Structural analysis of proteins by laser desorption and electrospray mass spectrometry

Structural analysis of proteins by laser desorption and electrospray mass spectrometry Terry D. Lee and John E. Shively Beckman Research Institute of the City of Hope, California, USA The development of electrospray and matrix-assisted laser desorption mass spectrometry has provided protein chemists with tools for peptide and protein structure analysis with unprecedented sensitivity and molecular weight range. The two technologies can be viewed as competitive with respect to their molecular mass determinations, but complementary with respect to their differences in instrumentation, sample preparation methods, and nature of spectra produced. Current Opinion in Biotechnology 1991, 2:52-60 Introduction The determination of protein primary sequences remains one of the most pervasive of first objectives in the majority of biological problems. Whether one is studying a biological activity or a biophysical parameter involving a biological system, the chances are high that a protein of some sort is responsible, directly or indirectly (at least by biosynthesis or association), for the activity studied. Two completely different, but complementary, approaches are available for the determination of protein primary sequence information; direct protein sequencing, or translation from the corresponding cDNA or genomic clone. Although preferences vary, many laboratories use a combination of both approaches, as the availability of even fragmentary direct protein sequence information can verify protein sequences deduced from corresponding cDNAs. Moreover, sequences deduced from the DNA contain little more than clues about the location and nature of post-translational modifications in proteins. For example, the absence of information about post-translational proteolytic processing will lead to the deduction of the wrong protein sequence. Lack of information about other post-translational modifications, such as glycosyiation and phosphoryiation, may seriously compromise expression or activity-function studies. Thus, the biologist is acutely aware of the need for structural studies to be performed directly on the protein. Because it is almost axiomatic that the more important the biological problem, the less protein will be available for the study, more highly sensitive methods for protein structural analysis are in great demand. Today, mass spectrometry (MS) offers sensitive and rapid approaches to protein structural analysis, including the

detection of post-translational modifications. However, the field is expanding and diversif3ang to such a great extent that the potential user may be confused about the possibilities it offers in terms of sample amounts and the nature of information obtained from a given method. Additionally, the mass spectrometrist may not be used to dealing with biological samples, thus necessitating an extensive dialogue with the biologist. Fortunately, many mass spectrometrists have become fascinated with biological molecules and have entered into this dialogue with enthusiasm. The purpose of this review is to make the everyday researcher aware of both the scope and limitations of two of the most promising techniques available; laser desorption (LD) time-of-flight (TOF) mass spectrometry, and electrospray (ES) quadrupole mass spectrometry. LD mass spectrometry has been chosen for its unique ability to allow high mass measurements on proteins using very simple sample preparation procedures with amounts in the subpicomole range. Additionally, the technique gives fast results and can be used to analyze protein mixtures even in the presence of common buffers and other molecules such as lipids and DNA. ES mass spectrometry will be considered because of its convenient interface (at atmospheric pressure) with popular liquid chromatography (LC) and capillary electrophoresis (CE) separation techniques, and its ability to perform complete structural analyses on peptides using the tandem MS approach. ES mass spectrometry is also able to give highly accurate mass measurements for proteins with molecular masses of up to 30 000 to 50 000, and fiequently even higher. Although this review will not discuss other ion sources such as fast atom bombardment (FAB) and plasma desorption (PD), or other analyzers such as tandem mag-

Abbreviations CAD---collision-activated decomposition; CE-capillary electrophoresis; ES clectrospray;FAB~fast atom bombardment; FTICR--Fourier transformer ion cyclotron resonance; FF/MS---Fouriertransformer mass spectrometer; HPLC--high-performance liquid chromatography; LC--liquid chromatography; LD~laser desorption; MS----massspectrometry; PD~plasma desorption; TOF~time-of-flight. 52

© Current Biology Ltd ISSN 0958-1669

Structural analysis of proteins Lee and Shively netic instruments, Fourier transformers or ion traps, these techniques still deserve a mention. However, studies that directly compare the results of using two different methods on identical samples are rare [1.,2..]. This review aims to acquaint the non-expert with two exciting new approaches in this field which, it is hoped, wiU increase the awareness and communication levels between biologists and mass spectrometrists.

Goals in protein structural analysis Molecular mass determination In order to understand how mass spectrometry can be used in protein structural analysis, it is important to review the overall objectives and challenges which face the protein chemist. The first challenge is to purify the protein, determine its molecular mass, and deduce the number of subunits, if any. Currently, SDS gel electrophoresis is the most widely used technique for purity and molecular weight determination of proteins. Advantages of this method include simplicity of operation, high resolution (which can be expanded further by two-dimensional gels), and sensitivity (10-50 ng amounts can be detected with the silver stain). Limitations include lack of quantification and poor mass accuracy. Although it is possible to estimate sample amounts from SDS gels, investigators have often been disappointed to learn in later analyses, such as amino acid and amino-terminal sequence analyses, that their estimates were high by a factor of two to three. Molecular mass estimation is an even more serious problem in cases where the mass calculated for a protein sequence is compared with that predicted by SDS gel electrophoresis. Even in fortunate circumstances, mass accuracies may be in the range of + 5%, which for a protein of mass 50 000 means an error of + 2500. This accuracy is simply not good enough to unravel product-precursor relationships or reveal the presence and nature of post-translational modifications. In the case of glycoproteins, molecular weights estimated by SDS gel electrophoresis are notoriously inaccurate. It is thought that glycoproteins bind less SDS than expected and thus migrate more slowly than predicted. Because the mass estimate depends on the binding of a constant amount of SDS per length of peptide chain, any structural feature which perturbs this relationship will result in anomalous results. There are also well known examples of protein species which differ by only a single amino acid, but are separated by over 1000 mass units on SDS gels.

