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Chapter 1

Electrospray: A Popular Ionization Technique for Mass Spectrometry A. Peter Snyder

Downloaded by 5.62.159.165 on October 1, 2016 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1995-0619.ch001

U.S. Army Edgewood Research, Development and Engineering Center, Aberdeen Proving Ground, MD 21010-5423

A comparison of major sample ionization techniques for mass spectrometry (MS) is reviewed in a quantitative and qualitative fashion. The Proceedings of the American Society for Mass Spectrometry (ASMS) for the years1979-1995were consulted with respect to ionization methods. Trends were established and comparisons noted for the total number and percentage of papers with selected ionization techniques. Electrospray ionization (ESI) and its derivatives are noted in particular, and the total number as well as percentage of ESI papers presented at the 1994 and 1995 ASMS conferences exceeded that of all ionization techniques including the ubiquitous electron ionization method. Descriptions of the contents of the book and overviews of the scientific fields are presented where analytical, biological, biotechnological, biochemical, environmental, immunological, microbiological and pharmaceutical applications of ESI-tandem mass spectrometry occupy central roles. The chapters are grouped around the following topics: tutorial and mechanisms of ESI, the internal and surface molecules and macromolecules of bacterial cells, non-covalent biomolecule association and interactions, nucleic acids, drugs, drug metabolites, marine toxins, man-made environmental contaminants, enzyme active sites, immunological processes, protein identification with database analysis, recombinant and post-translational protein investigations, and complementary protein structure and function information from ESI and matrix-assisted laser desorption/ionization (MALDI). If only Malcolm Dole could have known. That is, the great potential of a relatively simple technique known as electrospray ionization (ESI) was just that for more than a decade. But that is what the stuff of science is about. We only need to look in our own backyard for examples. Witness the length of time that it took to advance the

This chapter not subject to U.S. copyright Published 1996 American Chemical Society

Snyder; Biochemical and Biotechnological Applications of Electrospray Ionization Mass Spectrometry ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


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BIOLOGICAL AND BIOTECHNOLOGICAL APPLICATIONS OF ESI-MS

technique of gas chromatography (GC), from a very basic column system using, not mega, but literally macro-bore, rigid columns in the early 1950s (1,2), to the ubiquitous capillary variety of the eighties which are standard today. These changes in column usage occurred over a thirty year gestation and maturation period. Even more sobering is the time span for evolution of mass spectrometry (MS). The formative years for MS were 1900-1940 (3) to provide for commercially-available computerized quadrupole and sector mass spectrometers. In the forties and fifties, MS could be noted for its basic characterization concerning different analyzer geometries. In the late fifties, the beginnings of fundamental yet formulation-building applications on chemical compounds were taking effect, and it wasn't until the mid-to-late eighties that MS was taken from the oscilloscope to the personal computer. A few publications and presentations on electrospray ionization (ESI), mainly by Dole, can be found from the late sixties to the early eighties (4-10). This literature found ESI interfaced to ion mobility spectrometry (IMS) - a low resolution, poor man's, but cheap, pseudo-mass spectrometer which operates at atmospheric pressure. Even at his retirement in 1984, Dole employed IMS as a detector for ESI (11, 12). At that time he was attempting to use ESI to deliver large compounds into an IMS analyzer. Thus, a superior sample ionization technique was interfaced to a low resolution device, however, an important advantage here is that both the analyzer and sample introduction device conveniently operate at atmospheric pressure. But where was mass spectrometry with respect to ESI? Around that time, John Fenn must have had a higher form of resolution in mind, because he, along with Yamashita and Whitehouse (13-15), conducted experiments to show the usefulness of Dole's ESI sample transfer and ionization method to a mass spectrometer. The enticing aspect of ESI and MS as partners in science is that no heat, vacuum or sophisticated interfaces are needed. Just some pressure is required. Pressure pushes the liquid-containing analyte past the tip of a metal needle that has applied kilovolt voltages. The high electric field density places many charges on and/or in the many tiny droplets of liquid spraying from the ESI needle tip. The transition from highly charged, tiny droplets to gas phase ions doesn't seem like a big deal, but it is. It is a highly controversial subject. A number of theories have been offered and Chapter 3 is a very timely report on the subject. John Fenn has attempted to place the various theories into a rational perspective. The philosophy in preparing this book was that electrospray is ready to be adopted by the entire biological community. In practice, this philosophy is envisioned as two intertwined goals in that the contents of this book would appeal to the uninitiated as well as to the seasoned practitioner. A strength of this book is in the many applications of ESI-MS from analytical, biochemical, biological, biotechnoiogical, environmental, immunological, microbiological, and pharmaceutical perspectives as observed from a varied group of people. Biologists, biochemists, microbiologists, chemists and their analytical counterparts are all represented. A Survey of Electrospray Ionization In order to appreciate the scientific experience of these participants, an introduction to the practice of electrospray ionization and the major parameters that affect ESI performance appeared to be a useful addition. Tom Covey was one of the pioneers of



