with the surface and with other molecules (i.e., aggregation). Although the mechanisms involved in the use of the nitrocelluloae surface and glutathione matrix will continue to be a subject of intense study and interest, one cannot overestimate the important role these techniques have played in analyzing large molecules by plasma desorption. In all of the measures described below. either the nitro-
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Figure 5. Plasma desorption mass spectra of (a) carbohydrate-free allelic (Serle2)ovomucoid third domain and (b) fully glycosylated, polymorphic (7096 Sar162/30% G I Y ’ ~ovomucoid ~) third domain from Japanese quail. cellulose surface or the glutathione matrix has been employed. In addition, enhancements of molecular ion yields for SIMS (using KeV primary particles) have been reported for both nitrocellulose (44) and glutathione (45). Molecular weights ot peptides and protein fragments It is a common perception that plasma desorption mass spectra contain primarily molecular ions with little or no structurally informative fragment ions. To some extent this is true, because the TOF analyzer provides a “snapshot” (46) of ions very close to their time of formation, and it has been noted (14) that delayed ion extraction in TOF mass spectrometers can at times yield additional fragmentation. However, mass spectra of insulin obtained by PDMS (2) and FAB (47) are remarkably similar. Both show the appearance of primarily molecular ions, with smaller signals due to the A and B chains, but little other fragmentation. Thus it may be more correct to say that the heavy ions commonly analyzed by plasma desorption produce little useful fragmentation in any of the current techniques. For smaller molecules, plasma desorption (similar to other methods) produces abundant fragmentation. However, the ability to measure molecular ions of large molecules in com788A
bination with other techniques plays an important role in structure elucidation. The molecular weight of a peptide or protein fraction can be used to verify sequences obtained by other methods, to reveal heterogeneity, or to survey crude peptide extracts. Changes in molecular weight following enzymatic or chemical cleavages will be an effective protocol for observing the structural components that cannot be produced by fragmentation in the gas phase. An increasing number of these kinds of reactions will be carried out directly on the sample foils (48). In our laboratory and in collaboration with Christian Schwabe and Erika Billesbuch from the Medical University of South Carolina, the newiy acquired BIO-ION plasma desorption mass spectrometer was used to verify chemical modifications to relaxinspeptides that have tertiary but not sequence homology t o insulins ( 4 9 ) . Chemical deletions made to both the A and B chains were verified by mass measurements of molecular ions and those arising from the A and B chain fragments. Mass accuracy was sufficient to confirm the integrity of the disulfide bridges following chemical modifications. Working with R. E. Chance and B. H. Frank of Lilly Research Laboratories (Indianapolis, IN), we verified the molecular weights of a family of protamines by PDMS (42).In
ANALYTICAL CHEMISTRY. VOL. 60. NO. 13, JULY
1, 1988
both cases, the glutathione matrix was used. In Odense, a molecular weight measurement by PDMS was used to settle a structural problem. Sequence data for the structural protein from the cuticle of the migratory locust, Locusta migratoria, gave a molecular weight of 15,323 amu, whereas the molecular weight from SDS-gel electrophoresis indicated a molecular weight of 21.6 kDa. The presence of several repeat sequences caused some concern over the sequence results. Using 1271 primary ions from the tandem accelerator in Uppsala and commercial nitrocellulose membranes, Roepstorff et al. (50) determined the molecular weight to he 15,329 i 50 amu, thus verifying the sequence data. The limits of precision in the measurement were established by averaging the molecular weights determined from the measured masses of the singly, doubly, and triply charged molecular ions. Peptide processing in catfish pancreas has been studied by Philip Andrews a t Purdue University. PDMS was used to survey the crude peptide extract for the pre-, pro., connecting, and processed pancreatic peptides. In a single plasma desorption mass spectrum, molecular ions were observed for a somatostatin 28 fragment (1013 amu), proglucagon fragment (1374 amu), somatostatin 14 (1639 emu), glucagon-connecting peptide (2727 amu),