Molecular mass determination by mass spectrometry

Mass spectrometry represents an obvious choice for solving this problem. ES mass spectrometry can be used to obtain highly accurate mass measurements (0.01%) on samples of only a few picomoles. For a small protein, this mass accuracy would easily confirm a calculated mass for a sequence. Alternatively, it would alert the investiga-

tor either to an error in the sequence, or to the presence of a post-translational modification. For example, the molecular mass of the blue copper protein rustacyanin can be obtained to an accuracy of 16 552 ± 1 using an ES mass spectrometer (Fig. 1). A single mutation, such as Gly to Ala, could be detected easily as it would increase the molecular mass by 14 mass units. Similarly, the presence of a phosphate would increase the mass by 80 mass units. The phosphate group could be verified by treating the sample with alkaline phosphatase and repeating the mass measurement. Similar experiments can be designed to determine the presence and nature of glycosyl units on glycopeptides where a modest amount of microheterogeneity can be handled by the analytical system. In the case of proteins in the mass range 30-50000 (which is a reasonable upper limit for routine analyses on ES quadrupole mass spectrometers at this time), a mass accuracy of 0.01% is still sufticient to alert the investigator to errors in primary structure determination or to the presence of post-translational modifications. The work of Dorsselaer et al. [3 o] is a good illustration of the usefulness of mass spectral analysis in characterizing recombinant proteins. Ideally, this technique could also be used to quantify the amount of desired protein present as well as any impurities. Unfortunately, because every protein gives a unique response with ES, it would first be necessary to calibrate the signal response for a given protein with known amounts. Once this was done, it should be possible to rapidly quantify minor impurities in a variety of protein samples. LD/TOF mass spectrometry is an exciting-alternative to ES mass spectrometry because of its greater sensitivity and higher mass range. Proteins such as ~-galactosidase (116000), immunoglobulin G (150000), and catalase (236 000) have been analyzed using this technique. However, compared with an ES mass analysis, the mass accuracy is generally less by a range of approximately + 0.05%. Nevertheless, LD mass spectrometry is 20-100 times more accurate than SDS gel electrophoresis. Perhaps the most attractive feature of LD mass spectrometry is that it allows proteins to be analyzed directly from complex mixtures such as serum or milk [4"']. This lack of interference from common biological contaminants is a unique feature which should make LD mass spectrometry one of the most powerful tools available to the protein chemist.

Protein structural analysis Once purified, partial or complete structural analysis of a protein is required. This process usually begins with an amino-terminal sequence determination by Edman chemistry. However, this analysis is frequently hampered by the presence of amino-temainal blocking groups which, themselves, are important post-translational modifications of proteins. When successful, even the best Edman analysis cannot elucidate the structure of intact proteins with masses above 7500. The next step, therefore, is to break the sample down into smaller frag-

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Fig. 1. Electrospray mass spectrum of the blue copper protein rusticyanin. The numbers in parentheses indicate the number of charges for that ion. The spectrum was obtained using a Finnigan TSQ700 mass spectrometer. The inset figure shows the computer deconvolution of the spectrum to yield the molecular weight.

ments. This is usually achieved either by proteolytic digestion (using, for example, trypsin or chymotrypsin), or chemical digestion (by, for example, cyanogen bromide). The small fragments produced are then analyzed by Edman chemistry and, after the analysis of several overlapping peptide maps, the structure can be reassembled. Problems with this method include incomplete analysis of some of the peptides by Edman chemistry, loss of hydrophobic peptides (either during or after chromatography), and the large amount of time required to complete the analysis. Even a modest protein of molecular mass 50000 may require the analysis of over 100 peptides to determine its complete structure and, as each peptide may require one day per analysis, it is not unusual to devote 4-12 months to the analysis of a single protein. In addition, many post-translational modifications may be missed, or may demand additional time-consuming analyses.