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Electrospray: A Popular Ionization Technique for MS 3

the electrospray technique when in the laboratory of Jack Henion, an outer sheath of nebulization gas was added coaxially near the tip of the sample introduction capillary, thus, nebulization-assisted electrospray, or ionspray, was born. Tom provides an overview in Chapter 2 of the ESI process, what makes it tick, and the operational and logistic choices an operator has, and together, both Chapters 2 and 3 provide the reader with a concise report on the operational and theoretical aspects of ESI-MS. The vast array of problems that can be addressed by the relatively simple technique of ESI-MS make it a powerful, even revolutionary tool. A barometer for the previous statement can be argued as that of the Proceedings of the American Society for Mass Spectrometry Conferences on Mass Spectrometry and Allied Topics (ASMS Proceedings) (16). The ASMS Proceedings provides the mass spectrometry practitioner with the latest in research, applications and hardware development. These Proceedings can also provide a competitive database for sample introduction and ionization techniques for mass spectrometry systems. Hence, important clues can be obtained as to the trends of sample introduction/ionization techniques in terms of which have succeeded in a relative sense and for how long. By noting the trends, it may be possible to predict, postulate or at least to observe which relatively new ideas are worth watching and investigating. The ASMS Proceedings were consulted by noting every presentation, and some papers used multiple techniques. Table 1 describes the various abbreviations and names for each sample introduction/ionization technique found in the pages of the 27th-43rd ASMS Proceedings. Some techniques are known by a variety of names and acronyms. In certain instances, a number of different techniques have been combined into one category. For purposes of clarity, one abbreviation was chosen to represent each category, and that abbreviation is presented first. Provision for an explanation of each technique is outside the scope of this Introduction, however, the reader can refer to the appended references. Figures 1 and 2 represent trends of the seven most commonly used methods of sample ionization. Figure 1 plots the year vs. the total number of papers in the respective ASMS Proceedings featuring each ionization method, and Figure 2 plots the year vs. the percentage of papers of each of the ionization methods with respect to the total number of ASMS Proceedings papers in the respective year. Table 2 presents a survey of the number of papers containing other types of sample introduction and ionization techniques for MS. In Figures 1 and 2, ESI, MALDI and LIMS refer to the categories; EI refers to the EI category plus that part of the following categories of papers which use the EI technique: Py methods, SID, GC-EI, PB and SFC. In Figures 1 and 2, CI refers to the CI category plus that part of the following categories which use the CI technique: Py methods, heated nebulizer APCI, DCI, GC-CI, PB and SFC. In Figures 1 and 2, the FAB and SIMS categories have been combined, but are tabulated separately in Table 2. The SFC and Py categories in Table 2 represent the total number of ASMS Proceedings papers for each sample introduction method, regardless of ionization technique. Over the analyzed time span, it appears that the number of LIMS investigations have approximately doubled (Figure 1), yet Figure 2 shows that LIMS has not grown in overall stature compared to fifteen years ago. Thus, it can be assumed that since 1979, LIMS has retained a relatively constant percentage of investigators as the MS community has grown, while nevertheless, doubling in size. LIMS realizes a relatively


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Table 1. Glossary of sample ionization/introduction techniques to mass spectrometry analyzers. Abbreviation

Reference

Nomenclature

ESI, ESP, ES, ESPI ISP IE

electrospray ionization ionspray ion evaporation ultraspray

17, 18 17, 18 17, 18 19

API APCI

atmospheric pressure ionization atmospheric pressure chemical ionization high pressure ionization radioactive 63-Ni energetic (beta) electron emission source

18

heated nebulizer APCI atmospheric pressure spray ionization

18

APS MALDI

FD FI

matrix-assisted laser desorption/ ionization aerosol MALDI, laser spray ionization field desorption field ionization

18 18 18, 20

21 22, 23 22 24 24 Continued on next page


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Electrospray: A Popular Ionization Technique for MS

Table 1. Continued


Abbreviation EI NRMS SIFT FA EB flow EI-PD EI-PID EC

Reference

Nomenclature electron ionization neutralization-reionization MS selected ion flow tube flowing afterglow electron bombardment flow El-photodissociation El-photon-induced dissociation electron capture electron attachment

