Mass Spectrometry Michael Kinter Departments of Microbiology and Pathology, University of Wrginia Health Sciences Center, Charloflesville, Virginia 22908
Mass spectrometry continues to be an important contributor to the analytical methodologies used by clinical chemists. The rapid advancement of electrospray ionization and atmospheric pressure chemical ionization are making liquid chromatography/ mass spectrometry analyses more sensitive and more widely applicable. The past several years have also seen a maturing of the operating systems for the instruments that has dramatically improved the ease of instrument operation. These advances have given the clinical scientist renewed access to LC/MS methods and G U M S methods that are robust and reliable. One obstacle to wider incorporation of mass spectrometry into clinical laboratories has been economic issues such as the cost of the instrumentation and the relatively small number of samples that can be analyzed in a day. Although it is likely mass spectrometers will always be expensive pieces of equipment, economic factors should become less of a disadvantage as the instruments become easier to operate and more highly automated. As a result, the focus may shift to the scientific issue of what new or improved diagnostic information can be provided by a mass spectrometric experiment. In this regard, instrumental developments that are making mass spectrometric analyses applicable to new analytes are most exciting. Foremost of these developments are the advances in electrospray ionization and matrix-assisted laser desorption ionization that make analysis of polar compounds with molecular weights approaching 100 000 possible. Molecular weight measurements, peptide mass mapping experiments, and amino acid sequence analysis can be performed using these techniques to provide specialized information about protein structure that may compliment genetic analyses of human disease. This purpose of this section is to review the recent advances in mass spectrometry through a discussion of their application to clinically relevant analyses. The approach taken has been to select topics currently or potentially of interest to the clinical laboratory, protein characterization,the measurement of endogenous species, and stable isotope studies, with part of each discussion focusing on the instrumental advances that make these analyses unique. Papers published between October 1992 and October 1994 have been considered for this review. PROTEIN CHARACTERIZATION As our understanding of the contribution of genetic factors to human disease has increased, our appreciation of the effects of differences in the protein products that are encoded has also grown. For example, a series of genetic variations in the superoxide dismutase gene that produce corresponding mutations in the enzyme have recently been linked to familial amyotrophic lateral sclerosis (91).Posttranslational modifications of proteins can also affect protein function in a deleterious manner. For example, the oxidative moditication of low-density lipoprotein has been linked to accelerated deposition of cholesterol into the aortic intima in the initiation and progression of atherosclerosis (92). In general, it is clear that individual variations in protein structure may signiticantly contribute to a number of human diseases. Recent advances in mass spectral analysis of proteins are providing new ways of characterizing protein structure. These
techniques have overcome the fundamental problems of generating gas-phase ions of proteins and peptides and increasing the effective molecular weight ranges of the mass analyzers that have previously prevented the use of mass spectrometry for protein analysis. Mass spectrometric characterization of a protein is carried out in three primary ways: molecular weight measurements of the intact protein, peptide mass mapping of degradation products, and amino acid sequencing. Important features of these experiments are the sensitivity of the analyses and the precision of the molecular weight measurements. The measurement of the molecular weight of a protein by either matrix-assisted laser desorption ionization mass spectrometry or electrospray ionization mass spectrometry is routinely carried out in an experiment that consumes subpicomole amounts of the sample. The molecular weight measurements have relative standard deviations of 0.1% or better, making these data highly informative. Matrix-assisted laser desorption ionization (MALDI) is most frequently used with time-of-flight mass analysis O F') to measure protein molecular weights. Developmental work for these analyses have been directed toward improving the mass resolution that is achieved and discovering better matrices. Routine linear TOF mass analysis coupled with MALDI has a mass resolution of