Protein structural analysis by tandem mass spectrometry It has already been noted that peptide mass measurements are easily accomplished by mass spectrometry. This information alone can be of paramount importance in confirming peptide structures obtained by Edman chemistry. Additionally, because results can be obtained so rapidly (1-5min per peptide), several protein structure projects can be carried out at any one time. For instruments with multiple analyzers (tandem mass spectrometers), considerably more information than molecular weights can be obtained. With ES on a triple-sector quadrupole mass spectrometer, it is possible to select one molecular ion species from a mixture, fragment it by collision-activated decomposition (CAD), and then record the fragment ion spectrum. The analysis of the fragment ion spectrum is often enough to determine the complete sequence of a peptide, including the location and nature of post-translational modifications. The sen-

sitivity of the analysis is in the low picomole range and only a few minutes are required to achieve a spectrum. ES is not unique in its capacity to analyze peptides in this fashion. Indeed, most such analyses are carried out on either tandem magnetic sector or triple sector quadmpole systems using either FAB or liquid secondary ion mass spectrometry sources. The most common method for performing peptide mapping is on reversed-phase high-performance liquid chromatography (HPLC) columns. Because ES is easily coupled to HPLC, complete structures on the majority of peptides analyzed during a single HPLC run (30-90 minutes) may be obtained in this way. In practice, two HPLC runs are required; the first to identify molecular ions, and the second to perform CAD tandem MS on selected molecular ions. This technique promises to revolutionize the way in which protein chemists perform structural analyses on proteins. In theory, one could visualize the complete structural analysis of a protein in as little as several days to a week. In practice, this goal has not yet been realized became the technique is so new. Researchers are still exploring the limits of the technique and are engaged in spirited debates about the merits of a variety of ES ion sources and their advantages and disadvantages compared with other ionization methods. The remainder of this review describes the instrumentation and methodology of both of these techniques, with references to specific applications.

Electrospray mass spectrometry Instrumentation A full description of ES mass spectrometry is beyond the scope of this review and so only a very brief one will be given here. The reader is referred to recent reports by Fenn et al. [5"] and Smith et aL [6.] for more detailed

Structural analysis of proteins

tor and pass through the second sector gas collision cell, where CAD causes the ions to fragment. The spectrum of the fragment ions is then obtained by scanning the third quadrupole sector. For peptides, analysis of the fragment ions yields sequence information. Tandem mass spectral analysis using ES has been shown to be an effective method for sequencing peptides from proteins that have been digested with trypsin [7.,8o]. Because tryptic peptides have a basic moiety at the amino-terminus and a basic side chain on the carboxyl-terminal residue, they generally give strong doubly-charged ions. When subjected to CAD, these ions readily fragment into two singlycharged pieces with cleavage occurring primarily at the peptide bonds. The resulting spectra can be interpreted easily and usually yield complete sequences. Non-tryptic peptides yield spectra that are more difficult to interpret but st~ contain a great deal of useful sequence information. Smith and co-workers [9",10"] have described the CAD of multiply-charged ions for intact proteins. The observed spectra can be interpreted if the sequence of the protein is known and it may be possible to use them as fingerprints for identifying known protein structures. A single sector quadrupole instrument equipped with an ES source is sufficient for molecular weight analyses. Such systems can be obtained commercially for as little as $100 000 and would be a powerful analytical tool in a protein chemistry laboratory. Using only a single sector does not preclude obtaining fragment ion information on peptides. If ions are accelerated through the relatively high pressure near the opening between the desolvation region of the source and the high vacuum region of the analyzer, CAD can be done prior to mass analysis [10.,11.]. The resulting spectrum is sufficient to sequence the peptide. This approach would be suitable only for purified peptides. A single quad system would be incapable of sequencing one peptide in a mixture, without prior separation.

accounts of the history and theory behind the methodology. In a mass spectrometer equipped with an ES source, the sample solution is pumped through a small needle held at high voltage resulting in a fine spray of charged solvent droplets (Fig. 2). Collisions with the surrounding gas at atmospheric pressure cause the droplets to evaporate. Sample ions associated with varying amounts of solvent molecules are pulled into the vacuum environment of the mass spectrometer generally via two stages of pumping. Useful spectra are obtained only if sample ions are fully desolvated prior to mass analysis. This can be accomplished in different ways and, presently, there is no evidence to indicate that one source design is fundamentally superior to another. The key feature of ES that has revolutionized the analysis of large biomolecules is the phenomenon of multiple charging. This is illustrated in the spectrum of the blue copper protein rusticyanin (Fig. 1). In this example, the experimental mass is 16551.9 and the calculated average mass is 16552.0. The ions extending from the mass-to-charge ratio of 500-1500 are all derived from the intact protein with differing numbers of charges. Each one is an independent measure of the molecular weight of the protein and, when taken together, they provide a very accurate deterruination of the mass of the molecule. Thus, spectra for proton-sized molecules (5 to greater than 100 kD) can be obtained using mass spectrometers with an upper limit of mass-to-charge ratios in the range 1500-2000. Spectra having more than one component can be rather complex and difficult to analyze. Fortunately, computer algorithms have been developed that simplify data analysis. Such an algorithm was used to reduce the ES spectrum of rusticyanin to a single peak (inset of Fig. 1). Clearly, computer programs are essential for the efficient interpretation of ES mass spectral data. Triple-sector quadrupole h

tandem mass spectrometry High-performance liquid chromatography - electrospray mass spectrometry

An ES ion source is generally coupled to a quadrupole mass analyzer. The triple-sector quadrupole arrangement (Fig. 2) permits tandem mass spectral analysis. Ions of a given mass can be selected by the first quadrupole sec-

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piing to HPLC and capillary electrophoresis is readily accomplished and has been described in a number of papers [8°,12o,13 o, 14]. Of particular significance is the work of Steven Carr and colleagues [2 oo] who have compared on-line LC/MS analysis of peptides using ES and continuous flow FAB. It was found that a greater percentage of peptides could be detected with ES. This was particularly true for glycopeptides in that the carbohydrate heterogeneity of the peptide is clearly evident in the spectrum.