23 25 26 26 27 28 28 29, 30

The following differ in the method of sample introduction to an EI source HPLC

LD

High pressure liquid chromatography-moving belt direct liquid introduction Townshend discharge Knudson cell membrane inlet MS chemical reaction interface MS supersonic molecular beam laser desorption

CI

chemical ionization

DLI MIMS CRIMS

31 32 33 34 35 36 37 38 23

The following differ in the method of sample introduction to a CI source. HPLC DLI LD

High pressure liquid chromatography-moving belt direct liquid introduction laser desorption

31 32 39

GC-EI-MS

29, 30

GC-CI-MS

29, 30 Continued on next page


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Table 1. Continued Abbreviation

40 41 41

2

desorption chemical ionization

42

3

surface-induced dissociation

43

particle beam, Thermabeam Monodisperse Aerosol Generation Interface for Combining LC with MS

44

DEP DCI Downloaded by 5.62.159.165 on October 1, 2016 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1995-0619.ch001

Reference

pyrolysis thermal desorption thermogravimetric analysis direct insertion probe, direct probe, solids probe direct exposure probe

1

Py TD TGA DIP

SID

Nomenclature

4

PB MAGIC-LC

42 42

44

FAB Cf-FAB

fast atom bombardment continuous flow FAB

23, 45, 46 45, 46

SIMS LSIMS

secondary ion MS liquid SIMS gaseous SIMS fast ion bombardment pulsed ion bombardment liquid secondary ionization liquid ion MS liquid metal ionization cesium SIMS gallium SIMS Internal Ion Impact Ionization

23, 46, 46, 46, 46, 46, 46, 46, 46, 46, 46,

FIB PIB LSI LIMS LMI Cs Ga +

+

LIMS RIMS REMPI MPI/MUPI/MPRI LAMMA LD-LI PD/PI PIPICO PEPICO/PIPECO PD

47, 48 47 47 47 47 47 47 47 47 47 47

38 laser ionization MS resonance ionization MS 38 resonance-enhanced multiphoton ionization 38 38 multiphoton resonance ionization laser microprobe mass analysis 38 laser desorption or laser ablationionization 23, 38 23, 38 photodissociation/photoionization photoion-photoion coincidence spectrometry 49 photoelectron photoion coincidence spectrometry 50, 51 plasma desorption, C f 23, 52 Continued on next page 252


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Table 1. Continued Abbreviation GD HCP

Reference 23, 53 53 53

MIP SSMS

glow discharge hollow cathode discharge Penning ionization atmospheric sampling glow discharge ionization microwave-induced plasma spark source MS

ICP

inductively coupled plasma

23, 57

TSP LIMS EHD, EHDI

thermospray plasmaspray liquid ionization MS electroheterodynamic ionization

58, 59 58, 59 60 61

TIMS

thermal ionization MS

62

AMS

accelerator mass spectrometry

63

potassium ionization of desorbed species

64

supercritical fluid chromatography

65

ASGDI Downloaded by 5.62.159.165 on October 1, 2016 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1995-0619.ch001

Nomenclature

+

K IDS SFC or SCF

5

|

54 55 56

!

These methods are heating or sample introduction methods, not ionization techniques. EI and CI dominate as ionization techniques. 2

This is the analogue of the EI-DEP method, because the sample is heated on a probe in a torr pressure CI source.

3

This is a sample introduction method for EL

4

These are sample introduction techniques for EI or CI.

5

Sample introduction method for ionization methods including EI, CI, TSP, APCI, LD and photodissociation.


8


550-,

500Η


45θΗ

400-

350-]

•

ESI

Δ

MALDI

•

El

X

FAB/SIMS

•

•

CI Ο

LIMS

300H

•

•

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X

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S2 250H

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200-|

J

D

X

150H X

•

100-

Δ

x

χ

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ο

ο

9

I

· Δ

X

ο Ο

50H

Ρ

79

°

8

ο

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Α

80

ο ^ Α 83

Α

Α

—Δ

86

Δ.

I 89

Τ" 92

95

Year 19

Figure 1.

Calendar year vs. number of papers in the ASMS Proceed ings for the most common methods of sample ionization for λβ! analysis.



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_

•

60

50-

A

ESI

X

FAB/SIMS

Δ

MALDI

·

CI

•

El

Ο

LIMS

• • •

40-

• • • • •

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X

30-

A

χ Χ χ

20-

10-

f> ° δ ο ο

f ? 79 80

A à

ο

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86

r

89

92

95

Year 1 9 _

Figure 2.

Calendar year vs. the percentage of papers in the ASMS Proceedings for the most common methods of sample ionization for MS analysis.