somatostatin 22 (2943 amu), glucagon (3511 amu), a glucagon-like peptide (3785 amu), insulin (5506 amu), and prosomatostatin 22 fragment (6466 amu) (51). Some structural information can be obtained by taking the entire mixture through successive Edman cycles and surveying the changes in molecular weights. Roepstorff et al. (52) obtained the PDMS spectrum of the polycyclic peptide antibiotic, Nisin, from Streptococcus lactis. Using the electrospray technique, they observed considerable fragmentation between the cyclic domains created by the five disulfide bonds. The spectrum obtained with nitrocellulose gave primarily molecular ions, and a prototype plasma desorption instrument with an extended flight path and an electrhstatic particle guide (to maintain secondary ion transmission) were used to confirm the molecular weight of a minor component (identified by other techniques as C-des Dba-Lys Nisin) with higher precision. Ovomucoids are glycoproteins present in avian egg white and are strong inhihitors of serine proteinases. Ovomucoids consist of three tandem domains, each of which is homologous to single-domain pancreatic secretory trypsin inhibitors (53). A t Purdue University, Laskowski and co-workers (54) have determined the amino acid sequences and hypervariability of enzyme inhibitor contact residues of carbohydrate-free ovomucoid third domain from more than 100 avian species. The third domain of the ovomucoid from Japanese quail (OVBJPQ) exhibits serine/glycine polymorphism a t position 162 (53). The allelic forms of intact, carbohy-
drate-free third domain have been isolated and separated, and their secondary, tertiary, and crystallographic structures have been determined hy X-ray studies and crystallographic refinement (55). Recently crystals from the intact, fully glycosylated, polymorphic ovomucoid third domain have been ohtained for crystallographic studies. PDMS was employed to determine the number and possible heterogeneity of N-acetylglucosamineand mannose residues attached to the asparagine a t position 175. The PDMS spectra of the carbohydrate-free OV3JPQ containing serine a t position 162 is shown in Figure 5a; the spectrum of the glycosylated, polymorphic form is shown in Figure 5b. The molecular ion a t 6944.8 amu corresponds to the average weight of a 70%serine/30% glycine ovomucoid with two N-acetylglucosamines and three mannoses. The peak at 6782.8 amu corresponds to the loss of a single mannose, and the presence of correspondingly doubly charged ions in the same ratio as the singly charged ions suggests that this peak represents a minor component rather than a fragment ion. Degradation of insulin in long-term insulin preparations bas been of concern for some time. The degradation processes include aggregation, reduction of disulfide bonds in the presence of thiols, and dimerization (56). Ironically, as we have noted, the use of the thiol glutathione as a matrix for PDMS improves the molecular ion yield for insulins and other peptides. Similarly, the mixture dithierythritol/dithiothreito1 bas been employed as a highly successful matrix for FAB mass spectro-
metric analysis of peptides (57), and Unger et al. (58)have carried out deliberate disulfide reductions to the A and B chain of modified and degraded insulins directly in the dithiothreitol FAB matrix. Chait and Field (48) have carried out cleavage reactions of the disulfide bonds in bovine insulin, cyclic somatostatin, and conotoxin G1 by the addition of dithiothreitol directly on the sample foil. From these initial studies they have suggested a general approach for structural elucidation that employs enzymatic and chemical reactions directly on the foil. This suggestion has been followed by Craig e t al. (59),who obtained the plasma desorption mass spectrum of intact cecropin B, an antibacterial protein from the Chinese oak silk moth Antherea pernyi and determined the carboxy terminal amino acid sequence by Staphylococcus aureus V8 protease digestion directly on the sample foil. The Rockefeller group has carried out a number of other enzymatic reactions directly on the sample foil (60). Figure 6 shows the maw spectrum of unreacted bradykinin adsorbed to nitrocellulose (Figure 6a) and the mass spectrum of the same sample after incubation for 5 min with carhoxypeptidase Y (Figure 6b). Similar reactions were carried out using carboxypeptidase B, which removed the C-terminal arginine of 10-12 moles of bradykinin, absorbed on nitrocellulose and dipped for 12 min in a solution of the enzyme (60).Similarly, porcine proinsulin was bound to a nitrocellulhse-coated foil and its spectrum obtained; the peptide adsorbed to the foil was then incubated with trypsin, and its spectra revealed several structural fragments (60).