Scope and limitations ES mass spectrometry suffers from some of the same factors that plague other mass spectral methods: contamination by salts and other strong electrolytes can greatly reduce the sensitivity of the system or eliminate the signal entirely; the relative abundance of ions for different components in a mixture does not correlate directly with the relative amount of each component in the sample, and many compounds fail to yield any data at all despite efforts to purify the sample. On the plus side, sensitivities are very good, generally in the low pmole range for molecular mass determinations, and many analyses can be carried out on samples in the fmole range. Additionally, the accuracy of mass determination is excellent (usually to within 0.01% or better).

Fi& 3. Schematic

diagram of a laser desorption time-of-flight mass spectrometer. (See text for explanation).

Electrospray on other mass analyzers Although ES ion sources are ideally suited to quadrupole instruments, there are potential advantages to be gained from using other analyzers. In particular, quadrupoles are somewhat limited with respect to resolution. If an analyzer was capable of resolving the isotopes for a given multiply-charged ion, then the charge state, and thus the mass, could be assigned independently of other ions in the spectrum. This would greatly assist in the interpretation of CAD spectra obtained from multiply-charged ions where the charge states of the fragment ions are often unknown. In an attempt to achieve this, ES sources have been mated successfully to magnetic sector [15o,16 °] and Fourier transformer ion cyclotron resonance (FTICR) mass spectrometers [ 17°] A disadvantage is that magnetic sector and FTICR systems are far more expensive than quadrupole systems, with prices ranging from $500 000 to $1 500 000 for systems with tandem MS capability. Additionally, numerous technical problems exist that must be overcome before ES confers the same advantages to these systems as it does to quadrupole mass spectrometers. ES spectra have also been obtained using an ion trap mass spectrometer [18°]. This system has the potential to provide excellent tandem MS capability and greater sensitivity, and is far cheaper than a triple quadrupole. Unfortunately, mass accuracy is presently an order of magnitude less than for quadrupole analyzers. A further disadvantage is that ion trap systems with ES sources are not available commercially.

Commercial availability Prime concems of any new technique are the cost and availability of the instrumentation needed to perform the analysis. Fortunately, a number of commercial vendors market ES sources on quadrupole mass spectrometers. Prices range from $100 000 for a single-sector quadrupole with a dedicated ES source, to $300000-$400000 for a triple-sector quadrupole system. Because of the large number of existing quadrupole mass spectrometers, several vendors have found it profitable to supply ES sources for older instruments, thus allowing many investigators to take advantage of the new ES technology.

Matrix-assisted laser desorption mass spectrometry Instrumentation At present, LD analysis of proteins is only carried out on TOF mass spectrometers. The nsec time scale of the ionization event precludes its use with mass spectrometers that scan the mass range to record a spectrum. Additionally, observed ions have only a few charges which, for proteins, places the mass-to-charge ratio beyond the lhn-

Structural analysis of proteins its of most other mass analyzers. Two recent reviews by Cotter [19"] and HiUenkamp [20"] provide more complete descriptions of TOF mass spectrometers and LD analyses. The instrumentation is conceptually very simple (Fig. 3). The sample is introduced into the instrument in an appropriate matrix on the sample probe. A 5-10 nsec pulse from an ultraviolet laser strikes the sample. Ions are literally blasted into the gas phase and accelerated because of the potential difference between the sample stage and the acceleration grid. The time required for ions of a given charge to travel the length of the drift tube is a function of their mass. Because a large proportion of the ions formed with each laser pulse are collected at the detector, TOF analyzers are inherently more sensitive than scanning mass spectrometers. The mass range is limited by the size of ions that can be formed in the source as well as the ion detection efficiency. Ion detection efficiencies steadily decrease for larger slower moving ions. The actual ionization event is poorly understood. The sample matrix is absolutely essential and must have a chromophore that absorbs at the laser wavelength. Most results have been obtained using a nicotinic acid matrix and a frequency-quadrupled Nd-YAG laser tuned at 267nm [21,22",23"] or a sinapinic acid matrix and a frequency-tripled Nd-YAG laser ttmed at 355 nm [24,']. Beavis and Chait have surveyed a large number of matrix compounds [25",26-] and have concluded that sinapinic acid is the best choice because of its ability to yield good spectra for a wide variety of protein samples [27"]. Using a simple sample-washing technique, they have succeeded in obtaining spectra of complex samples, such as milk, in which the major protein components are clearly evident [4°.]. This technique also works very well for samples that are contaminated with salts or denaturing reagents such as guanidinium hydrochloride.