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low percentage of the MS ionization techniques (Figure 2) averaging 6.6% in 1995 and 9% over the past seventeen years. However, that should be considered a healthy percentage of the entire pool of MS ionization techniques. In the 1979-1988 time period the FAB/SIMS desorption techniques steadily gained in popularity, slowly leveled off in the early nineties, and then took a sudden drop in usage in 1994 (Figure 1). Figure 2 shows essentially that the relative use of FAB/SIMS took a somewhat bell-shaped curve where the techniques peaked in the late 1980s. Nevertheless, in 1995, the desorption techniques showed an 8% incidence of use in all reported MS investigations. The use of CI ionization techniques has varied in approximately three cycles from 70-100 papers per year between 1979-1993, and 1994 had a significant increase in the number of CI reports (Figure 1). However, Figure 2 shows a steady decline in the overall percentage of presentations, rangingfromapproximately 20% in the early 1980s to 9% in 1995. MALDI is a relatively recent phenomenon in mass spectrometry and since 1990 it launched into a steep, geometric increase in the number of reports (Figure 1). Likewise, as Figure 2 attests, its share in the amount of papers rose at an impressive rate to that of approximately 19% in 1995. Electron ionization has had a commanding lead over the years. EI clearly dominated as the ionization method of choice in the early to mid 1980s (Figure 1) despite a significant drop in the number of investigations. Then EI took a turn for the better in the mid-1980s and sustained a consistent increase up until 1993, all the while leading in the number of reports as the method for sample ionization. However, Figure 2 shows another analysis in that EI posted a steady downward trend. As stated, EI was the front-runner in total number and percentage of investigations in the ASMS Conferences. ESI, the star of this book, unseated EI in 1994 in the areas of this survey. It is surprising to note that ESI had the longest gestation period (acceptance? public relations?) of all major ionization techniques. An ESI-IMS report by Dole (10) as well as a paper on ion evaporation (66) were presented at the 1981 ASMS Conference. Since then, approximately 30 papers were presented up to 1988; this is an eight year time period. In 1983, there were no scientific reports using ESI as an ionization technique. ES (electrospray) did appear in one report by Beavis et al. however, not as an ionization technique (67). ES was used as a sample transfer technique to a flat plate which interfaced to a SIMS source. However, since 1989, ESI was taking hold in the MS community, and it was promoted in a geometric fashion up to the present. Figure 2 shows that ESI has climbedfromobscurity to a very impressive 35.8% of total ASMS presentations in 1995. However, even GC, a current workhorse of many a laboratory, followed a similar track record in the literature (68). Its literary gestation spanned the 1940s until 1955 when it started a geometric increase in the rate of publications. EI, in contrast, has steadily decreased in its share of scientific investigations in the ASMS Proceedings from a grand high (and probably never to be reached again by any MS method) of 64% in 1980 (nearly two-thirds of all papers) to 25.8% in 1995. A further analysis (Figure 2) reveals some interesting trends and noteworthy points. 1993 saw ESI and EI sharing essentially the same percentage of investigations at 26.5% and 27.3 %, respectively. In 1994, ESI (30.9%) jumped ahead of EI (28.7%) and increased its share in 1995. These relative and absolute percentage levels of sample ionization methods in the MS user community were matched only once, in 1988, 9



FD TIMS TSP AMS K+IDS GD ICP SFC Py FAB SIMS Total number papers

82

17 18 7 0 0 7 4 0 17 35 25 485

81

16 9 4 0 0 9 2 0 18 21 18 433

80

29 6 2 0 0 8 0 0 22 0 8 430

79

31 9 2 0 0 10 1 0 18 0 12 443

84 9 8 21 0 0 6 2 0 10 73 34 452

83 14 13 11 0 0 13 4 0 21 71 34 517

89 8 9 43 0 2 19 14 2 21 140 53 732

88 6 8 60 0 3 9 1 2 25 161 58 703

87 6 5 47 0 2 7 7 3 19 101 37 590

86 5 8 55 0 1 3 8 1 20 109 35 555

85 7 10 37 0 0 6 1 0 17 81 38 496

95 7 2 15 1 0 25 9 5 29 69 47 1444

94 2 3 19 0 1 20 9 1 24 69 54 1169

93 7 2 33 4 1 23 11 1 24 100 81 1099

92 4 0 37 0 0 19 8 0 14 123 81 994

91 4 3 46 0 1 15 5 5 24 136 57 862

90 1 4 55 0 1 20 5 1 26 142 49 760

Table 2. Number of papers of the relatively lesser used ionization and sample introduction techniques in the ASMS Proceedings in the years 1979-1995.