\RGPROPROOLY-PHE-SERPRHE-ARG 5
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Flgure 6. Plasma desorption mass spectrum of bradykinin (a) a carboxypeptidase Y. (Adapted with permission horn Re1-n.a BO.) ANALYTICAL CHEMISTRY, VOL.
BO, NO. 13,
JULY 1. 1988
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As we have noted, reductions of insulin have not been observed during the plasma desorption analysis of insulins in the presence of the glutathione matrix (42). It has been suggested that such reactions take place via the formation of an insulin-glutathione-mixed disulfide intermediate hut require an enzyme to .catalyze the reaction (61, 62). The mixed disulfide, however, was not previously isolated. Recently Alai and Fenselau (63) hound bovine insulin to a glutathione agarose column eluted with glutathione-ammonium acetate and obtained plasma desorption mass spectra that show the insulin-glutathione-mixed disulfide (both singly and doubly charged ions) and an increase in the amount of B chain along with its mixed disulfide. Another degradation reaction of insulin solutions is the formation of dimers, which is attributable mainly to the reaction between an N-termina amino group in one insulin molecule with a carhoxamide group of a glutamine or an asparagine residue in the A chain of another insulin molecule (56). For porcine insulin (MW 57771, the covalent dimer formed by such an amide linkage would have a molecular weight of 11,536. Plasma desorption mass spectra obtained by Unger et al. (58) have confirmed the formation of two isomeric covalent insulin dimers. The importance of mass spectrometry in biotechnology has been outlined by Richter et al. (5),who used a variety of techniques to elucidate the posttranslational modification of recombinant eglin c expressed in E. coli, which had the same biological and immunological behavior but a lower PI value than the native peptide. At Upjohn (Kalamazoo), Lohl and co-worker8 (64) have been involved in the total chemical synthesis of human interleukin-1 (IL-11.a 153 aminoacid (17.5 kDa) protein that has also been produced by recombinant techniques. Figure 7a shows the plasma desorption mass spectrum of the synthetic IL-170 residue with a calculated average mass of 8086.2 amu that was used to verify the sequence. Jardine et al. (65) used the PDMS technique to confirm the molecular weight (Figure 7b) of intact recombinant interleukin-2 (IL-2) obtained from Hoffman-LaRoche (Nutley, NJ). Additional structural confirmation was obtained from the “PD map” of the peptide fragments following cyanogen bromide cleavage of IL-2. Dithiothreito1 reduction directly on the PDMS foil generated signals corresponding to all five CNBr fragments anticipated from the DNA-derived sequence. The Rockefeller group has made several other outstanding contributions using the PDMS instrument developed in their laboratory. In collaboration with Fairlamb (66),they confirmed the 780A
*
._ a m c
e! .-c
.3 ib m
Flgure 7. Plasma desorption mass spectra of (a)the 70 residue of chemically synthesized human interieukin-1 and fb) . , recombinant interleukin-2. (Adapted wnh permission lrom Reference 65.)
molecular weight for a cofactor for glutathione reductase, which is found in trypanosomes and leishmanias and appears to be required for activity of the enzyme. Isolated from the insect trypanosomatid Crithidia fasciculata, the cofactor has been identified as a novel his(glutathiony1) spermidine conjugate (66). With Garcia-Bustos and Tomasz (63, this gronp obtained PDMS measurements for the peptide moieties in the peptidoglycan cell wall components of the gram-positive bacterium Streptococcus pneumonae. PDMS was also employed by Burman e t al. (68)for the partial assignment of disulfide pairs in the hormone-binding protein neurophysin. Mass spectra were obtained from subtilisin-digested tryptic producta of bovine neurophysin containing cysteines.
