Mass Spectrometry - Analytical Chemistry

Capillary Electrophoresis/ Mass Spectrometry Richard D. Smith, Jon H. Wahl, David R. Goodlett1, and Steven A. Hofstadler Chemical Methods and Separations Group Chemical Sciences Department Pacific Northwest Laboratory Richland, WA 99352

Many of the most difficult chemical, environmental, biochemical, and bio medical analytical problems require a combination of i n s t r u m e n t a l a t tributes, including speed, low detec tion l i m i t s , wide l i n e a r d y n a m i c range, good sensitivity, and high se lectivity. For such demanding appli cations, the on-line combination of separation methods with MS often provides the most practical or per haps the only approach. The orthogo nal n a t u r e of the selectivities pro v i d e d by a c h r o m a t o g r a p h i c or electrophoretic s e p a r a t i o n in con junction with MS has long been con sidered attractive. Indeed, GC/MS is firmly established as a definitive an alytical technique for many environ mental and clinical analyses. The h a l l m a r k s of GC/MS are its speed, selectivity, and sensitivity. U n f o r t u n a t e l y , however, both GC and the conventional ionization methods used in MS (primarily elec tron impact and chemical ionization) require sample volatilization. Thus, GC/MS is not amenable to many an alytical problems without invoking often complex and problematic chem ical d e g r a d a t i o n or d e r i v a t i z a t i o n procedures designed to modify sam ple components to "GC-able" forms. Interest in LC/MS has continued to grow, and the technique has begun to open new avenues for the charac terization of biological and biomedi cal samples (2). The 1980s saw t h e g e n e s i s a n d rapid development of a high-resolu tion s e p a r a t i o n method, capillary electrophoresis (CE), primarily be cause of the efforts of J. W. Jorgenson of the University of North Caro lina (2). He and his co-workers have 1 Current address: Immunobiology Research Institute, Route 22 East, P.O. Box 999, Annandale, NJ 08801

demonstrated t h a t CE can generate both rapid and very high resolution separations, based on differences in t h e e l e c t r o p h o r e t i c m o b i l i t i e s of charge-carrying species in an elec tric field, in small-diameter fused silica capillaries. The a d v a n t a g e s of the capillary format for electrophoresis are multi fold. First, small-diameter capillar ies (generally 50-100-μπι i.d.) gener ate less Joule heat and dissipate this h e a t more effectively, allowing h i g h e r electric fields t h a n can be used with conventional electrophore sis and providing faster and higher resolution separations. Second, the capillary format allows for easy au-

REPORT tomation of sample handling and in jection. The CE format allows ready i m p l e m e n t a t i o n of a r a n g e of oncapillary detection methods; most ef fectively a n d broadly used a r e UV absorption and fluorescence emis sion detectors. Since the first commercial CE in struments appeared in the late 1980s, CE technology and its applications have grown explosively. In fact, the rate of growth, use, and commercial implementation has considerably ex ceeded that seen earlier for LC meth ods. Improvements in injection meth ods, detector sensitivity, capillary surface d e a c t i v a t i o n , a n d coating technologies, as well as the introduc-