§

I

«2

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between FAB/SIMS (31.3%) and EI (35.7%). The relatively significant drop in percentage of investigations by EI in the 1980s could have been mostly due to the popularity of the FAB/SIMS desorption techniques. EI dropped from 60% to a 35% level while FAB/SIMS rose from a few percent to 25-30%. Thus it appears that FAB/SIMS pioneered the way as new and more versatile methods for sample ionization at the expense of EI. Then, FAB/SIMS decreased in relative popularity, and this appears to coincide with the relative increase in ESI and MALDI techniques. Even though FAB/SIMS usage decreased in the late 1980s and early 1990s, the popularity of the ESI and MALDI techniques appears to have not only made up for the loss of FAB/SIMS investigations, but they also caused further inroads in the overall share of the EI research programs. In this time frame, EI decreased from 35.5% in 1989 to 25.8% in 1995. Biological Applications of Electrospray Ionization Mass Spectrometry As the survey indicates, ESI has become one of the leading sample introduction/ionization techniques for MS, and the literature shows that it has made a major impact on the bio-related areas of pure and applied science and technology. Thus, it appeared desirable to gather a series of scientific contributions which presented timely biological applications with ESI-MS-MS. We start with the bacterial cell, a complex entity unto itself and entirely too formidable to be considered an analyte for an ESI-MS analysis. Thus, monomelic, oligomeric and polymeric components must be considered in an elucidation of structure as it relates to function as well as detection and characterization of the microbial entity. Chapters 4 and 5 present chemical and physical methods in the characterization of the peptidoglycan- associated sugar monomers and glycopeptide oligomers, respectively. Black and Fox show in Chapter 4 that it is possible to devise methods that can become a part of a comprehensive identification scheme for bacteria. This is based on the neutral and amino-sugar profiles of hydrolyzates of bacterial cells, and a large portion of the sugars derivefromthe the cell wall peptidoglycan and lipopolysaccharide layers. Comparisons are made on time-consuming derivatization methods with GC-MS and analyses consisting of ESI-MS/MS procedures on underivatized, direct hydrolyzates. Chapter 5 also discusses the peptidoglycan layer, however,froma structural perspective. Lee et al. analyze the glycopeptide components of peptidoglycan, because that is where antibiotic action resides. By probing antibiotic-resistant bacteria, such as E. faecalis and Leuconostoc, alterations were discovered in the peptide portion of the peptidoglycan from that of the native molecular architecture. These structural studies led to the development of an assay to determine the binding affinity of antibiotics such as vancomycin with the native and altered glycopeptide components of peptidoglycan. Chapters 6-10 are concerned with the surface of the organism where there exists complex arrays of biochemical entities. Organisms must communicate and interact in some fashion with other organisms, cellsfroma host body, and protein antibodies from host cells. Structure-function relationships enter into these complex recognition events by the bacterial cell with host components during infection. Reinhold and Reinhold (Chapter 6) explore the complex bacterial surface motif which includes polysaccharides and lipopolysaccharides. These surface entities contribute to toxic host responses, and as such, biochemical extraction and selected modifications of these macromolecules



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Electrospray: A Popular Ionization Technique for MS13