approximately 200. Resolution in these instruments can be improved to nearly 3000 with the use of post-ion source focusing to minimize the effects of translational energy spreads and the duration of ionization (93). Reflectron time-of-flight mass spectrometry and Fourier transform mass spectrometry offer better mass resolution than linear TOF instruments ( 9 4 , 95). These instruments also allow the observation of fragmentation reactions that can be utilized to determine additional structural features of a protein or peptide such as amino acid sequence (96-98). Descriptions have appeared of a number of new matrix compounds and systems. The desired features of improved matrices are better resolution and sensitivity (99, &IO), reduced alkali metal adduct formation ( Q I I ) , and the ability to utilize basic pH conditions (912). Experiments have been described in which proteins and peptides blotted onto nylon membranes were analyzed directly by MALDI-TOF (913). Investigators are also developing techniques that facilitate the recovery of proteins and peptides from acrylamide gels for both ESI and MALDI analyses. These protocols have used electroelution (914,electroblotting (&la, and pressure extrusion (916)to efficiently elute the analytes from the gels in a form that is amenable to mass spectrometric analysis. Immunoprecipitation has been used to prepare transferrin for MALDI analysis, taking advantage of the dissociation of the transferrin-antibody complex under the acidic conditions of the matrix (917). Electrospray ionization (ESI) is most frequently coupled with either quadrupole- or magnetic sector-based mass analysis. The ionization systems have improved significantly in terms of sensitivity and operating parameters such as the range of flow rates that can be used and the amenable solvent systems. Microscale ESI interfaces have been introduced that can operate at nanoliter per minute flow rates and achieve attomole- to zeptomole-level sensitivity (918,919).Hydrophobic proteins, which are dficult to analyze because of poor solubility in the acetonitrile/water or Analytical Chemistry, Vol. 67, No. 12, June 15, 1995
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methanol/water solvent systems most often used for ESI, have been analyzed using chloroform/methanol/water solvent systems (920). New interfaces employing heated capillary tips and heated coaxial gas flow have been found to produce strong, stable ion currents with low flows of 100%water (921). Array detector systems have also been found to increase the sensitivity of ESI analysis of proteins (922). Accurate molecular measurements are used to identdy structural features of proteins by characteristic mass differences. MALDI-TOF has been used to determine structural differences, including glycosylation patterns, in a series of bacterial lipases (923). Exoglycosidase treatment followed by MALDI-TOFanalysis has also been used to localize the glycosylation sites in a recombinant human tissue inhibitor of metaloproteinases (924). In another report, the determination that a neuromodulin protein contains a single phosphorylation site was based on deconvolution of the ESI mass spectrum that indicates two forms of the protein with a mass difference of 80 Da at a molecular weight of over 25 000 (925). Peptide mass mapping and amino acid sequence analysis by tandem mass spectrometry were subsequently used to describe two distinct phosphorylationsites in this protein. Three transferin isoforms in patients with carbohydrate-deficient glycoprotein syndrome have been distinguished by ESI mass analysis (926). The relative standard deviations of the molecular weight measurements in this experiment were 0.008%. Electrospray mass analysis has also been used to measure the molecular weights and confirm the correct structure of Fab fragments of antibodies with molecular weights in the 100 000 range (927). Peptide mass mapping experiments involve the measurement of molecular weights of peptides formed by chemical or enzymatic degradation of a protein. The peptide mass map produced in such an experiment is analogous to an electron ionization mass spectrum of an organic compound in that it provides mass spectral data about the component parts of the protein. Several computer algorithms have been developed that utilize the molecular weights of the peptides observed in the mass maps, along with the specificity of the degradation chemistry, to search protein data bases and identify unknown proteins (928, 929). Peptide mass mapping is particularly sensitive to modest changes in protein structure, since small molecular weight changes are more readily detected at the lower molecular weights of peptides. This type of information has been used to detect and characterize different posttranslational modications of proteins. For