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Overview of Biochemical Applications of Mass Spectrometry*
Overview of Biochemical Applications of Mass Spectrometry Victor E Vandell and Patrick A Limbach, Louisiana State University, Baton Rouge, LA, USA & 2010 Elsevier Ltd. All rights reserved. This article is reproduced from the previous edition, volume 1, pp 84–87, & 1999, Elsevier Ltd.
Mass spectrometry is a powerful tool for the characterization of various biomolecules including proteins, nucleic acids, and carbohydrates. The advantages of mass spectrometry are high sensitivity, high mass accuracy, and more importantly, structural information. Historically, biomolecules have proven difficult to characterize using mass spectrometry. Problems often arise with impure samples, low ion abundance for analysis due to inefficient ionization processes, and low mass accuracy for higher molecular weight compounds. Advances in the development of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) now permit the analysis of biomolecules with high sensitivity and good mass accuracy. These improvements now allow the use of mass spectrometry for the identification of unknown structures and are suitable for applications focused on acquiring sequence information on the samples of interest. This article provides a brief introduction to the various applications of mass spectrometry to biomolecule analysis. More detailed discussions of particular applications of mass spectrometry to such analyses can be found in other articles in this encyclopedia.
Ionization and Mass Analysis Mass spectrometric measurements of biomolecules involve the determination of molecular mass, or of the masses of various components of the original molecule, which can then be related to various structural properties, such as amino acid sequence. In either case, the crucial step lies in the conversion of liquid- or solidphase solutions of the analytes into gaseous ions. Common ionization sources used in mass spectrometry for biomolecule analysis experiments are: fast-atom bombardment (FAB), ESI and MALDI. FAB was historically the ionization method of choice, but has been largely replaced by ESI or MALDI. ESI- and MALDI-based methods have particularly benefited the analysis of biomolecules because these techniques accomplish the otherwise experimentally difficult task of producing gas-phase ions from solution species that are both thermally labile and polar. Table 1 summarizes the different instrument configurations used in the analysis of various classes of biomolecules. MALDI has extremely high sensitivities with reports of detection limits at the sub-femtomolar level. A higher
efficiency for protonated molecular ion production with MALDI has been observed relative to FAB. MALDI typically yields intact protonated molecular ions with minimal fragmentation and is therefore commonly referred to as a soft ionization process. MALDI sources are generally coupled to time-of-flight (TOF) mass analysers. TOF mass analysers are characterized by high upper mass limits with reduced resolution at the higher masses. The production of high molecular weight ions and subsequent analysis of these ions makes the MALDITOF combination a powerful tool for biomolecule analysis. ESI can be considered a complementary method to MALDI. As with MALDI, electrospray ionization of biomolecules yields protonated or cationized molecular ions with little or no fragmentation, and it is also referred to as a soft ionization process. A particular advantage of ESI compared to MALDI is that the analyte is sampled from the solution phase. Under these conditions, ESI is readily coupled to high-performance liquid chromatography (HPLC) or capillary electrophoresis (CE) separation systems. Such combinations permit online LC-MS or CE-MS experiments. The overriding feature of an ESI-generated mass spectrum is the appearance of multiply charged ions. Multiple charging results from the loss or addition of multiple hydrogen ions or metal ions (e.g. potassium or sodium) to the
Table 1 Summary of mass spectrometry techniques used in biomolecule analysis Analyte
Ionization source
Mass analyser
Proteins/peptides
FAB MALDI ESI
Oligonucleotides
FAB MALDI ESI
Oligosaccharides
FAB MALDI ESI
Lipids
FAB MALDI ESI
Quadrupole, sector TOF, FTICR Quadrupole, sector, FTICR, TOF Quadrupole, sector TOF, FTICR Sector, FTICR, TOF Quadrupole, sector TOF Quadrupole, sector, TOF Quadrupole TOF Quadrupole, FTICR, TOF
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Overview of Biochemical Applications of Mass Spectrometry
biomolecule. The multiple charging effect is advantageous because it allows for the analysis of high molecular weight biomolecules using mass analysers with low m/z limits. The disadvantage of multiple charging is that a spectrum has to be deconvoluted and thus spectral interpretation can be complicated, especially during the analysis of mixtures.
