Mutagenesis, Vol. 15, No. 5, 415-430,
September 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
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Biological mass spectrometry: a primer
Department of Drug Metabolism and Pharmacokinetics, Novartis Institute for Biomedical Research, East Hanover, NJ 07936, USA
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Abstract |
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 Top Abstract IntroductionMALDI-MS and ESI-MSProteomicsNon-covalent complexesChip-based technologies and...Post-translational modificationsConclusionsReferences |
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Biological polymers undergo numerous significant and fascinating interactions, such as post-translational modifications, non-covalent associations and conformational changes. A valuable parameter for the characterization of a biopolymer is molecular weight. Modern methods of mass spectrometry, including electrospray ionization and matrix-assisted laser desorption ionization mass spectrometry, are ideally suited for the accurate determination of the molecular weight of a biopolymer of interest. Molecular weight measurements are now routinely utilized in the qualitative and quantitative analysis of macromolecules. In many cases small sample quantities (i.e. a few micrograms) limit the utility of nuclear magnetic resonance spectroscopy and X-ray crystallography in obtaining structural information. Thus, mass spectrometry offers an attractive alternative to the more traditional bioanalytical methods for rapid and sensitive measurements. The ultimate goal of these experiments is to obtain sufficient information in order to map the complex molecular circuitry which operates within the cell. In the analysis of complex mixtures mass spectrometry is even more powerful when utilized in conjunction with separation methods. Herein we present some of the aspects of modern biological mass spectrometry for the investigation of large molecules. For more advanced or detailed technical descriptions we refer the reader to a number of recently published reports.
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Introduction |
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TopAbstractIntroductionMALDI-MS and ESI-MSProteomicsNon-covalent complexesChip-based technologies and...Post-translational modificationsConclusionsReferences |
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In 1898 Wien succeeded in deflecting charged rays using a combination of electric and magnetic fields. Thomson demonstrated the presence of two neon isotopes in 1912. More advanced mass analyzers were designed and constructed by Dempster and Aston in 1918 and 1919, respectively (Biemann, 1962
; Matsuo and Seyama, 2000). Although early mass spectrometry (MS) provided important information about stable isotopes and radionuclides, it was limited to lower mol. wt compounds that could be readily volatilized. The problems of involatility and high mass (>1000 Da) limited the scope of MS applications. Larger species simply could not be transferred to the gas phase without substantial degradation and/or fragmentation.
Recent advances in ionization methods (vide infra) have circumvented the limitations of traditional MS and it is now possible to analyze high mol. wt compounds of all types. Since Fenn and co-workers introduced electrospray ionization (ESI-MS) in 1984, the field of bioanalytical chemistry has seen explosive growth (Fenn et al., 1989; Loo et al., 1999a; McLafferty et al., 1999; Kelleher, 2000; Thomas et al., 2000). The compatibility of ESI with separation techniques such as capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) allows characterization of a large array of components, such as small organic molecules, peptides, proteins, DNA fragments, inorganic/organometallic complexes and synthetic polymers. In addition, matrix-assisted laser desorption ionization (MALDI) mass spectrometry, a complementary approach to ESI which was introduced by Hillenkamp and Karas (Hillenkamp et al., 1991), is now widely utilized for protein/peptide analysis and in characterization of combinatorial chemistry libraries, protein mapping and DNA sequencing (Murray, 1996; Hop and Bakhtiar, 1997; Brewer and Henion, 1998; de Jong, 1998; Altman et al., 1999; Costello 1999; Deng and Smith, 1999; Ding and Vouros, 1999; Feng et al., 1999; Gygi et al., 1999a; Harvey, 1999; Kiselar and Downard, 1999; Kuster and Mann, 1999; McCloskey et al., 1999; Pramanik et al., 1999; van Baar, 2000). These new approaches promise a stunning breath of perspective, driven by a continued pursuit of macromolecular structural information in proteomics and genomics research (Lamond and Mann, 1997; Lottspeich, 1999).
At the outset it should be noted that both techniques are sensitive and allow observation of intact biopolymers with a mol. wt of 100 000 Da or higher. Since the ESI and MALDI spectra of modified biopolymers show little or no fragmentation, these techniques can be useful in obtaining accurate identification of the specimen and both are referred to as `soft' ionization techniques. In this article we present a brief description of the ionization processes involved in ESI and MALDI as well as a number of examples, which will demonstrate some of the capabilities of the ESI-MS and MALDI-MS techniques.
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MALDI-MS and ESI-MS |
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TopAbstractIntroductionMALDI-MS and ESI-MSProteomicsNon-covalent complexesChip-based technologies and...Post-translational modificationsConclusionsReferences |
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In a typical MS experiment the sample of interest is ionized in the ionization source and guided via a series of electric and/or magnetic lenses to the detector. The three main events during MS analysis are ion production, ion transmission and ion detection. In order to control the motion of the ions during their transmission to the detector it is necessary to control the influences of pressure and temperature on ion mobility (kinetic energy). Therefore, a vacuum system with a pressure ranging from ~10–5 to 10–8 Torr is utilized in all mass spectrometers. The vacuum minimizes interference from collision of the analyte ions with the background neutral gaseous molecules and facilitates their transmission to the detector.