ANALYTICAL CHEMISTRY, VOL. 60, NO. 13. JULY 1, 1988
ol~co~tpids, ~ho~pholipids, and whole Celk
The capability for high mass measurements of peptide molecular weights has in some way obscured the real utility of this technique for the mass spectral analysis of other samples. In our laboratory it is often the method of choice for fmt analysis of samples available in low quantity, whereas higher resolution measurements on a sector instrument are carried out when needed for additional structural information. Takayama et al. (69) have used a variety of mass spectral, NMR, and chemical techniques to elucidate the structnres of lipid A obtained from the lipopolysaccharides (LPS) found on the outer wall of the outer membranes of gram-negative bacteria. Lipid A con-
sists of phosphorylated diglucosamines to which from four to seven fatty acids are attached by acyl or amide linkages and forms the anchor to the membrane for the intact LPS. Generally the stoichiometric quantities of phosphate, glucosamine,and the different fatty acids can he established hy analysis of hydrolized lipid A, but information about the positions of attachment are best determined by analysis of the fragmentation pattern from the intact molecule. In addition, these compounds often exhibit considerable heterogeneity. The plasma desorption mass spectra generally show cleavages of the acyllinked fatty acids on the 3- and 3'-positions and formation of an oxonium ion containing only the distal sugar and its fatty acid constituents. The intact LPS, containing lipid A-a core region composed (primarily) of several 2-ketooctanoate (KDO) moieties-and an antigenic portion containing neutral sugars, are a far greater challenge to MS (Figure 8). However, the intact LPS from the rough (Re) mutant of E. coli has a somewhat simpler structure. It has recently been isolated and purified by the Takayama group (70). The plasma desorption mass spectrum in Figure 9a was used to confirm the addition of two KDO units to the known structure of the lipid A portion. At the Mayo Clinic, Jardine et al. (72) have used PDMS for the structural analysis of lipooligosaccharide (LOS) antigens from Mycobacterium kansasii. FAB mass spectra had been obtained previously for the oligosaccharide portion of these trehalose-containingl i p liiosaccharides, and PDMS spectra of intact LOS revealed the attachment of groups. three 2,4-dimethyltetradec~noyl This group also used PDMS to analyze polysulfatedamino glycans (72)and other more complex bacterial antigen oligosaccharides, lipooligosaccharides,glycoproteolipids, and larger heparin fragmenta (73).Figure 9h shows the mass spectrum of a phenolicglycolipid from M. lepme. Townsend et al. (74) have carried out plasma desorption analyses of proteolytic glycopeptides from bovine fetuin to elucidate glycosylation site microheterogeneity. Whole cells from hackria and intact membranes from rat brain myelin have also been deposited on aluminized mylar foils and analyzed with PDMS by Heller et al. (75). The specific biomarkers phosphatidyl choline, phosphatidyl ethanolamine, cerebrosides, and sulfatides are observed depending upon species and the charge sign of the phospholipids when analyzed by positive and negative PDMS. Summary The future for PDMS, and for MS in general, is exciting. It is clear that MS will play an important role in biotech-
oapecifc chain
I
Lipid A
7
Figure 8. General structure of the R. to R. chemotype lipopolysaccharide structure from the rough mutants of gram-negative bacterla. (S = sugar; Glc = glucose: Gal = galactose: GIcNAc = Kaoetylplucosamine;P = phosphate: EtN = ethanolamine; KW = 2Xetooctonate moieties; R, and R2 are polar head groups.)
nology, and the PDMS technique has already proven to he a sensitive and accurate method for rapid (indeed, routine) comparisons among chemically synthesized, recombinant, and native peptides. A few years ago, mass spectroscopists would have been surprised a t the possibilities for making such large ions, but we have since appreciated that the ruggedness of these peptides, as they are transferred from the condensed to the gaseous phase, could not be extrapolated from o w prior experience with molecules with less
defined tertiary structure. At the same time, the remarkable stability of such ions poses a problem in obtaining the usual structural information from ionic fragments. In this regard, attempts to induce fragmentation in high-mass, high-performance (and high-cost) four-sector instruments will be paralleled by the use of chemical and highly specific enzymatic cleavages in situ (i.e., on the PDMS foil or in the FAB matrix), where structural information will he obtained by molecular ion measurements of the reaction products.