Lee and Shively

Intact multisubunit proteins Using ES there would seem to be no way to obtain spectra on intact proteins whose subunits are not covalently linked. This is because the multiple charging phenomenon that occurs with ES has a strong denaturing effect, destroying secondary and higher order interactions through simple charge-repulsion. LD, on the other hand, has the potential to preserve these interactions. Dimer, trimer and higher oligomer clusters are observed for some protein samples, particularly at higher sample concentrations. This implies that non-covalent interactions can survive the desorption event. It remains to be seen if authentic subunit interactions can be distinguished from simple clustering. Certainly this is one of the more interesting aspects of LD mass spectrometry that needs to be explored.

Scope and limitations The accuracy of the molecular mass measurement for moderate sized proteins (of less than 50 kD) is comparable with that obtained using ES on a quadrupole analyzer. For example, the mass of proteinase K (28 904 D) has been determined with an error of 0.04% using myoglobin as an internal standard (Fig. 4). For larger proteins, however, the mass accuracies are far less certain. Further disadvantages include the resolving power of a TOF analyzer using LD which is, at best, only a few hundred. Additionally, although matrix adducts are separated and observed for lower molecular weight samples (of less than 20 kD), these adducts cannot be resolved for samples with higher masses and, indeed, there is no way of knowing which species is responsible for the molecular ion signal. For samples with low masses, the mass scale must be linearly extrapolated from the well characterized ions of

Fig. 4. Laser desorption mass spectrum of proteinase K. Myoglobin was used as an internal standard to calibrate the mass scale. The spectrum was obtained using a Vestec Model 2000 LDMS. Sinipinic acid was used as the sample matrix and irradiated at 355 nm with a NdYAG laser. (Spectrum used with permission from Vestec Corp.)

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Analyticalbiotechnology low molecular weight proteins. Using high mass samples, it must be assumed that high molecular weight protein standards have the same degree of adduct formation as the sample molecule. Larger proteins are more likely to be heterogeneous, particularly those that contain carbohydrate. The broad peaks observed in the LD mass spectrum of, for example, the monoclonal antibody to carcinoembryonic antigen are due, at least in part, to carbohydrate heterogeneity. Despite these problems, matrixassisted LD is the only mass spectral method suitable for very high molecular weight compounds (of greater than 150kD), and the values obtained are far more accurate than those obtained by any other method.

spray Mass Spectrometry to the Characterization o f Rec o m b i n a n t Proteins up to 44 kDa. Biomed Environ Mass Spectrom 1990, 19:692-704. Mass measurement by ES mass spectrometry is used as a rapid preliminary verification o f the identity of various recombinant proteins ranging from 7--44 kD, with an accuracy o f 0.1-0.3%. When used in conjunction with other methods of protein analysis, ES improves not only the speed but also the reliability of protein structure determination. Modifications of these large molecules including the loss of carboxy-terminal amino acids, amino-terminal acetylation, addition of 2-mercaptoethanol to cysteine, and trace formation of a covalent dimer (3%), are easily detected both individually or in mixtures by mass measurement using ES; As an example, in a 15kD recombinant protein preparation, this technique enables the detection of various structurally-related protein contaminants, at levels as low as 1% of the mixture. 4. o•

Commercialavailability Matrix-assisted LD mass spectrometry is practiced in only a few laboratories, partly because commercial instrumentation has only recently become available and is relatively expensive ($300 000 -$400 000). As the market for these instruments expands, the prices should come down. The simplicity of the instmmenta0on and the sample preparation techniques make it accessible to researchers with no formal training in mass spectrometry.

Acknowledgement

BEAVlSRC, Cl-IArr BT: Rapid, Sensitive Analysis o f Protein Mixtures by Mass Spectrometry. Proc Natl Acad Sci USA 1990, 87:6873-6877. This paper describes a useful application o f LD/TOF mass spectrometry for the analysis of protein samples in complex mixtures. Sinapinic acid was used as the matrix. Samples analyzed included a delipidated human high density lip•protein fraction, bovine and human breast milk. Most

of the malorproteincomponentswererevealedin eachcase.Cyanogen bromide digests were also analyzed for human apolipoprotein A1, and the entire structure could be accounted for by virtue of partial cleavage creating overlapping peptides. 5.

FENN JB, MANN M, MENG CK~ WONG SF, WHrrEHOUSE CM: Electrospray Ionization for Mass S p e c t r o m e t r y o f Large Biomolecules. Science 1989, 246:64-71. This is an excellent review of the design and performance of ES ion sources, describing the generation of multiply-charged ions as a unique feature of this technique. Spectra are shown for small molecules, proteins and oligonucleotides. •

6.

The authors thank Steven A Carr of Smith Kline and French Laboratories for data on the CD4 receptor glycopeptides and Vestec Corporation for data on lencine enkephalin and proteinase K.