provide important information on their composition. This chapter also is an excellent primer in the understanding of these two classes of bacterial macromolecules. Thibault et al. (Chapter 7) tackle this problem through the determination of the "bare" oligosaccharide nucleus of the lipopolysaccharide (LPS) of Pseudomonas aeruginosa with subsequent determination of the various pendant groups and modified carbohydrate residues. An appreciation to detail is presented in the reconstruction of the intact LPS species. Gibson et al. (Chapter 8) concentrate on finding the select lipooligosaccharides (LOS), among many surface species, that are relevant to the disease processes in Haemophilus. To aid in this determination, various forms of chemically-processed LOS were investigated. Cole (Chapter 9) uses acid and base chemical procedures to determine the decomposition pathways of lipid A in the LPS endotoxin structure. Various chemical reaction parameters are implemented and yield chemical detoxification procedures which can be correlated with degrees of reduced toxicity for the lipid A fragments from Enterobacter agglomérons and Salmonella minnesota organisms with ESI-MS and PDMS. Chapter 10 discusses a class of surface biomarker that is relatively low in molecular weight and high in the number of different isomers and species. The major histocompatibility complex (MHC) consists of a broad class of glycoprotein surface immune markers which bind an extremely diverse array of peptides, and the latter act as antigenic determinants for cells. Yates et al. present studies on mutant cell surface markers in order to identify differences in the peptide arrays from that of the native cell. These differences can provide clues as to the cellular mechanisms and structure-antigenic activity of the MHC species and are important steps toward the goal of developing drugs to combat diseases. Chapter 10 also offers a scheme for the identification of these antigenic small peptides based on a comparison of the raw ESIproduct ion mass spectrum, a synthetic mass spectrum, and a search of a database containing known protein sequences. A different, automated method of protein identification is given in Chapter 11. The hallmark of these automated methods is that the investigator is relieved of having to manually interpret a complex peptide fragment mass spectrum. This process can be very time consuming, and expert knowledge is usually required for unraveling series of peptide ions and being alert to the fact that not all potential peptide ion sequences in a protein may be present in a mass spectrum. Stults et al. present a scheme for protein identification with ESI-MS/MS and twodimensional gel electrophoresis (Chapter 11) by digesting a series of proteins with the same enzyme and sorting the mass spectral sequences. As few as three or four peptides from a complex protein digest can be used to differentiate one protein from another, and this technique givesriseto peptide mass fingerprinting. Complex protein extracts from normal and enlarged heart cells, the latter of which leads to congestive heart failure, is illustrative of this identification method. Lipids represent an important class of compounds in cellular systems. The control of the mosquito insect pest is usually accomplished by chemical pesticides, however, it may be possible to control mosquito presence in certain environmental situations by the introduction of fungal species. Lipids could play an important role, because they are required for successful invasion of the mosquito by the fungal parasite. Kerwin provides an analysis on the Lagenidium giganteum water mold that is a parasite on mosquito larva (Chapter 12). Both need phospholipids and eicosanoids, yet both do not synthesize or produce these necessary metabolic compounds. Thus ESI-MS/MS was used to study the metabolic profile of these compounds during water fungal mold



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infection under controlled diet and growth conditions. Chapter 13 explores the major classes of phospholipid in glia cells and rodent brain membrane tissue. Kim et al. conduct investigations with respect to the qualitative and quantitative aspects of the dynamics of phospholipid turnover and maintenance in these cells using ESI-MS/MS. All cellular functions ultimately focus on DNA, the biological molecule which encodes life as we know it. ESI-MS has created in-roads into the structure and function aspects of nucleic acids by tackling such complex molecules as high molecular weight single and double stranded DNA (69). Both transfer ribonucleic acid (t-RNA) (70) and, incredibly, the 110 megadalton DNA from coliphage T4 (71) have yielded to molecular weight studies. Polymerase chain reaction, or PCR, is a technology which amplifies or increases the amount and concentration of a specific sequence of DNA which can be present in trace levels. Only one DNA strand containing a specific sequence of nucleotides is required. PCR has received recent coverage in the popular and scientific news because its forensic utility has signficant use in a judicial court of law (72, 73). Analysis is usually accomplished by staining the DNA and performing gel electrophoresis. ESI-MS/MS has recently been shown to be able to analyze for PCR gene products (74). In order for the reader to appreciate these concepts, Iden (Chapter 14) presents a primer on the basics of an understanding of the primary sequences of DNA oligomers with ESI-MS/MS. The four primary DNA bases can also be found in modified states, and strategies for their characterization are also presented. Biochemical enzyme reactions (refer to Chapter 19) as well as electrostatic, noncovalent biomolecule interactions are the lifeblood for cellular function and recognition events. The latter interaction finds immense importance in cellular functions such as immunological antigen-antibody events, the protective histone protein coating of DNA, drug-biomolecule interactions, enzyme processes requiring co-factors, and the MHC recognition events (Chapters 10 and 20). The non-covalent biomolecule association phenomenon is a new field for ESI-MS/MS and is explored by Smith et al. (Chapter 15) using DNA. Evidence for three types of non-covalent complex interactions is shown for oligonucleotides with their complementary strands, multimeric proteins, and complexes between an oligonucleotide strand and a small drug molecule. Drug therapy for combatting human disease and poisoning agents are topics which focus on a wide variety of chemical compounds. Disease and poisoning agents can be very debilitating, and as such, ESI-MS/MS has provided great strides in the understanding of the biochemical origins of these assaults on the quality of human life. Gillespie (Chapter 16) takes us on a tour of a systematic analysis of the muscarinic M drug agonists of Alzheimer's disease. This disease is primarily responsible for memory loss in the aged population, and has received a heightened awareness in the public over the last few years. Biotransformation products from the drug analyte are observed. However, urine is shown to provide effective roadblocks in the separation of drug metabolites due to the overwhelming amount of endogenous interferences, and methods are suggested to alleviate this problem. Gilbert then provides a treatise (Chapter 17) on the investigations of drug analyses in human subjects from a quantitative perspective by ESI-MS/MS. Advantages/disadvantages of LC-MS and LC-MS/MS are presented with respect to sensitivity and selectivity, and comparisons to the established radioimmunoassay and HPLC-ultraviolet detection methods for quantitative drug determination in mammalian fluids are discussed. Cross validation methods are shown to be useful in correlating drug test l