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 13. JULY 1, 1988 * 7 9 1 A
MW 2987 (c34.24)
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In addition to cleavages, other enzymatic reactions carried out directly on the foil can be expected, provided that the reaction can he monitored by the measurement of a change in mass. The observation of covalent dimers suggests the possibility for observing noncovalently bound complexes as well; in our laboratory, noncovalent dimers formed in the presence of divalent cations have been observed with correct stoichiometry (76).The use of the peptide-binding surface, nitrocellulose, suggests the possibility for using more specific (e.g., antibody) binding surfaces mmplexed with substrate. After exposing a surface to a solution containing a second substrate, one might measure the binding by the relative intensities of the two substrates desorbed from the surface, as a kind of immunoassay technique. The lower mass resolution of the PDMS technique, when compared with other mass spectrometric methods, has been of concern to some, although it has been noted that resolution of the rather broad isotopic molecular ion clusters from heavy molecules does not necessarily improve the information content of a mass spectrum (773. We note also that the remarkable resolutions (l/l,OOO,OOO) and mass ranges (25,000) cited in the Pimentel diagram (Figure 2) were not achieved with the same instrument, but rather by FT-MS and PDMS, respectively. At the same time, the mass range of the FT-MS technique has been improved (78) with some sacrifice in resolution. Array detectors for double-focusing instruments (79) have improved the ability to record ions simultaneously over a narrow mass range (sensitivity), but with some sacrifice in mass resolution and cost. TOF measurements have been made for bovine insulin that resolve the molecular ion cluster (80) with some sacrifice in sensitivity and cost. Currently, however, it is difficult to beat the high sensitivity a t high mass capability of the PDMS technique. The cost and simplicity of operation of this instrument will make it a popular tool for quality control in biotechnology firms as well as an important research tool for biochemists.
24b
Figure 9. Examples of PDMS of glycolipids and phospholipids. (a) Plasma deswptlon mass spenrum of the hexamelhy derivative of me llpOp01ygac~rldeobtain= from the Re mutant of E. mil, showlng t h molecular ion (2370 amu). 10sof dimethyl phosphate lrom C, (2261). and bss of me acyl-linked fatty add at Cs(2034). (b) Piasma dewption mass spechum of a phenolkglycolipidObtained from M. leprae (R = Cs0. Caz, M CW fatty acid group). (Adapted with permisslm born Reference73.)
792A
ANALYTiCAL CHEMISTRY, VOL. 60. NO. 13, JULY 1. 1988
&5;azg.
G.
in Chemistry; Pimentel, E.,ortunities Ed.; National Academy Press:
Washington, DC, 1985, pp. 56-68. (5) Raschdorf, F.; Dahinden, R.; Domon,
B.; Muller, D.; Richter, W. J. In Moss Spectrometry in the Analysis of Large Molecules; McNeal, C. J., Ed.; John Wiley: Chichester, England, 1986, pp. 49-65. (6) Sundqvist, R.; Macfadane, R. D. Mass Spectrom. Re". 1985,4,421. (7) Macfarlane, R. D.; Torgerson, D. F. Science 1976,191,920. (8) Macfarlane, R. D.; Torgerson, D. F.; Fares, Y.; Hussel, C. A. Nuel. Instrum. Methods 1974,116,381. (9) Macfarlane, R. D.; Uemura, D.; Ueda, K.; Hirata, Y. J. Am. Chem. Soc. 1980, I"*
O I C
l " I , 0 , d.
(10) McNeal,
C. J.; Ogilvie, K. K.; Ther-
Thurston, E.L. Anal. Chem. 1979, 51, 2036. (37) Macfarlane. R. D.: Toreerson. D. F. Phys. Re". Lett. 1976,'36,4&. (38) Cooks, R. G.;Buseh, K. L. Int. J. Mass Spectrom.1onPhys. 1983,53,111. (39) Macfarlane. R.G. Ace. Chem. Res. 1982,15,268. (401 Jordan, E. A.; Macfarlane, R. D.; Mar-
tin, c. R.; McNeal, c. J. Int. J. Mass Spectrom. Ion Phys. 1983.53,345. (41) Jonsson, G.; Hedin, A,; Hakansson, P.; Sundqvist, B.U.R.; Save, G.; Nielsen, P. F.; Roepstorff, P.; Johansson, K. E.; Kamensky, I.; Lindberg, M. Anal. Chem. 1986.58.1084.
iault, N. Y.;Nemer, M. J. J. Am. Chem. SOC.1982,104,981. (11) McNeal, C. J.; Macfarlane, R. D. J. Am. Chem. Soc. 1981,103,1609. (12) Chait, B.T.; Field, F. H. Int. J. Mass Spectrom. Ion Phys. 1981.41,17. (13) Chait, B. T.; Field, F. H. J. Am. Chem. SOC.1984,106,193. (14) Demirev, P.; Olthoff, J. K.; Fenselau, C.; Cotter, R. J. Anal. Chem. 1987, 59,
fin imfi .., .