example, analysis of a recombinant antigen from Schistosoma mansoni detected several covalent modifications of cysteine residues including glutathione and 2-mercaptoethanol addition (930). A comparison of mass mapping data before and after alkaline phosphatase treatment has been used to locate protein phosphorylation sites (931). A similar approach has been taken to determine glycosylation sites in proteins by evaluating mass mapping data before and after glycosidase treatment (924, 932). It is likely that any moditication of a protein that produces a molecular weight change can be detected in a well-designed mass mapping experiment. Amino acid sequencing experiments represent a detailed examination of the most basic aspect of a protein’s structure. The advantages of mass spectrometric protein sequencing are the sensitivity, the ability to detect modified amino acid residues, and the fact that sequence information can be obtained from massselected peptides without the rigorous purification generally needed for Edman sequencing. Sequence analysis by mass 494R
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spectrometry is most often carried out in a tandem mass spectrometry experiment with collision-induced decomposition (CID) of the mass-selected peptide species. For example, mass spectrometric sequencing of tryptic peptides from adenylate cyclase toxin of Bordetella pertussis using ESI-tandem mass spectrometry identified the palmitoylation of a single lysine residue later shown to be a required component of the toxin activity (933). Sequence data obtained by CID analysis of intact proteins has been used to differentiate eight hemoglobin ,&chain proteins each differing by a single amino acid residue (934). These types of experiments are generally carried out in-line with liquid chromatography so that the high sensitivity of mass spectrometric methods can be applied to peptides present in mixtures. Microcapillary reversed-phase HPLC with ESI-tandem mass spectrometry has been used to sequence an extensive series of peptides isolated from major histocompatibility complex molecules in extracts of cultured tumor cells (935-937). In these experiments, peptides present at levels between approximately 100 and 500 fmol in a mixture containing approximately 2000 peptides were sequenced. The disadvantages of using tandem mass spectrometry for peptide sequencing are the difficulty and expense of operating tandem mass spectrometer system and the difticulty of interpreting the CID spectra. Other sequence analysis experiments utilize MALDI-TOF systems. One approach has been to observe metastable decompositions in reflectron-TOF instruments (96-98). A second approach has been to trap the ions prior to injection into a TOF instrument (938). In these experiments, the energetics of the desorption ionization process produces sufficient excess internal energy to fragment the peptides at the amide bonds. Reflectron TOF optics allow for proper mass analysis and produce a spectrum of daughter ions that allows deduction of the amino acid sequence. Another novel approach to protein sequencing with mass spectrometery has been to modify Edman chemistry in a manner that takes advantage of mass analysis of the products. One such method incorporates 5%phenyl isocyanate into the E d ” reaction to block 5%of the degradation reaction at each cycle (939). This chemistry produces a mixed series of peptides products that differ by one amino acid residue. In the mass spectrum of this mixture, the difference between molecular weights of these peptides corresponds to different amino acid residues. A variation of this method has been reported that utilizes trifluoroethyl isothiocyanate, a volatile analogue of the Edman reagent, to simplify the degradation protocol (940). These methods based on Edman chemistry require that the peptide of interest be purified so they cannot be applied to complex mixtures. However, the advantages of these approaches are that they utilize TOF instruments rather than tandem mass spectrometry systems and that the interpretation process is significantly simpliied. DETERMINATION OF ENDOGENOUS SPECIES The sensitivity and specificity that characterize mass spectrometric analyses are ideal for qualitative and quantitative analysis of amenable compounds in modest amounts of complex specimens such as blood and urine. The challenge of developing these analyses has generally been to expand the types of compounds that can be analyzed while ensuring that the methods are reliable, accurate, precise, and as inexpensive as possible. Gas chromatography/mass spectrometry (GC/MS) has been the mass spectrometric method most frequently applied to clinical analyses. The advantage of GC/MS in these assays is the