Molecular Weight Determinations of Biomolecules Relative mass is an intrinsic molecular property which, when measured with high accuracy, becomes a unique and unusually effective parameter for characterization of synthetic or natural biomolecules. Mass spectrometry-based methods can be broadly applied not only to unmodified synthetic biomolecules, but also to modified synthetic and natural biomolecules (e.g. glycosylated proteins). The level of mass accuracy one obtains during the measurement will depend on the capabilities of the mass analyser used. Quadrupole and TOF instruments yield lower mass accuracies than sector or Fourier transform ion cyclotron resonance (FTICR) instruments. High mass accuracy is not only necessary for qualitative analysis of biomolecules present in a sample, but is necessary to provide unambiguous peak identification in a mass spectrum. The primary challenge to accurate molecular weight measurements of biomolecules is reduction or complete removal of salt adducts. In the majority of situations, the buffers used to prepare or isolate the analyte of interest contain Group 1 or Group 2 metal salts. These metal salts can potentially interfere with the accurate mass analysis of the analyte due to the gas-phase adduction of one to several metal cations to the analyte. Optimal results are obtained only after substantive (and in some cases, exhaustive) removal of these contaminants. Recent developments for sample purification involve the use of solution additives or online purification cartridges which reduce the presence of interfering salts while retaining the ability to characterize minimal amounts of analyte. Measurement of molecular mass of biomolecules is now a suitable replacement for prior methods based on the use of gel electrophoresis. Molecular weight measurement for all classes of biomolecules is a relatively routine procedure, and the results obtained are typically of greater accuracy than those previously obtained by gel electrophoresis. A common application of mass spectrometry and molecular weight measurement is for the identification of unknown proteins. The experimentally obtained molecular weight value can be searched against the available protein databases (e.g. Swiss–Prot or PIR) to potentially identify the protein. This application becomes particularly useful for the identification of unknown proteins when used in conjunction with enzymatically generated peptide fragments (see below).
Determination of the Primary Sequence of Biomolecules One of the most popular and productive uses of mass spectrometry for biomolecule analysis is the sequence or structure determination through analysis of smaller constituents of the original molecule. Two different approaches for generating the smaller, sequence informative constituents from the original molecule are available. An indirect approach is to generate sequence-specific information by solution-based chemical or enzymatic reactions. Chemical or enzymatic digestion of an intact biomolecule results in a number of smaller constituents that are amenable to mass spectrometric analysis. The so-called direct approach involves fragmentation of the analyte in the gas phase. Fragmentation can be induced by the desorption or ionization process, or can be induced by collisions with neutral target molecules or by collisions with surfaces. The analysis of fragment ions initiated by gasphase dissociation resulting from collisions with neutral molecules or surfaces are broadly referred to as tandem mass spectrometry (MS/MS) experiments. With both approaches, the sequence of the original molecule is obtained by interpreting the resulting fragments. Enzymatic Approaches Enzymatic digestion followed by mass spectrometric analysis of the resulting products is a popular and powerful approach to sequence determination. A number of enzymes are available which either are specific for a particular substituent of the biomolecule or are nonspecific but sequentially hydrolyse the analyte of interest. Trypsin is an enzyme commonly used to digest proteins into smaller peptides. Trypsin selectively cleaves proteins at the C-terminal side of lysine and arginine amino acid residues. Digestion of a protein using trypsin will generate a tryptic digest whose components are amenable to mass spectrometric analysis. In many cases, the masses of the tryptic fragments can be measured more accurately than the mass of the original molecule, thereby improving the identification of unknown proteins. As mentioned earlier, the masses of tryptic peptides, used in conjunction with molecular weight measurements of intact proteins, can be used to search known protein databases for efficient and accurate identification of unknown proteins. Sequential digestion of biomolecules is an alternative approach to determining sequence information. The utility of any mass spectrometric sequencing method that relies on consecutive backbone cleavages depends on the formation of a mass ladder. The sequence information is obtained by determining the mass difference between successive peaks in the mass spectrum. For example, phosphodiesterases are enzymes which sequentially hydrolyse the phosphodiester linkage between oligonucleotides and nucleic acids. In the