A majority of commercial mass spectrometers utilize an electron multiplier detector, which provides an internally amplified electrical current subsequent to exposure to charged ions. The ion current output corresponding to each specific analyte is then processed by the instrument electronics and translated to a mol. wt. In the resulting mass spectrum the ordinate indicates the relative intensity or abundance while the abscissa shows the observed ratio of mass to the number of charges on the ions. The latter is referred to as the mass-to-charge ratio or m/z. Regardless of the ionization source, it is the m/z that is measured by the mass spectrometer.
MALDI (Figure 1) uses pulses of laser light (e.g. a nitrogen laser at 337 nm) to desorb the analyte from a solid phase surface (the analyte co-crystallized with a light-absorbing matrix; Figure 2) and yield gaseous ions. A laser is a device that can deliver coherent and high density energy (photons) to a small space. Pulsed laser radiation tuned to the absorption maximum of the matrix is used to initiate the desorption/ionization event and to simultaneously generate a packet of ions of different m/z values. The laser may be tuned to UV, visible or infrared wavelengths (Zenobi and Knochenmuss, 1998). The matrix is typically a small organic molecule which has an absorption band that closely coincides with the energy of the laser radiation. Figure 2 depicts the molecular structures of some of the commonly used matrices (Siuzdak, 1996). The matrix is generally co-crystallized in large excess over the analyte and facilitates ionization of the analyte as well as minimizes sample degradation due to the laser radiation. For MALDI the sample preparation procedure can be extremely crucial because the ion population depends upon the type of matrix and the presence of impurities (Chapman, 1996; Stimson et al., 1997; Jespersen et al., 1998; Breaux et. al., 2000; Garner, 2000; Landry et al., 2000). Depending on the specialist's experience and instrumentation, it is now possible to acquire mol. wt information on a biopolymer using picomole quantities of samples. However, in some cases additional sample quantities may be required for detailed analyses, such as peptide or oligonucleotide sequencing.
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Figure 3
shows a representative MALDI spectrum obtained from an 8mer oligonucleotide, d(GGAGGCCT), containing the codon 249 sequence (AGG) of the p53 gene (Jones et al., 1999). In this experiment, the sample (200 fmol) was mixed with 1% 
-cyano-4-hydroxycinnamic acid in 1:1 acetonitrile/deionized water, loaded onto the MALDI sample holder and evaporated to dryness. A nitrogen laser using 4 ns pulses at 337 nm was used to desorb and ionize the sample, yielding a signal corresponding to a [M–H]– species. Since MALDI is considered a mild ionization process, the sample experiences little or no fragmentation during analysis (Muddiman et al., 1997). Therefore, the mixture can be analyzed in a single experiment, because each component generally produces only one predominant signal ([M+H]+ or [M–H]–).
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A complementary technique to MALDI is ESI (Figure 4
), which produces single or multiply charged gaseous ions directly from solution by generating a fine spray of highly charged droplets in the presence of a strong electric field. There are two widely proposed theories for ion formation in ESI (Gaskell, 1997; Bruins, 1998; Constantopoulos et al., 2000; Fernandez de la Mora, 2000; Gamero-Castano and Fernandez de la Mora, 2000; Kebarle and Peschke, 2000). One theory suggests that ionized sample molecules are expelled from the droplets. Alternatively, it has been proposed that individual ionized sample molecules remain after solvent evaporation and droplet fragmentation.
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As shown in Figure 4
, a solution of the analyte(s) and the solvent are introduced into a sampling metal capillary (~100 µm in internal diameter) which is charged by application of an electrical voltage (4–5 kV). The voltage polarity of the metal capillary is positive or negative for positive or negative ion generation, respectively. At some point mutual repulsion between the ions at the surface becomes greater than the surface tension of the liquid, which gives rise to formation of the so-called Taylor cone (Wilm and Mann, 1994). If the electrical field is sufficiently strong, spraying commences and small charged droplets form. The ions generated by ESI carry multiple charges, provided the sample molecules have a mol. wt of more than ~1000 Da. The characteristic feature of ESI that distinguishes it from other ionization techniques is that it generally imparts multiple charges to larger analyte molecules and the extent of multiple charging increases in near proportion to mol. wt. The resulting highly charged molecular ions are thus within the m/z range in which most conventional mass spectrometers function quite well (Bakhtiar et al., 1996; Bakhtiar and Nelson, 2000). It is the multiple charging phenomenon that allows assay of high mass ions by mass analyzers with only a modest m/z range.
For the sake of clarification, let us dissect a hypothetical positive ion ESI-MS spectrum (Figure 5) of a biopolymer with a mol. wt of 5 kDa. Unevenly spaced signals corresponding to charge states 1+ to 5+ are evident. In comparison with a MALDI spectrum, the ESI spectrum shown in Figure 5 clearly appears rather complex and convoluted. Thus, ESI spectra require deconvolution algorithms, which are commonly utilized on all commercial MS instruments. Assuming that a positive ion series represents different protonation states, then the m/z of two successive peaks can be denoted P1 and P2, corresponding to (m/z)1 and (m/z)2, respectively (Figure 6). The objective is to extract the charge state of each individual signal in order to deconvolute the spectra and obtain the mol. wt of the biopolymer. This can be achieved easily by setting up two equations and two unknowns using at least two adjacent signals in the spectrum (Figure 5). For example, solving for z1 and mol. wt yields values of 5 Da and 5000 Da, respectively. Analogous outcomes could be obtained using any two sets of signals in the spectrum (Siuzdak, 1996). Of course, additional mathematical procedures, such as smoothing, background subtraction, noise filtering and automated algorithms, have been introduced as options on most modern MS computer workstations (Bruenner et al., 1994; Bonner and Shushan, 1995; Horn et al., 2000). Figure 7 shows a `real life' example of a positive ESI-MS spectrum acquired for a sample of bovine serum albumin in 0.1% formic acid solution. The top and bottom panels represent the originally acquired convoluted and subsequently processed deconvoluted spectra, respectively.