specificity of the analysis, enhanced by capillary gas chromatography, that provides unambiguous identification and quantitation of the species of interest. The difficulty of applying GC/MS to biological analyses is that the protocols must include laborious isolation and derivatization steps to remove the analyte from the complex, aqueous matrixes and convert it to a form that can be volatilized without thermal degradation. Therefore, although GC/ MS has been used in clinical analyses for over a decade, it has been applied to only a few classes of compounds. Steroid analyses frequently use GC/MS in part because the immunoassays are plagued by poorer specificity due to crossreactivity of the antibodies. Dehydroepiandrosterone sulfate has been quantitated by isotope dilution in human plasma using GC/ MS (941). In these experiments, correlation with radioimmunoassay indicated that the immunoassay overestimated the proper concentration. Other examples of isotope dilution assays for steroids include cortisol (942, 943),cortisone (943),7a-hydroxykholesten-Sone (944),and 7a-hydroxycholesterol (945). Profiling of urinary steroids by G U M S can be used as a tumor marker and indicator of endocrine disease. For example, a recent clinical study of the effectiveness of surgical treatment of adrenocortical carcinoma used urinary steroid profiling data in the followup phase of the study as part of the routine evaluation of recurrence (946). A steroid profiling assay for the detection and classification of steroid related endocrine disorders has also been described (947). Features of this program are its current use of a highly automated, including report preparation, tabletop GC/MS system and its developing use of LC/MS analyses. Another common application of GC/MS analyses is the detection and quantitation of carboxylic acids indicative of inborn errors in fatty acid metabolism. For example, D- and L-2hydroxyglutaric acids have been quantitated in urine, plasma, cerebrospinal fluid, and amniotic fluid by an isotope dilution assay that uses chiral capillary gas chromatographycoupled to ammonia chemical ionization mass spectrometry (948). The urinary assay was found to be useful for the detection of 2-hydroxyglutaric acidemias while the amniotic fluid assay is potentially applicable to prenatal diagnosis. Isotope dilution GC/MS assays have been developed for N-acetyl-L-aspartic acid in urine associated with Canavan-van Bogaert syndrome (949) and 3-methylglutaconic acid in urine, plasma, and amniotic fluid associated with 3-methylglutaconic aciduria (950). Another investigator has used GC/ MS profiling of infant urine to detect a series of dicarboxylic acids (951). The presence of 3-hydroxydicarboxylic and 3-ketodicarboxylic acids was found to be symptomatic of a previously undescribed defect in 3-ketoacyl-CoAthiolase activity. The use of fast atom bombardment @AB) in clinical determinations is expanding. The primary advantage of FAB is its ability to ionize polar species without the derivatization chemistry needed for GC/MS analyses. The introduction of continuous-flow FAB has allowed FAB to become a useful LC interface as well. Both FAB and continuous-flow FAB are relatively mature techniques, in use since the early to middle 1980s. However, continuing technical improvements such as the description of a multisample probe design that simplXes the analysis of multiple samples (952) and a matrix modifier that improves sensitivity for negative ion analyses (953)are indicative of ongoing interest in this technique. FAB-tandem mass spectrometry has been used to quantitate amino acids in the blood of newborns (954). This method detects a number of amino acids in methanolic extracts of human blood
using a neutral loss scan. Butyl ester derivatives of the amino acids were formed because they facilitate a characteristic fragmentation reaction for all the amino acids. The method was applied to the detection of abnormal phenylalanine and tyrosine levels for the diagnosis of phenylketonuria and tyrosinemia. This paper also contains an interesting functional description of a clinical mass spectrometry laboratory that analyzed approximately 200 sampledweek by tandem mass spectrometry at the time the paper was written (954). It is important to note that the use of the more advance technique of tandem mass spectrometry actually facilitates these analyses by simplifying the sample preparation protocols. These investigators have also developed isotope dilution FAB-tandem mass spectrometry quantitation of acylcarnitines in blood for diagnosis of medium-chain acyl-CoA dehydrogenase deficiency (955) and in amniotic fluid for prenatal detection of propionic acidaemia (956). Urinary bile acids have also been determined by FAB for the detection of inborn errors of metabolism. As with the acylcarnitines, FAB can ionize the polar bile acids without chemical derivatization to enhance