Overview of Biochemical Applications of Mass Spectrometry
case of oligodeoxynucleotides, the expected mass differences between successive peaks will correspond to the loss of: dC ¼ 289.5, dT ¼ 304.26, dA ¼ 313.27, and dG ¼ 329.27 Da. Mass ladder methods have a distinct advantage for sequence determination, because it is the difference in two mass measurements that results in the desired information. A drawback to this approach is the limited size of the analyte that is amenable to sequential digestion. Tandem Mass Spectrometry Approaches The gas-phase approach to determining structural information about biomolecules is through tandem mass spectrometry (MS/MS). Tandem mass spectrometry involves isolation of the ion of interest (commonly referred to as the parent ion in MS/MS) and then dissociating this ion via collisions with neutrals or surfaces to produce fragment ions (commonly referred to as product ions in MS/MS) from which the primary sequence of the molecular ion of interest can be determined. Generally, the fragmentation process in MS/MS experiments generates product ions which contain sequence-specific information from throughout the molecular ion of interest. Tandem mass spectrometry is applicable to all types of biomolecules but typically requires specialized mass spectrometry instrumentation for implementation. The most common tandem mass spectrometer is a triple quadrupole instrument, wherein the first and third quadrupoles are used as mass analysers and the middle quadrupole is used as a collision chamber. MS/MS can be performed with double focusing sector instruments and in some cases with specialized TOF mass analysers. Quadrupole ion traps and FTICR mass spectrometers are ideally suited for MS/MS experiments, and due to the operational characteristics of these mass analysers additional MS/MS experiments can be performed, allowing for MSn studies of biomolecules.
Higher Order Gas-phase Structure and Noncovalent Interactions Studied Using Mass Spectrometry Similar to studies performed using NMR, hydrogen/ deuterium (H/D) exchange experiments on biomolecules are feasible with mass spectrometry. Labile hydrogens can be exchanged in solution prior to mass spectrometric analysis, or alternatively H/D exchange experiments can be performed in the gas phase. The former studies have been used to localize sites of H/D exchange on
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biomolecules of interest and are used for mechanistic studies of biomolecule function. The latter studies typically involve ESI-MS and permit investigations into the gas-phase conformation of biomolecules in the absence of the solvent. The majority of H/D exchange experiments have focused on peptide and protein analysis, although the method is amenable to other classes of biomolecules. A particular advantage of ESI-MS for biomolecule analysis is realized by generating the analyte ions from solution conditions that retain the secondary, tertiary and even quaternary structure of the biomolecules. Noncovalent binding of biomolecules has been observed in the ESI mass spectrum and, when operated under the appropriate conditions, the mass spectral data are a direct probe of the solution-phase biomolecule assembly. Protein assemblies, protein–nucleic acid complexes, duplex DNA, and other noncovalently bound biomolecule assemblies have been studied using mass spectrometry. See also: Chromatography-MS, Methods, Computer Methods in Mass Spectrometry for Chemical Structure Assignment, Fast Atom Bombardment Ionization in Mass Spectrometry, Fragmentation in Mass Spectrometry, Hyphenated Techniques, Applications of in Mass Spectrometry, MALDI Techniques in Mass Spectrometry Imaging, MS Based Metabonomics, MS–MS and MSn, Nucleic Acids and Nucleotides Studied Using Mass Spectrometry, Peptides and Proteins Studied Using Mass Spectrometry, Proteomics, Quadrupoles, Use of in Mass Spectrometry, Sector Mass Spectrometers, Surface Induced Dissociation in Mass Spectrometry, Historical Perspective, Time of Flight Mass Spectrometers.
Further Reading Fenn JB, Mann M, Meng CK, Wong SF, and Whitehouse CM (1989) Electrospray ionization for mass spectrometry of large biomolecules. Science 246: 64--70. Karas M, Bahr U, and Hillenkamp F (1989) UV laser matrix desorption/ ionization mass spectrometry of proteins in the 100,000 Dalton range. International Journal of Mass Spectrometry and Ion Processes 92: 231--242. Loo JA (1995) Bioanalytical mass spectrometry: Many flavors to choose. Bioconjugate Chemistry 6: 644--665. McCloskey JA (ed.) (1990) Methods in Enzymology, vol. 193. San Diego: Academic Press, Inc. Senko MW and McLafferty FW (1994) Mass spectrometry of macromolecules: Has its time now come? Annual Review of Biophysics and Biomolecular Structure 23: 763--785. Smith RD, Loo JA, Edmonds CG, Barinaga CJ, and Udseth HR (1990) New developments in biochemical mass spectrometry: Electrospray ionization. Analytical Chemistry 62: 882--899.