(66) Fairlamb, A. H.;Blackburn, P.; Which, P.: Chait. B. T.: Cerami. A. Science 1985. 227,1485.
(67) Garcia-Bush. J. F.; Chait, B. T.; Tomasz, A. J. Biol. Chem. 1987,262,1540. (68) Burman, S.; Breslow, E.; Chait, B. T.; Chsudhaw. T. Bioehem. Biophvs. Res. . . Commun.~l987,I48.827. (69) Qureshi. N.; Cotter, R. J.; Takayama,
K . J . Mzrrokol. Methods 1986,5.65.
(701 Cotter. H.J.: Hunovich. J.:Qureshi. N.:
Takayama, K. Biomed. Enur'ron. Mass Spectrom. 1987,14,591. (71) Jardine, I.; Hunter, S. W.;Brennan, P. J.; MeNeal, C. J.; Macfarlane, R.D. B$p:-d. Enuiron. Moss Speetrom. 1986,
1951. (15) King, B.; Ziv, A.; Ling, S.; Tsong, I. J. Chem. Phys. 1985,82,3641.
(16) Chait, B.T. Int. J. Mass Speetrom.
Ion Processes 1987,78,237. 0.;Furstenau, N.; Krueger, F. R.; Weiss, G.; Wien, K. Nuel. Instrum. Methods 1976,139,195. (18) LeBeyec, Y.; Della Negra, S.; Deprun, C.; Vigny, P.; Gmot, Y.M. Reu. Phys. Appl. 1980,15,1631. (19) Della Negra, S.; Deprun, C.; Jscquet, D.; LeBeyec, T. Zeitschrift fiir Fysik A, 19.42. . . ,107 ...,-Rns (20) Duck, P.; Treu, W.; Frohlich, H.; Galster, W.; Voit, H. Surf. Sci. 1980, %, 603. (21) Seiberling, E.; Griffith, J. E.; Tom. brello, T. A. Radiat. Eff. 1980.52,201. (22) Jungclas, H.; Danigel, H.; Schmidt, L. Org. Mass Spectrom. 1982.17.86, (23) Hakansson, P.; Sundqvist, B. Radiot. Eff. 1982,61,179. (24) Della Negra.S.;Ginot,Y. M.; LeBeyec, Y.; Spiro, M.; Vigny, P. Nuel. Instrum. Methods 1982,198,159. (25) Jungclas, H.; Danigel, H.; Schmidt, L. J. Chromatogr. 1983,271,35. (26) Danigel, H.; Schmidt, L.; Jungclas, H.; Pfluger, K.-H. Biomed. Mass Speetrom. (17) Becker,
.
1985,12,542. (27) Hakansson, P.; Kamensky, B.; Sund-
qvist, B.; Fohlman, J.; Peterson, P.; McNeal, C. J.; Macfarlane, R. D. J. Am. Chem. Sac. 1982,104,2948. (28) Hakansson, P.; Kamensky, I.; Kjellberg, J.; Sundqvist, B.; Fohlman, J.; Peterson, P. Biochem. Biophys. Res. Commun. 1983,110,519. (29) Fales, H. M.; Pannell, L. K.; Sokoloski, E.A.; Carmeci, P. Anal. Chem. 1985,57,
".
R7G I.
(30)Yang, Y. M.; Lloyd, H.A.; Pannell, L. K.; Fales, H. M.; Macfarlane, R. D.; McNeal. C. J.: Ito. Y. Biomed. Enuiron. Mass Spectrom. 1986,13,439. (31) Yang, Y.M.; Sokoloski, E. A.; Fales, H. M; Pannell, L. K. Biomed. Enuiron. Mass Speetrom. 1986.13.489, (32) Visri, A.; Ballini, J.-P.; Vigny, P.; Shire, D.; Dousset, P. Biomed. Enuiron. Mass Spectrom. 1987,14,83. (33) Hunt, J. E.; Salehpour, M.; Kanter, E.; Kutschera, W.; Zabransky, B.; Pardo, R.; Dunford, R. In Proceedings of the 35th ASMS Conference on Mass Spectrometry and Allied Topics: Denver, Colo., 1987, pp. 275-76, (34) Tabet. J.-C.; Rapin, J.; Poretti, M.; Gaumann, T. Chirnio 1986,40,169. (35) Roepstorff, P. Eur. Spectrosc. News ,OQ, .""I,
71 1 Q I",
I".