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ESI does have limitations in that it is not very tolerant of the presence of salts, detergents and inorganic buffers (MALDI has proven to be more amenable in such cases). Thus, in order to minimize signal suppression effects, ESI is often exploited as an interface between CE or HPLC and a mass spectrometer (Niessen, 1999
). Currently, HPLC-MS is an attractive tool in the analysis of complex mixtures in biochemical research and medical/diagnostic analysis. The up-front chromatographic separation aids in sample purification/enrichment from most common laboratory buffers and endogenous salts and provides additional useful parameters, such as retention time (Cole, 1997). For example, Figure 8 illustrates the influence of an organic buffer, HEPES, which is commonly used between pH 6.8 and 8.2, on the positive ion ESI-MS spectrum of horse skeletal muscle myoglobin (Mb). The typical working concentration range for HEPES is between 20 and 100 mM. While a solution of Mb in 0.1% formic acid yielded a satisfactory spectrum (Figure 8a), the same solution containing 10 mM HEPES exhibited significant ion suppression (Figure 8b). Similar ESI-MS analyte signal suppression has also been observed with several commonly used detergents and surfactants in protein chemistry (Ogorzalek Loo, 1996). The above example (Figure 8) and reports from other laboratories (vide supra) serve to exemplify the need for proper on- or off-line sample clean-up and preparation prior to MS analysis.
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As an alternative to HPLC purification, several laboratories have explored the utility of on-line protein (DeGnore et al., 1998
; Xu et al., 1998) or oligonucleotide (Liu et al., 1996; Huber and Buchmeiser, 1998) dialysis prior to mass spectral analysis. In our laboratory we have adapted a similar strategy to that reported by Liu et al. (1996) to perform on-line sample dialysis and clean-up for several protein biomarkers. We chose hemoglobin (Hb) as our test model. The Hb experiment was performed with two objectives in mind. First, the performance of our fabricated microdialysis device could be evaluated. Second, we could demonstrate that a relatively simple ESI-MS experiment is amenable to differentiation of a single amino acid substitution.
There are more than 700 Hb abnormalities known to be the result of a single amino acid variation (due to mutations in the coding sequence). In recent years a number of Hb variants have been successfully characterized by MS (Shackleton and Witkowska, 1996; Kaneko et al., 1999; Gatlin et al., 2000). In this regard, genetically transmitted sickle cell anemia is characterized by thin and elongated red blood cells. Sickle cell anemia is generally accompanied by cardiac enlargement, swelling of lymph nodes, jaundice and anemia. The topological alteration in individuals with sickle cell anemia is due to a single amino acid mutation in the ß-chain of their Hb. In a normal adult, position six of the ß-chain is occupied by Glu while in a patient with sickle cell anemia this amino acid is substituted by Val. This mutation dramatically reduces the solubility of the deoxygenated form of Hb. Sickle cell Hb is referred to as Hb S to distinguish it from the normal adult Hb (Hb A). Figure 9 shows the deconvoluted positive ion ESI-MS spectra for Hb S (top) and Hb A (bottom) subsequent to on-line dialysis. The result clearly indicates the mol. wt difference of 29 Da between the two ß-chains corresponding to the substitution ß6Glu
Val. There are no amino acid sequence differences in the -subunit of Hb S and Hb A and, therefore, the observed mol. wt values (mass accuracy 0.01% or ±1.5 Da) for both samples are similar. In other experiments high resolution instruments have identified a mass difference of 1 Da in proteins having mol. wts of ~12 kDa (Marshall et al., 1997).
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Proteomics |
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TopAbstractIntroductionMALDI-MS and ESI-MSProteomicsNon-covalent complexesChip-based technologies and...Post-translational modificationsConclusionsReferences |
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The human body is estimated to contain ~70 000–100 000 genes (the entire human genome is composed of ~3 000 000 000 bp) potentially encoding 100 000 different proteins (Rowen et al., 1997
). Furthermore, post-translational modification, mutation, degradation and other cellular processes increase the number of proteins. This extremely high degree of complexity warrants the need for a conglomerate of sensitive and rapid analytical techniques to yield qualitative and quantitative information with high efficiency. The science of proteomics involves the detection and identification of proteins, and complements genomics. The proteome is a highly dynamic system which can be influenced by environmental variations, such as quantitative changes in protein expression as a result of drug administration. For example, a drug may elicit inhibition or overexpression of a specific enzyme (hepatic cytochrome P450), which may yield alterations in the therapeutic outcome. Enzyme induction can decrease drug levels or increase the formation of toxic metabolites (Guengerich, 1999; Whitlock, 1999). The information obtained from proteome analysis can aid in identification of therapeutic targets or surrogate markers in understanding the initiation and progression of a disease state. Thus, proteomics research can be a valuable tool in drug discovery and for the first time offers the scientist an integration of genomics, mRNA analysis and protein expression (Blackstock and Weir, 1999).