References and recommended reading Papers of special interest, published within the annual period of review, have been highlighted as: • of interest 0• of special interest 1. .

LOO JA, EDMONDSCG, SMrrH RD, IACEYMP, KEOUGHT: Comparison o f Electrospray Ionization and Plasma Desorption Mass Spectra of Peptides and Proteins. Biomed Environ Mass Spectrom 1990, 19:286-294. This paper describes the comparison of more than 20 peptide and protein samples using ES on a quadrupole analyzer and PD on a TOF analyzer. The mass measurement accuracy of the ES system was 5-10 times greater than that obtained using the PD system. The ES system was generally more sensitive, but highly compound-dependent. Analyses could be carried out much faster using the ES system. 2. •0

HEblHNGME, ROBERTS GD, JOHNSON W, CARR SA: Analysis of Proteins and Glycoproteins at the Picomole Level by On-line Coupling o f Microbore High-performance Liquid Chromatography with F l o w Fast A t o m B o m b a r d m e n t and Electrospray Mass Spectrometry: a Comparative Evaluation. Biomed Environ Mass Spectrom 1990, 19:677-691. This article compares flow FAB LC/MS on a magnetic sector and ES LC/MS on a triple quadrupole instrument using proton digest mixtures. The authors conclude that the ES LC/MS interface is superior in terms of mass range for identified peptides, sensitivity, and lack of interfering substances. 3. •

DORSSELAERAV, BITSCH F, GREEN B, JARVlS S, LEPAGE P, BISCHOFFP,, KOIBE HVJ, RorrscH C: Application of Electro-

SMITH RD, I.oO JA, EDMONDS CG, BARINAGACJ, UDSETH HR: N e w Developments in B i o c h e m i c a l Mass S p e c t r o m e t r y m Electrospray Ionization. Anal (2bern 1990, 62:882-899. A review of the development of the ES ionization source and its use on a triple quadrupole mass spectrometer. The mass accuracy demonstrated for a variety of peptides and proteins is about 0.01%. Protein samples with masses of up to 77 500 are shown. The sensitivity for cytochrome c is as low as 23 fmoles. A tandem MS spectrum is shown for a 39-residue proton dernonstrat~ag mostly the B and Y series ions. Fragment ions are also seen for proteins such as serum albumin, but interpretation is difficult, The CE/ES interface is demonstrated for myoglobin and leuenkephalin. •

7. •

CHOWDHURYSK, KA'rrA V, CHAIT BT: Electrospray Ionization Mass Spectrometric Peptide Mapping: a Rapid, Sensitive Technique for Protein Structure Analysis. Biochem Biophys Res Commun 1990, 167:686--692. The authors describe an ES single quadrupole mass spectrometer and its use in determining molecular weights for tryptic peptides from human apolipoprotein A1. The tryptic digest was analyzed as a mixture without prior HPLC separation. All of the expected fragments were observed with the exception of several mono- and dipeptides. HUANGEC, HENIONJD: LC/MS and LC/MS/MS Determination o f Protein Tryptic Digests. J Am Soc Mass Spectrom 1990, 1:158-165. An ES triple quadrupole mass spectrometer was used to analyze protein tryptic digests on-line. The proteins analyzed included 13-1act•globulin A and B, and cytochrome c. The fragments were detected by total ion currents and mass analyzed to identify the sequence. Tandem MS spectra were performed on several peptides, revealing abundant B and Y series ions which could be used to deduce the peptide sequence. 8.

•

LOO JA, EDMONDSCG, SMITH RD: Primary Sequence Information from Intact Proteins by Electrospray Ionization Mass Spectrometry. Science 1990, 248:201-204. Intact proteins were analyzed in a tandem (triple quadrupole) mass spectrometer equipped with an ES ion source. The MH12+ and MH13 + species for RNase A were selected for collision-induced dissociation revealing a number of characteristic B and Y series ions which enabled structural information about the protein to be obtained. Such 9.

•

Structural

experiments may be used to fingerprint proteins and to obtain limited sequence information. 10. •

SM1THRD, LOO JA, BARINAGACJ, EDMONDSCG, UDSE'H-IHPc Collisional Activation and Collision-Activated Dissociation of Large Multiply Charged Polypeptides and Proteins Produced by l~ectrospray Ionization. J Am Soc Mass Spectrora 1990, 1:53-65. The authors describe CAD spectra for peptides and proteins using an ES source on a triple quadmpole mass spectrometer. Samples may undergo CAD either at the sldmmer nozzel before the first quadrupole, or at the second quadrupole by the introduction of a collision gas. B and Y series ions are shown for mellitin. CAD spectra am also shown for several intact proteins including myoglobin and cytochrome c. These spectra may be used to fingerprint proteins. MENGCK, MCEWENCN, LARSENB8: Peptide Sequencing with Electrospray Ionization on a Magnetic Sector Mass Spectrometer. Rap/d Commun Mass Spectrom 1990, 4:151-155. Sequence information for pept~des is obtained by fragmentation in the ES source on a magnetic sector mass spectrometer. The method involves collision-induced fragmentation of multiply-charged ions in the source and does not involve tandem mass spectrometric instrumentation. The high resolution capability of the magnetic sector analyzer is used to determine the charge state of the fragment ions by resolving the isotope peaks. 11. •