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resultsfromthe different detection methods, and isotope addition to samples highlights the quantitative aspect. Quilliam provides a quantitative and qualitative presentation of the poisoning agents in plankton shellfish (Chapter 18). An interesting observation was that different toxin preparation/extraction procedures resulted in different mass spectral responses. In addition, different culture ages produced different toxin levels which suggested endogenous esterase action over time on the toxins. ISP-MS/MS was used to resolve these phenomena, and this investigation presents numerous, almost seemingly random, problems in bio-analysis scenarios that can be solved in a systematic fashion. The biochemical dynamics of cells is the next subject for discussion in Chapters 1922. A deliberate analysis of events in the cell such as enzymatic processes, complex interactions of a killer Τ cell with host tissue, and post-translational products of regulatory enzymes presents a unique evolving picture in the depth of ESI-MS/MS as applied to the elucidation of biological proceses. Withers starts our journey by investigating the active sites of Bacillus subtilis and Cellulomonasfimiglycosidases and human lysosomal glucocerebrosidase in Chapter 19. Covalent labeling with active site inhibitors and affinity labels was done, and subsequent peptic digests allowed ESI-MS/MS to accurately map out which amino acid residues are involved. The relative ease of this procedure has obvious implications for enzymes of unknown structures. Complex intracellular signal transduction pathways are essential for various biochemical processes. Aebersold shows in Chapter 20 how one can track tyrosine phosphorylation pathways on protein tyrosine kinases and Τ cell receptors. These series of complex immunochemical reactions are essential for successful Τ cell recognition of surface peptides complexed to the MHC molecule described by Yates, et al. in Chapter 10. Attempts to unravel the phosphorylation pathways are accomplished by chemical, enzymatic and immobilized metal affinity chromatography - HPLC - ESI-MS/MS methods. Chapter 21 presents the analysis of recombinant proteins with a slightly different objective than just straightforward structure characterization. It is well known that bacterial cell-produced recombinant protein can sometimes be different with respect to the native- produced protein from mammalian cells in vivo. Salient features between the mammalian-derived protein may not be expressed in the bacterial-derived recombinant. Some of these features include proper disulfide pairs and posttranslational carbohydrate attachment sites and sequences. Mammalian Chinese hamster assay cells produce recombinant proteins that are usually faithful replicates of the host mammalian cell protein, and Rohde et al. attempt to confirm this with ESI-MS/MS experiments on glycosylated proteins. Another topic that has much public awareness is the human immunodeficiency virus (HIV), because it is a disease state that is eventually fatal and no cure exists. Fenselau (Chapter 22) investigates the polypeptides produced from the human and bovine immunodeficiency viruses, HIV and BIV, respectively, with respect to their molecular weight distributions. Cellular processing and post-translational modifications incurred by the polypeptides are investigated with respect to protein multiplicity by mutations during viral replication. ESI and MALDI team up in the next two chapters and provide the core technologies in a suite of methods so as to provide structural characterization for complex proteins and the heterogeneous glycoprotein biomolecule. Chapter 23 finds Hancock et al