One of the most commonly utilized techniques for protein separation has been based on gel separation. Simple protein mixtures (<100 components) are normally separated using 1-dimensional (1D) SDS–PAGE. On the other hand, for complex protein mixtures (i.e. cell or tissue extracts) the resolving power of 2-dimensional (2D) SDS–PAGE is required. In this approach proteins are separated by isoelectric point in the first dimension and subsequently by their mol. wt in the second dimension. Visualization of the gel is easily achieved by Coomassie blue staining, silver staining, fluorescent tagging or radioactive labeling, with some approaches having a detection limit of ~10 ng of protein (Rabilloud, 2000). However, visualization does not provide unambiguous protein identification and, therefore, scientists typically use western blotting or classical Edman sequencing for this purpose. Fortunately, integration of the inherent benefits of MS (i.e. sensitivity, selectivity and speed) with those conferred by protein and expressed sequence tag (EST) databases has contributed to significant advances (Blackstock and Weir, 1999; Yates, 2000).
Figure 10 depicts a simplified strategy which is being widely utilized in high throughput polypeptide characterization. Typically, samples obtained from different cellular fractions are processed by 2D SDS isoelectric focusing gel electrophoresis. Each cell or tissue type may require a specific visualization approach, such as Coomassie blue or silver staining or fluorescence tagging, for protein detection (Hancock et al., 1999; Lottspeich, 1999; Wang and Hewick, 1999; Williams,K.L., 1999). Subsequently, gel images are electronically retrieved by high resolution scanners and analyzed (spot finding) using pattern recognition techniques against 2D gel database queries. Sophisticated computer software packages can be employed to enhance contrast, subtract background, align images, remove artifacts and perform gel comparisons. Proteome maps are compared against databases for identification of up- or down-regulation in a disease state. The resulting information could be utilized to identify biomarkers in clinical and toxicological studies.
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Conceptually, a similar and complementary approach but with higher accuracy (i.e. better than 10 p.p.m.), sensitivity and speed can be implemented with the aid of MS-based techniques. The gel `spots' can be excised, washed, subjected to proteolytic digestion and characterized by MALDI-MS or ESI-MS (Bantscheff et al., 1999
; Jungblut and Thiede, 1997; Keough et al., 1999; Loo et al., 1999b; Neubauer and Mann, 1999; Schrotz-King et al., 1999). Sometimes, affinity chromatography techniques are necessary to enrich a specific class of proteins prior to additional sample manipulations (Link et al., 1999; Gruninger-Leitch et al., 2000). Commonly, on-line CE-MS or HPLC-MS analysis can be employed to further separate complex protein or peptide mixtures (Cao and Stults, 1999; Jensen,P.K. et al., 1999; Tong et al., 1999). A number of software packages are currently available to query large databases and enhance the speed of the MS protein identification process (Jaffe et al., 1998; Clauser et al., 1999; Demirev et al., 1999; Green et al., 1999). Non-redundant protein databases with ~350 000 entries and human EST databases with ~1 200 000 entries can yield more sophisticated and accurate identification output compared with 2D gel analysis (Mann, 1996; Jensen,O.N. et al., 1999). In addition, 2D gel sample components below a mol. wt of 10 000 Da or above 100 000 Da that are not easily characterized can be readily observed by MS (vide infra). The following examples will clarify the above discussions.
Recently, a high throughput protein identification (double parallel digestion, DPD) method was reported by Sanchez and co-workers (Bienvenut et al., 1999). In the DPD approach partially digested proteins were obtained using an immobilized trypsin membrane and transblotted. The resulting peptides were trapped on a polyvinylidene difluoride membrane and scrutinized by MS. The DPD approach was successfully applied to a mini-2D gel electrophoresis of Escherichia coli extract. Several critical issues, however, need to be further improved in order to realize the full potential of 2D gel analysis in conjunction with MS analysis. These include a reduction in background chemical noise (i.e. due to keratins), which can mask the detection of lower abundance or `low copy number' proteins. Keratin interference can even originate from inadequately purified trypsin, which is widely used for peptide mapping in MS experiments (Zhang,Y. et al., 1998). Thus, a clean sample preparation environment, automation, minimum sample handling procedures and higher quality gel materials compatible with MS analysis could alleviate the problem of possible contamination. In addition, post-translational modifications, oxidation of protein during sample preparation and poor recovery of large proteins from the gel have the potential to extend the duration of the unambiguous analyte(s) identification process.
Smith and co-workers (Pasa-Tolic et al., 1999) demonstrated the utility of ultra-high resolution MS measurements in conjunction with the resolving power of capillary isoelectric focusing for characterization of the cadmium stress response in E.coli K-12 strain MG1655 cells. The cells were cultured in normal as well as rare isotope (i.e. 13C, 15N and 2H) depleted media in order to provide internal calibrants for all detected proteins subsequent to cadmium-mediated stress. The abundance of several intact and isotopically distinctive proteins was qualitatively monitored for up to several hours using this method.