(36) McNeal, C. J.; Macfarlane, R. D.;
Biophys.'Res. Cammu". 1986,134,420. S.; Turnell, W. G.;Blundell, T.L.; Schwabe, C. Nature 1977, 270,
(49) Bedarkar,
5636. (50) Roepstorff, P.; Hojrup, P.; Sundqvist,
B.U.R.; Jonsson, G.; Hakansson, P.; Andersen, S. 0.; Johansson, K. E. Biomed. Enuiron. Mass Spectrom. 1986.13,689. (51) Andrews, P.; Alai, M.; Cotter, R. J., submitted for publication in J. Bid.
..
Cham ~. .
(52) Roepstorff, P.; Nielsen, P. F.; Ka-
mensky, 1.; Craig, A. G.; Self, R. Biomed. Enoiron. Moss Speetrom.. in mess. (53) Bogard, W. C.; Jr.; Kato, I.;Laskowski, M., Jr. J. Bid. Chem. 1980,255,6569. (54) Laskowski,M., Jr.;Kato,I.;Ardelt, W.; Cook,J.;Denton,A.;Empie,M. W.;Kohr, W. J.; Park, S. J.; Parks, K.; Schatzley, B. L.; Sehoenberger, 0.L.; Tashiro, M.; Vichot, G.;Whatley, H. E.; Wieczorek, A.; Wieczorek, M. Biochemistry 1987, 26, 7n7-71 ._. (55) Papamakos, E.; Weber, E.; Bode, W.;
Huber, R.; Empie, M. W.; Kato, I.; Laskowski, M., Jr. J. Mol. B i d . 1982. 158,
515. (56) Brange, J.; Langkaer, L.; Havelund, S.;
Sorensen. E. Presented at the 20th Annual Meeting of the European Association for the Study of Diabetes, London, September 1984. (57) Whitten, J. L.; Schaffer, M. H.; O'Shea, M.; Cook, J. C.; Hemling, M. E.; Rinehart, K. L., Jr. Biochem. Biophys. Res. Commun. 1984,124,350. (58) Unger, S. E.; Brange, J.; Lauritano, A,; Demirev, P.; Wang, R.; Cotter, R. J. Rapid Commun. Mass Speetrom, in press. (59) Craig, A. G.;Engstrom, A.; Bennich, H.; Kamensky, I. Biomed. Enuiron. Mass Spectrom. 1987,14,669. (60) Chait, B. T.; Chaudhary, T.; Field, F. H. In Methods in Protein Sequence Analysis; Walsh, K. A,, Ed.; Humana Press: Clifton, NJ, 1987, pp. 483-92. (61) Morin, J. E.; Dixon, J. E. Methods Enzymol. 1985,113,541. (62) Varnadani,P. R.; Nafz,M. A. Diabetes 1976,25,173. (63) Alai, M.; Fenselau, C. Bioehem. Biophys. Res. Commun. 1987,146,815. (64) Lobl, T. J.; Deibel, M. R.; Yen, A. W.
Anal. Biochem., in press.
(65) Jardine, 1.; Scanlan, G. F.; Tsarbopoulous, A.; Liberato, D. J. Anal. Chem. 1988.
Robert J. Cotter is associate professor of pharmacology and molecular sciences at T h e Johns Hopkins Uniuersit y and director of the Middle Atlantic Mass Spectrometry Facility. His interest in TOF-MS dates back to 1970, when he used an instrument equipped with a photoionization source. Recentl y he has been inuolued in the design, construction, and application of a laser desorption TOF instrument and a secondary ion mass spectrometer employing a liquid matrix, using timedelayed ion extraction t o enhance the obseruation of fragment ions. His interests are the deuelopment of instrumentation and the interaction of ionization techniques with peptides, glycopeptides, and glycolipids.
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