volatility. The detection of 7a-hydroxy3-oxo-4cholenoic acid and 7a,l2a-dihydroxy-3-oxo-4-cholenoic acids by FAB in urine has been found to indicate 4-3-oxosteroid5-reductase deficiency (957). The urinary bile acid pattern in children with liver disease due to 3p-hydroxy-Cz~steroiddehydrogenase/isomerase deficiency has been determined by FAB (958). In the case of this disorder, early diagnosis made by timely screening allows therapy with ursodeoxycholic acid that can correct liver function and possibly prevent liver failure. Other investigators have described such a method for screening urine specimens for indicators of errors in bile acid metabolism that uses continuous-flow FAB, with either direct injection or as an HPLC interface (959). The specimens were screened by the direct injection technique capable of analyzing approximately 15 samples/h. As needed, the conknation analyses were then made by more detailed examinations using the HPLC-based technique. Newer interface techniques for liquid chromatography/mass spectrometry such as electrospray, ion spray, and atmospheric pressure chemical ionization (APCI) are now being incorporated into biological analyses. Electrospray and ion spray are closely related techniques that tend to be used with either HPLC systems employing flow rates in the 1 pL/min to approximately 50 pL/ min range or with postcolumn splitting. Atmospheric chemical ionization is better suited to the approximately 1 mL/min flow rates used in more standard HPLC systems. These interfaces are more sensitive and provide better chromatography than continuos-flow FAB. In fact, it would appear that these new interfaces are capable of performing on any of the clinical analyses currently performed by both GC/MS and FAB. For example, steroid conjugates in urine have been determined without hydrolysis by microbore HPLC-ESI for the detection of steroid disorders (947) while organic acids in urine have been determine by HPLC-APCI for the detection of inborn errors of metabolism (960). Other investigators have described the analysis of bile acids using ion spray (961). As a result, one would speculate that HPLC/MS will become the method of choice for clinical analyses of endogenous species by mass spectrometry. STABLE ISOTOPE STUDIES
A unique capability of mass spectrometry is the independent determination of specific isotopic species. This capability can be Analytical Chemistry, Vol. 67, No. 12, June 15, 1995
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used in fundamental measurements of biochemical processes in vivo such as cholesterol and fatty acid trafficking, protein and amino acid metabolism, and glucose production, as well as in measurements of body composition. These experiments require administration of a known quantity of a stable-isotopelabeled analogue of the species of interest with subsequent determination of the degree of isotope enrichment. While similar measurements can be made with radioisotopes, the use of stable isotopes is always preferable if methods for determining stable isotope incorporation are acceptable. Studies using stable isotopes require the best possible precision for the isotope ratio measurements because the quantity of the stable-isotopelabeled species added to the system must be small compared to endogenous amounts of that species. As the precision of the isotope ratio determination increases, the quantity of the tracer that is administered can decrease. These studies frequently use G U M S analyses with selected ion monitoring to determine the isotope ratios. For example, cholesterol adsorption has been calculated by determining the ratio in the blood of [2H~lcholesterol,administered orally, to [13C51cholesterol, administered intravenously, by GC/MS analysis of the acetate ester derivative (962). Other investigators measured the rate of glucose turnover by determining both 13C and 2H enrichment of blood glucose by G U M S following administration of [U-13C61glucoseand [2Hzlglucose (963). The rate of gluconeogenesis was measured by determining the labeling pattern of glucose following administration of [13C]lactate(964) or glutamate following administration of [13C]lactate and [13C]pyruvate (965). Investigations of the biochemistry of trace metals with stable isotopes are typically carried out using thermal ionization mass spectrometry to determine the isotope ratios (966, 967). Thermal ionization mass spectrometry makes the isotope ratio measurements with excellent precision, but the instruments suffer from low sample throughput. As a result, FAB and GC/MS methods have been developed for these determinations. For example, FAB analysis of iron in erthyrocytes has been used to q u a n the ~ incorporation of 58Feinto the cells (968). Multiple isotope ratios were accurately measured with an average relative standard