Another related area in proteome analysis is protein expression mapping, which is defined as the quantitative measurement of protein dynamics in a specimen (i.e. a cell, tissue, or body fluid) of interest. In this approach proteome analysis is typically performed in a subtractive fashion whereby alterations in individual proteins for two or more states are compared. These so-called `cell states' could refer to a cell prior to and subsequent to treatment with a xenobiotic or cells obtained from normal and pathological states. Recently an elegant quantitative microcapillary-LC-ESI-MS strategy for the analysis of protein mixtures in Saccharomyces cerevisiae was reported by Aebersold and co-workers (Gygi et al., 1999b). An isotope-containing affinity tag (ICAT), which consisted of an affinity tag (biotin), a linker containing a stable isotope and a reactive moiety with a propensity to react with free sulfhydryl groups (i.e. cysteines), was utilized (Figure 11). Two sets of cell states (or tissue extracts) were independently treated with isotopically light and heavy (8 Da higher in mol. wt due to incorporation of 2H) ICAT reagents. The cells were combined and subjected to proteolytic cleavage. The ICAT-labeled peptides were isolated using the biotin tag and analyzed by microcapillary-LC-ESI-MS. Peptide sequence information was obtained by tandem mass spectrometry experiments and identified by computer searches against protein data banks. Quantitation of proteins was performed using the ratios of the respective light and heavy ICAT-labeled peptides, which were generated by enzymatic digestion. The stable isotope labeling procedure was a clever approach to assist in the identification of two peptides with identical sequences and mol. wts from two different cell states. Since all the physical characteristics of the two identical protein samples from the two cell states remain the same, the resulting peptide fragments obtained by enzymatic cleavage yield identical mass spectra. Thus, incorporation of specific stable isotopes in one cell state results in mass shifts, which in turn serve as an internal standard for all other cell states within the same experiment (Mann, 1999). Since the light and heavy ICAT-tagged peptides are chemically identical, one can safely assume that they would yield analogous MS detection (ionization) responses and behave as mutual internal standards for quantification purposes.
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In general, the above strategies for qualitative and quantitative identification of key cellular proteins could have great potential in several areas of drug development, such as pharmacogenomics (Evans and Relling, 1999
). In the science of pharmacogenomics genetic polymorphisms in transporters, drug metabolizing enzymes (e.g. cytochrome P450s and uridine 5'-triphosphate glucuronosyltransferases), receptors and therapeutic target proteins have been postulated to be one of the underlying reasons for variable responses to drug treatment in patients. Currently these investigations are rather tedious and empirical. In most cases the human genetic variations resulting in different drug responses are realized in large studies at the post-marketing stages (i.e. subject sizes exceeding 100 000). Some of these idiosyncratic responses are toxic and thus it is essential to identify them prior to Phase III clinical studies (Gould Rothberg et al., 2000). Therefore, it is of interest to elucidate the identity and pharmacogenomic traits (i.e. polymorphically expressed enzymes) of key cellular proteins and to design optimum medication for individual patients. MS technology offers a viable platform which can be utilized to assay differential protein expression following drug treatment.
Considering the recent report by Sweedler and co-workers (Li et al., 1999; Rubakhin et al., 2000), the application of MS measurements to the identification of proteins in individual organelles will not be far in the future. In their findings Sweedler and colleagues devised an elegant direct approach to the identification of peptides in 1–2 µm diameter vesicles from the exocrine atrial gland of Aplysia californica as a model system. MALDI-MS was used to analyze sample volumes of as low as 300 al (300x10–18 l) and identified a wide range of bioactive polypeptides. This technology offers an exciting window of opportunity to study discrete small cells and processes such as synaptosomes, vesicular transport between the endoplasmic reticulum and the various Golgi compartments, hepatocytes and protein packaging pathways.
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Non-covalent complexes |
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TopAbstractIntroductionMALDI-MS and ESI-MSProteomicsNon-covalent complexesChip-based technologies and...Post-translational modificationsConclusionsReferences |
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A growing body of literature has been devoted to the application of MS to the detection of non-covalent complexes (Przybylski and Glocker, 1996
; Loo, 1997; Jespersen et al., 1998; Coyle et al., 1999; Griffey et al., 1999; Last and Robinson, 1999; Nordhoff et al., 1999; Rostom and Robinson, 1999; Rostom et al., 2000). Using inherently `gentle' ionization, MALDI-MS and ESI-MS have provided valuable information on structurally specific biomolecular interactions, including direct stoichiometry determinations for specific protein–drug, DNA–drug, protein–DNA, RNA–drug and protein–protein complexes (Bakhtiar and Stearns, 1995; Bakhtiar and Bednarek, 1996; Hop and Bakhtiar, 1997; Pramanik et al., 1998; Loo, 2000; Van Berkel et al., 2000). Furthermore, mass spectrometric determination of 1H-2H exchange has provided complementary information to existing nuclear magnetic resonance (NMR) data on water soluble peptide and protein tertiary structures and conformations (Gross, 1999; Weber-Ban et al., 1999; Bouchard et. al., 2000; Demmers et al., 2000; Jager and Pluckthun, 2000).
For example, interaction of the HIV protein gp120, on the exterior of the HIV virus, with CD4 glycoprotein, on the surface of T helper cells, is of interest in understanding the tissue specificity of HIV virus infection. In humans the main cells infected by the HIV virus are CD4 T helper lymphocytes and macrophages via CD4 surface receptor interactions. CD4 is a surface glycoprotein which is involved in the T helper cell reaction with other cells (cell–cell adhesion). As glycoproteins, gp120 and CD4 both show a high degree of oligosaccharide heterogeneity. The complex pattern of the non-covalent complex of gp 120 and CD4 poses difficulty in spectral deconvolution during ESI-MS analysis. Recently MALDI-MS was utilized in the characterization of a non-covalent complex of recombinant gp120 with the extracellular part of its primary cellular receptor, CD4 (Borchers and Tomer, 1999). MALDI-MS experiments clearly showed a 1:1 stoichiometry between gp120 and CD4, with a combined mol. wt of ~145 kDa. This approach yielded higher resolution and accuracy in comparison with sugar gradient sedimentation experiments. However, to determine the stoichiometry of a macromolecular non-covalent assembly by MALDI-MS, several key issues need to be considered. In MALDI-MS of intact non-covalent complexes the nature of the matrix, the laser power and the laser penetration depth in the target sample are among the most critical parameters. In the presence of organic solvents and acidic MALDI matrices denaturation of protein complexes is likely to occur (Glocker et al., 1996; Little et al., 1997a; Jespersen et al., 1998). In addition, repeated laser pulses on a given spot may dissociate the non-covalent complex.