Mass spectrometry is a powerful tool for the characterization of various biomolecules including proteins, nucleic acids, and carbohydrates. The advantages of mass spectrometry are high sensitivity, high mass accuracy, and more importantly, structural information. Historically, biomolecules have proven difficult to characterize using mass spectrometry. Problems often arise with impure samples, low ion abundance for analysis due to inefficient ionization processes, and low mass accuracy for higher molecular weight compounds. Advances in the development of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) now permit the analysis of biomolecules with high sensitivity and good mass accuracy. These improvements now allow the use of mass spectrometry for the identification of unknown structures and are suitable for applications focused on acquiring sequence information on the samples of interest. This article provides a brief introduction to the various applications of mass spectrometry to biomolecule analysis. More detailed discussions of particular applications of mass spectrometry to such analyses can be found in other articles in this encyclopedia.
Ionization and Mass Analysis Mass spectrometric measurements of biomolecules involve the determination of molecular mass, or of the masses of various components of the original molecule, which can then be related to various structural properties, such as amino acid sequence. In either case, the crucial step lies in the conversion of liquid- or solidphase solutions of the analytes into gaseous ions. Common ionization sources used in mass spectrometry for biomolecule analysis experiments are: fast-atom bombardment (FAB), ESI and MALDI. FAB was historically the ionization method of choice, but has been largely replaced by ESI or MALDI. ESI- and MALDI-based methods have particularly benefited the analysis of biomolecules because these techniques accomplish the otherwise experimentally difficult task of producing gas-phase ions from solution species that are both thermally labile and polar. Table 1 summarizes the different instrument configurations used in the analysis of various classes of biomolecules. MALDI has extremely high sensitivities with reports of detection limits at the sub-femtomolar level. A higher
efficiency for protonated molecular ion production with MALDI has been observed relative to FAB. MALDI typically yields intact protonated molecular ions with minimal fragmentation and is therefore commonly referred to as a soft ionization process. MALDI sources are generally coupled to time-of-flight (TOF) mass analysers. TOF mass analysers are characterized by high upper mass limits with reduced resolution at the higher masses. The production of high molecular weight ions and subsequent analysis of these ions makes the MALDITOF combination a powerful tool for biomolecule analysis. ESI can be considered a complementary method to MALDI. As with MALDI, electrospray ionization of biomolecules yields protonated or cationized molecular ions with little or no fragmentation, and it is also referred to as a soft ionization process. A particular advantage of ESI compared to MALDI is that the analyte is sampled from the solution phase. Under these conditions, ESI is readily coupled to high-performance liquid chromatography (HPLC) or capillary electrophoresis (CE) separation systems. Such combinations permit online LC-MS or CE-MS experiments. The overriding feature of an ESI-generated mass spectrum is the appearance of multiply charged ions. Multiple charging results from the loss or addition of multiple hydrogen ions or metal ions (e.g. potassium or sodium) to the
Table 1 Summary of mass spectrometry techniques used in biomolecule analysis Analyte
Ionization source
Mass analyser
Proteins/peptides
FAB MALDI ESI
Oligonucleotides
FAB MALDI ESI
Oligosaccharides
FAB MALDI ESI
Lipids
FAB MALDI ESI
Quadrupole, sector TOF, FTICR Quadrupole, sector, FTICR, TOF Quadrupole, sector TOF, FTICR Sector, FTICR, TOF Quadrupole, sector TOF Quadrupole, sector, TOF Quadrupole TOF Quadrupole, FTICR, TOF
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Overview of Biochemical Applications of Mass Spectrometry
biomolecule. The multiple charging effect is advantageous because it allows for the analysis of high molecular weight biomolecules using mass analysers with low m/z limits. The disadvantage of multiple charging is that a spectrum has to be deconvoluted and thus spectral interpretation can be complicated, especially during the analysis of mixtures.