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SMITHRD, LOO JA, BARINAGACJ, EDMONDSCG, UDSETH HR:

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Capillary Z o n e Electrophoresis and Isotachophoresis Mass

Spectrometry of Polypeptides and Proteins Based upon an Electrospray Ionization Interface. J Chromaiogr 1989, 480:.211-232. A description of ES spectrometry coupled to capillary electrophoresis. The CE/ES interface includes a sheath electrode to make the CE electrolytes compatible with the electrospray source. Acetic acid in the sheath electrode also ensures that the samples will be positively charged. The CE flow rote is 0.5uL/min and the sheath electrode 3 uL/min. Proteins analyzed by ES alone included cytochrome c, carbonic anhydrase, lactate dehydrogenase, ovalbumin, and serum albumin. Samples analyzed by CE/ES were Leu-enkephalin and myoglobin, and both of these required selected ion monitoflng. Capillary isotachophoresis was performed on cytochrome c, myoglobin, bradykinin and angiotensin I. SMITHRD, LOO JA, EDMONDS CG, BARINAGACJ: Sensitivity Considerations for Large Molecule Detection by Capillary Electrophoresis-electrospray Ionization Mass Spectrometry. J C~romat 1990, 516:157-165. This report details various factors that affect the ondine analysis of proteins separated by capillary electrophoresis. Sensitivity is enhanced by operating at very low flow rates. Peak intensity declines significantly for larger protein molecules. EDMONDSCG, LOO JA, FIELDSSM, BARAINAGACJ, UDSETHHR, SMm-t RD: Capillary Electrophoresis Combined with Electrospray Ionization Mass Spectrometry and Tandem Mass Spectrometry. In Biological Mass Spectrometry edited by Burlingame AL and McCloskey JA. Amsterdam: Elsevier, 1990, pp 77-100.

15. •

GALLAGHERRT, CHAPMANJR, MANN M: Design and Performance of an Electrospray Ionization Source for a DoublyFocusing Magnetic Sector Mass Spectrometer. Rap/d Corn. m u n Mass Spectrom 1990, 4:369-372. A report of the construction of an ES source for a magnetic sector in. strument. The instrument was modified to contain two extra stages of vacuum pumping. Spectra were shown for proteins with masses up to 66000 (up to 60+). The mass accuracy for myoglobin (16948) was disappointingly low at + 2.8. 16.

MENGCK, MCEWENCN, LARSENBS: Electrospray Ionization o n a High-Performance Magnetic-Sector Mass Spectrometer. Rapid Commun Mass Spectrom 1990, 4:147-150. A description of an ES ion source for a magnetic sector instrument. Results am presented for peptides and small proteins (e.g. myoglobin, 16950D). Sensitivity and mass accuracy are comparable to that obtained with a quadrupole analyzer. •

proteins

Lee and Shively

17.

HENRY KD, WnJagMs ER, WANG BH, MCLAFFERTY FW, SHABANOWITZ J, HUNTDF: Fourier-Transform Mass Spectrometry of Large Molecules by Electrospray Ionization. Proc Natl Acad Sci USA 1989, 86:9075-9078. The authors have interfaced an ES source to a Fourier transformer mass spectrometer (FT/MS) equipped with a quadrupole for initial mass selection, and in order to maintain the high vacuum required for the FT/MS. The mass accuracy for ribonudease A (13669 D), horse myoglobin (16950 D), and carbonic anhydrase (29022 D) was about 0.01%. A major advantage of the method is that trapped ions can be subjected to photcxtisse~on by a laser to reveal further structural information. In this example, Gramacidin S is shown to exhibit a number of fragment ions which can be used to deduce its structure. ,

18.

VAN BERKELGJ, GUSH GL, MCLUCKEYS& Electrospray Ionization Combined with Ion Trap Mass Spectrometry. Anal (~bem 1990, 62:1284-1295. Ions from an ES source were analyzed on a quadrupole-ion trap mass spectrometer. Proteins analyzed include cytochrome c, myoglobin, and bovine serum albumin. As little as 124 fmole of albumin were required to obtain a spectrum. In the case of mellitin, one of the peaks (4 + ) was chosen for MS/MS analysis, and this revealed abundant Y-series ions. This mass spectrometer is simple in construction, has high sensitivity, and has a major advantage over other systems in obtaining MS/MS spectral ,

19. •

COTTERRJ: Time-of-flight Mass Spectrometry: An Increasing Role In the Life Sciences. Biomed Environ Mass Spectrom 1989, 18:513-532. This is an excellent review of TOF mass spectrometers and their role in the analysis of biomolecules. In particular, major improvements in resolution and the availability of new ion sources such as LD, PD, and pulsed ion beams are considered. Both the design and function of these instruments are described in detail. 20. •

HILLENKAMPF: Laser Desorption Mass Spectrometry: Mechanisms Techniques, and Applications. Adv Mass Spectrom 1989, 11A:354-362. A review of LD mass spectrometry with a major emphasis on biomolecules. Matrix-assisted LD spectra of proteins are also included. 21.