The analysis of specific interactions of nucleic acid sequences with repressors and other regulatory proteins has commonly been performed using electrophoretic gel mobility shift assay. However, this technique requires radiolabeled DNA and does not provide accurate (i.e. within 0.01% of the theoretical value obtained from the known sequence) mol. wt determinations. In recent years MS techniques have provided a complementary approach to the existing methods for deciphering the mol. wt and stoichiometry of DNA–protein complexes (Veenstra, 1999). For example, an innovative method involving a high throughput determination of DNA-binding proteins using an immobilized DNA probe and subsequent read-out by MALDI-MS was recently reported (Nordhoff et al., 1999). Specific sequences of DNA strands were immobilized (i.e. using biotin) on Dynabeads, incubated with protein mixtures, washed and subjected to MS analysis. Several known DNA-binding proteins, such as cAMP receptor protein, rat retinoid X receptor and poly(ADP-ribose) polymerase, were identified.
The specific binding of aminoglycoside antibiotics to rRNA subdomains was examined by Griffey et al. (1999) using a high resolution mass spectrometer equipped with an ESI interface. Aminoglycoside antibiotics (i.e. apramycin, ribostamycin, tobramycin, bekanamycin, paromomycin and lividomycin) are a class of compounds that inhibit protein synthesis and RNA splicing both in vivo and in vitro. Elegant MS experiments demonstrated specific binding of several antibiotics to rRNA subdomains, provided estimated binding affinities and determined their respective binding sites.
Robinson and co-workers (Last and Robinson, 1999; Rostom and Robinson, 1999) presented a collection of elegant work on protein folding and multi-protein complexes using ESI-MS. Figure 12 depicts an example of nanoflow (2–10 nl/min) ESI-MS spectra for human transthyretin with retinol-binding protein (R) obtained from chicken plasma. Retinol-binding protein (~21 kDa) is a plasma protein which is a specific carrier of retinol (vitamin A). Transthyretin (~55 kDa) is a serum protein which plays a role in transport of thyroid hormones. Non-covalent complexes of transthyretin and R (binding affinity ~1.1x10–7–1.5x10–7 M) have been identified in both human and chicken. Generally, a few microliters of the solution (~1–5 µM) of interest sufficed for obtaining satisfactory ESI-MS results. Signals corresponding to the transthyretin tetramer (4T) and non-covalent complexes with one (4TR) and two (4TRR) molecules of retinol-binding protein were reflective of previously known solution chemistry observations.
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Although the majority of published MS work has been on previously characterized non-covalent complexes, it is clear that MS offers enormous possibilities (in conjunction with NMR and X-ray crystallography data) for the investigation of less well defined macromolecular complexes. Nonetheless, it is imperative to perform proper control experiments to rule out the possibility of false positive observations during the MS analysis of non-covalent complexes. Variations in solution pH, instrument parameters (ionization interface temperature and voltages) and chemical modification of the analyte under investigation are some of the measures that can be taken to distinguish between specific interactions and non-specific aggregations (Loo, 1997
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Chip-based technologies and microfabricated devices |
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TopAbstractIntroductionMALDI-MS and ESI-MSProteomicsNon-covalent complexesChip-based technologies and...Post-translational modificationsConclusionsReferences |
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Chip and other miniaturized technologies are rapidly being adapted to analyze changes in gene expression (Epstein and Butow, 2000
). DNA microarray technology (e.g. the Affymetrix GeneChip®) has shown great promise in toxicological studies, gene mapping, gene polymorphism and transciptional analysis (DeRisi et al., 1997; Kurian et al., 1999; Voehringer et al., 2000). DNA microarrays consisting of thousands of individual gene sequences can be printed in a high density format on a glass microscope slide or deposited on a miniature matrix by a photolithographic process (Knapp et al., 2000). These DNA microarrays can then be utilized to obtain a global view of changes in expression of genes during drug development and provide a vivid `snapshot' of how cells respond to a drug in vivo (Afshari et al., 1999).
Many diseases stem from gene mutations, which may take place via various routes such as replacement, deletion, insertion or duplication of a single or multiple base units that are the building blocks of the gene. In the area of gene expression, where many human genes have already been characterized, the sensitivity of detection of mRNA at the level of one in thousands is of interest. Driven by chemistry, the process of drug research and discovery is increasingly led in the post-genomic era by advances in biotechnology and bioinformatics (Drews, 2000; Sander, 2000). The trend towards sensitive and high speed microfabricated devices has been reinforced by a recent initiative to construct a mammalian gene collection (Strausberg et al., 1999). In this regard, MS is becoming a viable platform for rapid genomic DNA characterization (Laken et al., 1998; Ross et al., 1998; Griffin et al., 1999; Tang et al., 1999; Cantor, 2000).