deviation of approximately 0.9%. GC/MS methods suitable for isotope ratio measurement have been reported for several trace metals (969, 970). The key to the use of GC/MS for metals analysis is the availability of volatile metal chelates suitable for gas chromatographic analysis. One recent application has been the determination of total blood volume using j3Cr (971). Erthyrocytes in 15 mL of blood were labeled with 53Crand injected back into the subject. The dilution of the tracer was measured by GC/MS after conversion of the Cr to the trifluoroacetylacetone chelate, The use of the stable isotope is favored in making this measurement in pregnant women for evaluation of appropriate blood volume expansion. A recent development in the area of isotope ratio analyses is the advancement of combustion interfaces. These interfaces continually convert compounds eluting from a gas or liquid chromatograph into gaseous species suitable for high-precision isotope ratio determinations. One such interface uses a furnace containing Cu and Pt catalysts to combust the carbon-containing compounds eluting from a gas chromatography column to form CO2 and H20 (972). The products are carried by the helium carrier gas into a dual-inlet gas isotope ratio mass spectrometer equipped with three detectors that continually measure ion current 496R
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at m/z 44,45, and 46, respectively. As a result, these instruments combine gas chromatographic separation technology with isotope ratio mass spectrometry to measure isotope ratios in the compound of interest in complex mixtures with high precision. A comparison of gas chromatographycombustion isotope ratio (GCCIR) analysis with G U M S analysis using selected ion monitoring (SIM) for isotope ratio analyses in cholesterol found GC-CIR able to detect a 0.004 mol 36 enrichment compared to 0.06 mol % enrichment with SIM (973). An analogous liquid chromatography interface has also been described (974). This interface uses a moving wire to transfer the effluent into the combustion furnace. The ability to use liquid chromatography will facilitate the analysis of polar species by minimizing the processing steps associated with gas chromatographic analysis of polar compounds. Isotope ratio determinations are also being made with chemical reaction interfaces (975). This interface is similar to the combustion interface in that the compounds eluting from the chromatograph are converted on-line to low molecular weight products that are mass analyzed. This interface, however, can use a variety of reactive gases in this process with a corresponding variety of reaction products (975,976). As a result of this flexibility, a more conventional mass analyzer system is used so that the precision of the isotope ratio analysis is not as good as that obtained with the combustion interfaces (977). Moving belt chemical reaction interfaces for LC/MS analyses have also been described (978). SUMMARY AND SPECULATION The past five years have seen accelerating growth in the application of mass spectrometry to biological analyses. The use of electrospray ionization and matrix-assisted laser desorption ionization for the mass analysis of compounds that were once not amenable to mass spectrometry has contributed to this growth. The continued application of GC/MS, LC/MS, and FAB and the development of combustion reaction interfaces for isotope ratio analyses have also contributed. The analyses cited in this review were selected for either a current relevance to laboratory medicine or an indication of future utility. Other applications such as therapeutic drug monitoring and forensic testing also utilize mass spectrometric techniques. Although these subjects are not reviewed in this section, the reader should be aware that many of the advances discussed here can and have benefited these applications as well. As diagnosis and treatment techniques become more technically advanced, mass spectrometry will be well suited to provide novel analyses. The rapid advancement of diagnostic imaging techniques may serve as a model for future clinical applications of mass spectrometry. Where magnetic resonance spectroscopy was once a specialized laboratory tool, it is now used for intensive, high-tech structural studies of an individual's body. Similarly, while mass spectrometry is now a specialized laboratory tool, it may one day be part of intensive, high-tech characterization of an individual's biochemistry. Michael Kinter is a Research Assistant Professor of Microbiology and Pathology at the Universi of Virginia Health Sciences Center and the Program Leader in the &ass Spectromety Laboratoy in the Health Sciences Center Biomolecular Research Facility. He received his B.S. in chemisty fiom James,Madison University in 1982 and his Ph.D. in chem