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analysis o f

HR.ENKAMPF, KARAS M, INGENIX)H A, STAHL B: Matrix Assisted UV-laser Desorption/ionizatior~ a New Approach to Mass Spectrometry of Large Biomolecules. In Biological Mass Spectrometry edited by BurlinomameAL and McCloskeyJ.A. Amsterdam: Elsevier, 1990, pp 49-60.

22. .

KARASM, BAHR U, Hn~NKAMP F: UV Laser Matrix Desorption Ionization Mass Spectrometry of Proteins In the 100000 Dalton Range. Int J Mass Spectrom Ion Process 1989, 92:231-242. Molecular ions of proteins up to mass 120000 were observed using nicotinic acid as a matrix with an ultraviolet laser tuned at a wavelength of 266 nm. Spectra were obtained with 20-100 rig sample amounts with a mass accuracy of about 1%. 23. •

SPENGLERB, COTYERRJ: Ultraviolet Laser Desorption Ionization Mass Spectrometry of Proteins Above 100 000 Daltons by Pulsed Ion Extraction Time-of-flight Analysis. Anal 1990, 62:793--796. The authors describe an ultraviolet LD/TOF mass spectrometer which can be used for the analysis of proteins above the mass range 20 000-120 000. The laser is a frequency.quadrupled Nd-YAGwith maximum output at 266 rim. The sample matrix was nicotinic acid. 24. ••

BEAVlSRC, CHArt BT: High-accuracy Molecular Mass Deterruination of Proteins Using Matrix-assisted Laser Desorption Mass Spectrometry. Anal Gbem 1990, 62:1836-1840. This paper describes, in detail, the procedure for obtaining very accurate (to within 0.01%) mass measurements by matrix-assisted LD. Of particular value is the description of the sample preparation methods which are essential for a successful analysis. The work indicates that high concentrations of organic and inorganic contaminants, such as 1 M urea, do not strongly affect either the signal intensity or the mass assignment~ All measurements were done using sinapinic acid as the sample matrix and 355 um frequency-tripled output from a Nd-YAG laser.

59

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Analytical biotechnology 25. •

BEAVlSRC, CHArr BT: Factors Affecting the Ultraviolet Laser Desorption of Proteins. Rap/d Commun Mass Spectrom 1989, 3:233-237. The authors have tested several new matrices on an ultraviolet LD/TOF mass spectrometer of their own design. Besides nicot£rtic acid, strong pseudomolecular ion peaks are shown for the following matrices: 2- or 3- pyrazinecarboxylic acid, thymine, and 3-methoxy,4-hydroxybenzoic acid. Other matrices such as 2,4-pyridinedicarboxylic acid gave no signal, and matrices such as 2-pyridinecarboxylic acid gave weak signals. A major problem encountered with some matrices was the formation of protein-matrix adducts. Spectra were obtained for proteins up to the size of ~-galactosidase (molecular mass, 116 000). 26. •

BEAVISRC, CHhrr BT: Cinnamic Acid Derivatives as Matrices for Ultraviolet Laser Desorption Mass Spectrometry. Rap/d Commun Mass Spectrom 1989, 3:432-435. A report of the discovery o f three new matrices for matrix-assisted ultraviolet LD of proteins on a TOF mass spectrometer. The matrices are all derivatives o f cinnamic acid - - sinapinic, ferulic, and caffeic acids. The chief advantage of these matrices over nicotinic acid is a reduced formation of sample-matrix adducts which contribute to peak broadening. Excellent spectra were obtained for bovine insulin (5733 D), human

milk lysozyme (14693 D), and bovine carbonic anhydrase (20019 D). The mass accuracies were in the range of 0.02%. The authors describe analyses on over 50 samples, suggesting that these matrices are suitable for all types of proteins. 27. •

BEAVlSRC, CHAIT BT: Matrix-assisted Laser-desorption Mass Spectrometry Using 355 n m Radiation. Rapk/Commun Mass Spectrom 1990, 3:436-439. The authors describe matrices suitable for the frequency-tripled output (355 nm) Nd:YAG laser on a TOF mass spectrometer. The best matrices for obtaining pseudomolecular ions of proteins were derivatives of cinnamic acid with sinapinic acid (3,5-dimethoxy-4-hydroxycinnamic acid) being the most successful. This matrix produced extremely sharp peaks, suggesting that the formation of sample-matrix adduct peaks had been minimized. Spectra were shown for apomyoglobin (16950 D), RNase S (11 500 D), and tropomyosin dimer (65472 D versus a calculated 65 476).

TD Lee and JE Shively, Division of Immunology, Beckman Research In stitute of the City of Hope, Duarte, California 91010, USA.