In one strategy Tang et al. (1999) demonstrated the immobilization of reduced thiol DNA strands on silicon chips using N-succinimidyl(4-iodoacetyl)aminobenzoate chemistry. Each silicon chip contained 36 wells with a surface area of 6.25 mm2/well (~1 µl/well). DNA fragments containing the polymorphic sites (i.e. codons 163 and 33 in human platelet alloantigens 2 and 1, respectively) were amplified by PCR and detected by this method. Parallel primer annealing, extension and termination were performed on a 1 µl sample scale and directly subjected to MALDI-MS.
One of the emerging disciplines of human genetics is the detection of DNA polymorphisms to identify the components involved in complex genetic diseases. To put this in perspective, consider that the nucleotide sequence difference between humans and chimpanzees is estimated to be ~1.5% (Liyanage and Xanthopoulos, 2000). Clearly, this seemingly small variation has led to profound differences between the two species. The so-called single nucleotide polymorphism (SNP) refers to a position at which two alternative bases occur at a frequency of ~1% in humans. This corresponds to approximately 1 in every 1000 nt. The significant implications of SNP include the presence of a particular SNP allele that could be the underlying reason for a genetic disorder along with variations in protein coding sequences. Furthermore, SNPs could be useful as genetic markers for mapping purposes and localization of important functional genes. A combination of MALDI-MS and microfabricated devices for DNA minisequencing and characterization could obviate the need for labeling studies. Several laboratories have recently reported the use of MALDI-MS for detection of SNPs for direct genetic analysis (Haff and Smirnov, 1997; Griffin et al., 1999). For example, Haff and Smirnov (1997) discussed an assay for single base variations at specific locations within the DNA sequence using MALDI-MS. In this approach a primer oligonucleotide was annealed to a target DNA upstream of the polymorphic location and was extended by a single base in the presence of a thermostable DNA polymerase (ThermoSequenase) and all four dideoxynucleotide triphosphates. The resulting extension products were desalted using a POROS reversed phase column and subjected to MALDI-MS. The base at the polymorphic site was easily detected by the mass added onto the primer oligonucleotide. Similar approaches could be employed using `MALDI on a chip' technology in which only nanoliter amounts of sample are deposited on the target with piezoelectrical pipettes (Little et al., 1997b; Griffin and Smith, 2000).
Although DNA immobilization through biotin–streptavidin (i.e. on magnetic or controlled pore glass beads) or via covalent linkage (i.e. on a silicon surface) is compatible with high throughput MALDI-MS (Ekstrom et al., 2000) analysis, several ESI-based microfabricated devices have also been presented. Lazar et al. (1999) designed a microchip nano-ESI device with a fluid delivery of 20–30 nl/min and sub-attomole sensitivity for detection of several peptide and protein mixtures. Zhang,B. et al. (1999) reported the coupling of an electrophoretic device for on-chip CE (constructed using photolithographic/wet chemical etching techniques) separation followed by ESI-MS analysis. A sample volume of 15 µl could be electrosprayed for up to 20 min at a flow rate of ~200 nl/min. Similarly, Chan et al. (1999) used polydimethylsiloxane to design soft polymer chips that were successfully employed in the identification of rat serum albumin separated by 2D gel electrophoresis. A lower limit of detection of 100 fmol/µl was consistently achieved.
Affinity isolation is undoubtedly the most specific of separation techniques and, when coupled with MS, offers an extremely powerful method for the selective isolation and concentration of a desired ligand. Recently, ProteinChipTM technology based on surface-enhanced laser desorption/ionization (SELDI)-MS was introduced by Hutchens and co-workers (Kuwata et al., 1998; Paweletz et al., 2000). In SELDI-MS affinity chips are tailor designed to capture a specific small molecule or biopolymer. An antibody, a receptor or a DNA fragment with defined arrays of binding surfaces is selected to modify a chip surface. Subsequently, a crude protein sample or a combinatorial ligand library is applied to the chip surface, washed and subjected to SELDI-MS analysis. The resulting mass spectrum typically corresponds to all the analytes captured by the affinity chip with minimal sample clean up. SELDI-MS was recently applied to profiling the site of in vitro phosphorylation of caspase-9 (Cardone et al., 1998). Figure 13 depicts an application of the ProteinChipTM in conjunction with SELDI-MS that was successfully utilized in `fishing out' a high affinity ligand from a proprietary combinatorial ligand library. An immobilized `receptor' ProteinChipTM array (Figure 13, top) and a control ProteinChipTM array (Figure 13, middle) were incubated with a combinatorial library of 100 compounds of mol. wt < 600 Da. Only one of the ligands was retained specifically on the receptor and not the control chip. The bottom panel (Figure 13) depicts the differential ligand binding plot.