Molecular Weight Determinations of Biomolecules Relative mass is an intrinsic molecular property which, when measured with high accuracy, becomes a unique and unusually effective parameter for characterization of synthetic or natural biomolecules. Mass spectrometry-based methods can be broadly applied not only to unmodified synthetic biomolecules, but also to modified synthetic and natural biomolecules (e.g. glycosylated proteins). The level of mass accuracy one obtains during the measurement will depend on the capabilities of the mass analyser used. Quadrupole and TOF instruments yield lower mass accuracies than sector or Fourier transform ion cyclotron resonance (FTICR) instruments. High mass accuracy is not only necessary for qualitative analysis of biomolecules present in a sample, but is necessary to provide unambiguous peak identification in a mass spectrum. The primary challenge to accurate molecular weight measurements of biomolecules is reduction or complete removal of salt adducts. In the majority of situations, the buffers used to prepare or isolate the analyte of interest contain Group 1 or Group 2 metal salts. These metal salts can potentially interfere with the accurate mass analysis of the analyte due to the gas-phase adduction of one to several metal cations to the analyte. Optimal results are obtained only after substantive (and in some cases, exhaustive) removal of these contaminants. Recent developments for sample purification involve the use of solution additives or online purification cartridges which reduce the presence of interfering salts while retaining the ability to characterize minimal amounts of analyte. Measurement of molecular mass of biomolecules is now a suitable replacement for prior methods based on the use of gel electrophoresis. Molecular weight measurement for all classes of biomolecules is a relatively routine procedure, and the results obtained are typically of greater accuracy than those previously obtained by gel electrophoresis. A common application of mass spectrometry and molecular weight measurement is for the identification of unknown proteins. The experimentally obtained molecular weight value can be searched against the available protein databases (e.g. Swiss–Prot or PIR) to potentially identify the protein. This application becomes particularly useful for the identification of unknown proteins when used in conjunction with enzymatically generated peptide fragments (see below).
Determination of the Primary Sequence of Biomolecules One of the most popular and productive uses of mass spectrometry for biomolecule analysis is the sequence or structure determination through analysis of smaller constituents of the original molecule. Two different approaches for generating the smaller, sequence informative constituents from the original molecule are available. An indirect approach is to generate sequence-specific information by solution-based chemical or enzymatic reactions. Chemical or enzymatic digestion of an intact biomolecule results in a number of smaller constituents that are amenable to mass spectrometric analysis. The so-called direct approach involves fragmentation of the analyte in the gas phase. Fragmentation can be induced by the desorption or ionization process, or can be induced by collisions with neutral target molecules or by collisions with surfaces. The analysis of fragment ions initiated by gasphase dissociation resulting from collisions with neutral molecules or surfaces are broadly referred to as tandem mass spectrometry (MS/MS) experiments. With both approaches, the sequence of the original molecule is obtained by interpreting the resulting fragments. Enzymatic Approaches Enzymatic digestion followed by mass spectrometric analysis of the resulting products is a popular and powerful approach to sequence determination. A number of enzymes are available which either are specific for a particular substituent of the biomolecule or are nonspecific but sequentially hydrolyse the analyte of interest. Trypsin is an enzyme commonly used to digest proteins into smaller peptides. Trypsin selectively cleaves proteins at the C-terminal side of lysine and arginine amino acid residues. Digestion of a protein using trypsin will generate a tryptic digest whose components are amenable to mass spectrometric analysis. In many cases, the masses of the tryptic fragments can be measured more accurately than the mass of the original molecule, thereby improving the identification of unknown proteins. As mentioned earlier, the masses of tryptic peptides, used in conjunction with molecular weight measurements of intact proteins, can be used to search known protein databases for efficient and accurate identification of unknown proteins. Sequential digestion of biomolecules is an alternative approach to determining sequence information. The utility of any mass spectrometric sequencing method that relies on consecutive backbone cleavages depends on the formation of a mass ladder. The sequence information is obtained by determining the mass difference between successive peaks in the mass spectrum. For example, phosphodiesterases are enzymes which sequentially hydrolyse the phosphodiester linkage between oligonucleotides and nucleic acids. In the
Overview of Biochemical Applications of Mass Spectrometry
case of oligodeoxynucleotides, the expected mass differences between successive peaks will correspond to the loss of: dC ¼ 289.5, dT ¼ 304.26, dA ¼ 313.27, and dG ¼ 329.27 Da. Mass ladder methods have a distinct advantage for sequence determination, because it is the difference in two mass measurements that results in the desired information. A drawback to this approach is the limited size of the analyte that is amenable to sequential digestion. Tandem Mass Spectrometry Approaches The gas-phase approach to determining structural information about biomolecules is through tandem mass spectrometry (MS/MS). Tandem mass spectrometry involves isolation of the ion of interest (commonly referred to as the parent ion in MS/MS) and then dissociating this ion via collisions with neutrals or surfaces to produce fragment ions (commonly referred to as product ions in MS/MS) from which the primary sequence of the molecular ion of interest can be determined. Generally, the fragmentation process in MS/MS experiments generates product ions which contain sequence-specific information from throughout the molecular ion of interest. Tandem mass spectrometry is applicable to all types of biomolecules but typically requires specialized mass spectrometry instrumentation for implementation. The most common tandem mass spectrometer is a triple quadrupole instrument, wherein the first and third quadrupoles are used as mass analysers and the middle quadrupole is used as a collision chamber. MS/MS can be performed with double focusing sector instruments and in some cases with specialized TOF mass analysers. Quadrupole ion traps and FTICR mass spectrometers are ideally suited for MS/MS experiments, and due to the operational characteristics of these mass analysers additional MS/MS experiments can be performed, allowing for MSn studies of biomolecules.