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exploiting both mass spectrometry methods, as well as LC and capillary electrophoresis, and they are found to be complementary in the understanding of the structure and integrity of the complex single chain plasminogen activator glycoprotein. Burlingame and associates (Chapter 24) present a classical approach at solving complex biochemical structural problems by using an array of modern analytical and biochemical techniques. Polyacrylamide gel electrophoresis, select enzyme digests, HPLC, ESI-MS/MS, MALDI time-of-flight MS, LSIMS and low resolution quadrupole and high resolution electric/magnetic sector mass spectrometers comprise the tools. These biochemical and analytical methods provide levels of information that, when taken together, provide a relatively complete picture of complex biochemical structures, including the isomer and stereoisomer issues. The signal recognition particleribonucleoprotein,human melanoma A375 cell proteins, and bovine fetuin glycoproteins are investigated by the techniques. This chapter serves as a reminder that in many cases various scientific avenues are necessary in order to provide a comprehensive, informative analysis, and depending on the degree of complexity of a substance, some scientific systems provide more complete information than others. Chapter 25 is more fundamental in nature and finds molecules such as proteins/peptides, oligosaccharides and single-stranded RNA as the targets of investigation by ESI-ion trap MS analysis with photodissociation as the collision-induced dissociation (CID) route. Stephenson et al. show that this method has advantages over the conventional ion-molecule CID procedure, and photodissociation CID is used to search for salient mass spectral details in the characterization of these small molecules. Of great importance is the interface between man and the environment. Environmental factors can take the form of the contamination of soil, water and air with a host of harmful or irritant chemicals and by-products through inadvertant or deliberate means. Short as well as long term exposure and the effects of a host of man-made chemicals on humans are a significant part of the Environmental Protection Agency's responsibilities. GC/MS is a premier technology in the field screening and determination of many environmental contaminants. Voyksner (Chapter 26) provides descriptions of analyses concerning environmental contaminants such as dyes, pesticides, herbicides, amines, oxygenated compounds and hydrocarbons, and it appears that LC-ESI-MS/MS is a powerful tool in the discovery and identification of a host of environmental contaminants. Especially for these important classes of compounds, the ESI-MS technique provides information that often cannot directly be obtained from the conventional method of GC/MS. The Future of Electrospray Ionization Mass Spectrometry This is a tough question. Despite its success and predictions that could be made (75), one must keep in mind the phenomenon of the FAB/SIMS desorption technologies as quantitatively documented in Figures 1 and 2. One could have easily suggested that 1989 should have been the year for FAB/SIMS to take the lead from EI in the percentage of total ionization techniques. In 1988, ESI was a mere shadow of its present utility when FAB/SIMS were knocking on the door of EI. Thus, what of the future of ESI? Clues to this question should be found in what current methods are "shadows" or latest technologies for possible future increased exploitation. MALDI already has passed through its latent



1.

SNYDER


period and this occurred a few years after FAB/SIMS had their peak (Figure 2). MALDI is a sample desorption/ionization method that is constantly gaining in popularity, virtually at the same rate as ESI (Figure 2). Other than MALDI, the ASMS Proceedings provide no tangible evidence of other techniques on the horizon that could yield success stories like FAB/SIMS, MALDI or ESI. However, on an optimistic note, there is no reason to suspect that the popularity of ESI-MS will decline in the near future. ESI has firmly established itself as an easy to use, atmospheric pressure sample introduction and ionization system (75). It can handle virtually any analyte in a gentle manner and in almost all cases faithfully transforms a neutral of interest into nothing less than an ion of equal interest. Interpretation of ESImass spectra for the most part is straightforward, and even if it is not, the resulting clues and information more than make up for any tedious data analysis. This can be said because information generated by ESI-MS, as well as MALDI, may not be evident or even be impossible to observe with other ionization techniques (76). ESI-MS is exquisitely sensitive as is evident with the recent advent of micro-spray technology (Chapter 2). A revolutionary method can manifest itself by having an impact on pre-existing methods, and usually it suppresses or supplants these methods. The ESI technique invented by Dole a quarter of a century ago and reapplied by Fenn appears to be revolutionary, at least with respect to its acceptance and broad array of applications in many fields of science. Thus, the significant advantages that drive the large body of work at the present equally fuel the increasing amount of research that is trying to solve major disadvantages ôf ESI, such as ion signal suppression by dissolved salts and the unambiguous interpretation of the charge states of CID product ions from a multiply charged precursor ion. The future looks good for electrospray ionization! Literature Cited 1. James, A.T.; Martin, A.J.P. Biochemical J. 1952, 50, 679-690. 2. James, A.T.; Martin, A.J.P. Analyst 1952, 77, 915-932. 3. Brunnee, C. Intl. J. Mass Spectrom. Ion Physics 1982, 45, 51-86. 4. Dole, M.; Mack, L.L.; Hines, R.L.; Mobley, R.C.; Ferguson, L.D.; Alice, M.B. J. Chem. Phys. 1968, 49, 2240-2249. 5. Mack, L.L.; Kralik, P.; Rheude, Α.; Dole, M. J. Chem. Phys. 1970, 52, 49774986. 6. Clegg, G.A.; Dole, M. Biopolymers 1971, 10, 821-826. 7. Gienic, J.; Cox, H.L. Jr.; Teer, D.; Dole, M . Proc. 20th Am. Soc. Mass Spectrom. Conference on Mass Spectrometry and Allied Topics, Dallas, TX, 1972, 276-280. 8. Teer, D.; Dole, M . J. Polymer Sci. 1975, 13, 985-995. 9. Dole, M.; Gupta, C.V.; Mack, L.L.; Nakamae, K. Polymer Preprints 1977, 18, 188-193. 10. Nakamae, K.; Kumar, V.; Dole, M . Proc. 29th Am. Soc. Mass Spectrom. Conference on Mass Spectrometry and Allied Topics, Minneapolis, MN, 1981, 517-518.



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AND

BIOTECHNOLOGICAL

APPLICATIONS

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