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Other novel approaches, such as matrix-free MALDI using a porous silicon target (Wei et al., 1999
) and chip-based surface plasmon resonance biomolecular interaction mass spectrometry (Nelson and Krone, 1999; Williams,C. and Addona, 2000), have been reported. Furthermore, microfluidic chip technology for direct analysis of proteins using ESI-MS (Licklider et al., 2000; Pinto et al., 2000; Wen et al., 2000) has shown potential in characterization of minute (i.e. femotmole quantities) amounts of samples in an automated fashion. |
Post-translational modifications |
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TopAbstractIntroductionMALDI-MS and ESI-MSProteomicsNon-covalent complexesChip-based technologies and...Post-translational modificationsConclusionsReferences |
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Post-translational modification of proteins plays a pivotal role in functional activity and signal transduction in all living organisms. The mass changes due to post-translational modification (i.e. acetylation, farnesylation, glycosylation, phosphorylation, methylation and sulfation) can be easily detected by ESI (Verma et al., 1997
; Abraham et al., 2000) or MALDI (Vinh et al., 1997; Kirpekar et al., 2000; Zhou et al., 2000). Identification of the protein fragment of increased mass subsequent to enzymatic digestion allows determination of the site of modification. For example, in the case of phosphorylation radiolabeling with 32P is not required and low levels of phosphopeptides (i.e. 200–300 fmol/µl) can be readily identified with high efficiency and speed. Additional information can be obtained by performing tandem mass spectrometry or collision-induced dissociation (CID) experiments (Hop and Bakhtiar, 1997; Bakhtiar and Hop, 1999). Ease of application with most types of mass spectrometers, along with its experimental simplicity, account for the wide popularity of CID for structural analysis. The interpretation of CID spectra of peptides of unknown sequence is facilitated by the use of computer-aided database searching/matching algorithms. In a typical CID experiment a beam of ions with a specific m/z (denoted the precursor or parent ion) is selected and collided with a neutral and non-reactive gas phase target such as argon. These collisions result in subsequent fragmentation and product ions that are a direct consequence of dissociation of the precursor ion. Generally, the resulting fragmentation pattern is unique for all ions having a particular structure. CID experiments are particularly useful in peptide and DNA sequencing (Little et al., 1996; Roskey et al., 1996; Zhu et al., 1997; Kelleher et al., 1999).
Recently, Chait and co-workers reported quantitative measurements of protein expression and site-specific phosphorylation using a combination of MALDI, ESI and whole cell stable isotopic labeling (Oda et al., 1999). Cells were separately cultivated in media enriched with 14N and 15N, combined and subjected to SDS–PAGE. Gel spots of interest were then excised, digested by a protease and analyzed by MS. The ratio of 14N- to 15N-labeled peptides reflects their abundance. Protein expression and in vivo phosphorylation in the wild-type versus cells mutant for PAK-related yeast Ste20 protein kinase were investigated utilizing this approach. This technology is somewhat analogous to that described by Gygi et al. (1999b) and yields a quantitative visualization of cellular protein phenotype expression and modification (i.e. phosphorylation).
Cortez et al. (1999) conducted CID experiments using capillary LC-MS to locate the phosphorylation sites (Ser1423 and Ser1524) of the breast cancer gene 1 tumor suppressor protein. Likewise, automated sequence database searching algorithms were used on the resulting CID marker product ions to map the in vivo phosphorylation sites of endothelial nitric oxide synthase by Figeys et al. (1999). An immobilized metal affinity chromatography method was used to obtain fractions of tryptic digests, which were subsequently subjected to CE-MS and CID. A broader experimental set-up capable of identification of 22 different types of post-translational modification was reported by Wilkins et al. (1999). MALDI-MS analysis of proteins from a 2D gel of E.coli and sheep wool were studied using 5153 entries of post-translational modifications recorded in the SWISS-PROT data bank. This approach also shows promise in high throughput mapping of modified regulatory proteins.
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Conclusions |
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TopAbstractIntroductionMALDI-MS and ESI-MSProteomicsNon-covalent complexesChip-based technologies and...Post-translational modificationsConclusionsReferences |
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In the past decade dramatic progress in the field of MS has resulted in a large increase in the number of commercially available MS instruments. Based on the large number of published manuscripts, it is clear that MS is becoming an important bioanalytical tool in many biotechnology and biochemistry laboratories. MALDI-MS and ESI-MS allow the characterization of a large number of small and large molecules with high sensitivity, speed, accuracy and efficiency. MS-based techniques are becoming a permanent component of studies involving functional genomics, proteomics, early drug discovery, clinical diagnostics and combinatorial chemistry. In addition to large pharmaceutical corporations, a number of small start-up companies have begun to embark on the ambitious path of establishing large scale MS-based proteomics and genomics facilities (Service, 2000
; Stipp, 2000).
The literature on the above subject matter is growing exponentially. Consequently, specialized journals are now devoted to the area of MS, including Journal of the American Society for Mass Spectrometry, Rapid Communications in Mass Spectrometry, Mass Spectrometry Reviews, European Mass Spectrometry, Journal of Mass Spectrometry and International Journal of Mass Spectrometry. Thus, some related topics [e.g. complex sequencing and characterization of polysaccharides (Venkataraman et al., 1999) and lipopolysaccharides (Ernst et al., 1999), integration of MS into drug development and quantitative analysis (Lee and Kerns, 1999; Watt et al., 2000)] have not been included in this review but citations are provided for interested readers throughout the manuscript.
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Acknowledgments |
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R.B. is grateful to Prof. C.V.Robinson and Dr A.A.Rostom (Oxford University, Oxford, UK) for providing Figure 12
, Drs E.A.Dalmasso and M. Sha (Ciphergen Biosystems, Palo Alto, CA) for kindly providing Figure 13 and Dr J.Ashby (Astra-Zeneca) for encouragement and support of this initiative. We also thank the Finnigan Corporation (San Jose, CA) for providing Figure 4. The constructive comments of the reviewers and the Editor-in-Chief were extremely helpful in improving the quality of this contribution. |
Notes |
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1 To whom correspondence should be addressed at: Building 405, Room 229, 59 Route 10, Novartis Pharmaceuticals Corporation, East Hanover, NJ 07936, USA. Tel: +1 973 781 3562; Fax: +1 973 781 6076; Email: ray.bakhtiar{at}pharma.novartis.com
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Received on January 5, 2000; accepted on May 9, 2000.
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