Higher Order Gas-phase Structure and Noncovalent Interactions Studied Using Mass Spectrometry Similar to studies performed using NMR, hydrogen/ deuterium (H/D) exchange experiments on biomolecules are feasible with mass spectrometry. Labile hydrogens can be exchanged in solution prior to mass spectrometric analysis, or alternatively H/D exchange experiments can be performed in the gas phase. The former studies have been used to localize sites of H/D exchange on
2057
biomolecules of interest and are used for mechanistic studies of biomolecule function. The latter studies typically involve ESI-MS and permit investigations into the gas-phase conformation of biomolecules in the absence of the solvent. The majority of H/D exchange experiments have focused on peptide and protein analysis, although the method is amenable to other classes of biomolecules. A particular advantage of ESI-MS for biomolecule analysis is realized by generating the analyte ions from solution conditions that retain the secondary, tertiary and even quaternary structure of the biomolecules. Noncovalent binding of biomolecules has been observed in the ESI mass spectrum and, when operated under the appropriate conditions, the mass spectral data are a direct probe of the solution-phase biomolecule assembly. Protein assemblies, protein–nucleic acid complexes, duplex DNA, and other noncovalently bound biomolecule assemblies have been studied using mass spectrometry. See also: Chromatography-MS, Methods, Computer Methods in Mass Spectrometry for Chemical Structure Assignment, Fast Atom Bombardment Ionization in Mass Spectrometry, Fragmentation in Mass Spectrometry, Hyphenated Techniques, Applications of in Mass Spectrometry, MALDI Techniques in Mass Spectrometry Imaging, MS Based Metabonomics, MS–MS and MSn, Nucleic Acids and Nucleotides Studied Using Mass Spectrometry, Peptides and Proteins Studied Using Mass Spectrometry, Proteomics, Quadrupoles, Use of in Mass Spectrometry, Sector Mass Spectrometers, Surface Induced Dissociation in Mass Spectrometry, Historical Perspective, Time of Flight Mass Spectrometers.
Further Reading Fenn JB, Mann M, Meng CK, Wong SF, and Whitehouse CM (1989) Electrospray ionization for mass spectrometry of large biomolecules. Science 246: 64--70. Karas M, Bahr U, and Hillenkamp F (1989) UV laser matrix desorption/ ionization mass spectrometry of proteins in the 100,000 Dalton range. International Journal of Mass Spectrometry and Ion Processes 92: 231--242. Loo JA (1995) Bioanalytical mass spectrometry: Many flavors to choose. Bioconjugate Chemistry 6: 644--665. McCloskey JA (ed.) (1990) Methods in Enzymology, vol. 193. San Diego: Academic Press, Inc. Senko MW and McLafferty FW (1994) Mass spectrometry of macromolecules: Has its time now come? Annual Review of Biophysics and Biomolecular Structure 23: 763--785. Smith RD, Loo JA, Edmonds CG, Barinaga CJ, and Udseth HR (1990) New developments in biochemical mass spectrometry: Electrospray ionization. Analytical Chemistry 62: 882--899.