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tion of new electrophoretic buffer sys tems, continue to drive further devel opments in CE for chemical, biological, and environmental applications. The growth of CE as a viable ana lytical tool is primarily the result of advances in detection methods and a n i n c r e a s i n g r e c o g n i t i o n of i t s unique capabilities. CE would not be practical without the sensitivity im provements t h a t have been demon s t r a t e d w i t h o n - c a p i l l a r y UV a n d fluorescence detection. Detectable a m o u n t s in t h e femtomole (10~ 1 5 mol) range can be obtained routinely, although optimized and specialized detection schemes have been re ported for which detectable amounts extend to attomole (10~ 1 8 mol) and zeptomole (10~ 2 1 mol) levels. Thus, for CE with typical capillary diame ters, in which effective injection vol umes are generally in the range of 1-10 nL, routinely detectable con centrations are typically on the order of 10~ 6 M for the injected sample. Specialized detection systems allow these detectable concentrations to be extended to < 10" 1 0 M, which is well into the regime of trace analysis. Im proved detection limits can be ob tained by using electrophoretic meth ods to concentrate sample components during injection. The ability to manipulate and in ject extremely small sample volumes, steps that are generally problematic with LC, provides a basis for using CE to confront extreme analytical challenges (e.g., the analysis of, or s a m p l i n g from, s i n g l e biological cells). In addition, CE has the flexi bility provided by a range of formats (free-zone electrophoresis, electrokinetic micellar chromatography, isotachophoresis, gel electrophoresis, etc.) and a plethora of methods for m a n i p u l a t i n g injection conditions and separation specificity. Moreover, methods have been de veloped or are being investigated for CE a p p l i c a t i o n to t h e a n a l y s i s of practically any substance that can be dissolved or suspended in a liquid. Finally, from a pragmatic viewpoint, the small sample, buffer, and waste volumes required and generated by CE are much less than those used by 0003 - 2700/93/0365 -574A/$04.00/0 © 1993 American Chemical Society

LC methods; small volumes are at tractive because of the trend toward treating all LC (and CE) effluents as hazardous w a s t e s t h a t require ex pensive tracking and disposal. Development of CE/MS The early work of Jorgenson and co workers clearly d e m o n s t r a t e d t h e potential power of CE methods, but combining t h e m with MS required the solution of several conceptual and p r a c t i c a l p r o b l e m s . F i r s t , all early CE experiments, and nearly all today, involve p l a c e m e n t of b o t h ends of the capillary into reservoirs of the conductive buffer, where elec trical contact is established to define the CE field gradient. Second, any CE/MS interface must be compatible with low CE flow rates (< 1 μΕ/ππη at most) a n d should not i n d u c e a p r e s s u r e - d r i v e n (laminar) flow in t h e c a p i l l a r y t h a t would d e g r a d e separation quality. Moreover, detec tion sensitivities must extend to subpicomole levels for CE/MS to be of practical value. The early 1980s saw the introduc tion of the thermospray interface for LC/MS ( i ) , b u t this approach was impractical for CE because of t h e necessary flow rates (> 100 μϋι/ηιίη) and inadequate sensitivity. In 1984 Fenn and co-workers presented their initial results on electrospray ioniza tion (ESI) combined with MS (3). In the ESIMS method, liquid solutions are nebulized in a high electric field from the end of a capillary at flow rates in the Ι-ΙΟ-μΕ/πιίη range (4-6). More i m p o r t a n t , t h e early E S I M S studies suggested t h a t solute sensi tivity was exceptional (4). This work s t i m u l a t e d efforts a t Pacific Northwest Laboratory (PNL) to develop on-line CE/MS based on ESI w i t h t h e use of an e l e c t r o phoretic capillary, one end of which functioned as the electrospray source r a t h e r t h a n b e i n g i m m e r s e d in a buffer reservoir. After constructing ESIMS instrumentation at PNL, re sults were obtained in 1986 and pub lished in 1987 (7).

ure 2a). With this interfacing method it was necessary to select CE condi tions giving rise to a net electroosmotic flow in t h e direction of t h e m a s s spectrometer, t h a t is, a flow arising at the electric double layer of the capillary surface and creating a flat " p l u g - l i k e " flow profile away from the point of injection. The met allized capillary terminus also served as the electrospray source by having a 3 - 6 - k V difference in voltage be tween the t e r m i n u s a n d t h e m a s s spectrometer sampling aperture, which was 1-2 cm away. In ESI the liquid is nebulized from

a capillary to produce highly charged d r o p l e t s , typically only a few m i crometers in diameter. The charged droplets drift in the electric field be t w e e n t h e capillary a n d t h e m a s s spectrometer sampling aperture. In transit to the sampling orifice and in transport through the MS interface, the droplets experience conditions t h a t cause evaporation. Because the droplets initially are highly charged and close to t h e physical limit for their size (the Rayleigh limit), they s h r i n k by evaporation and quickly reach a point at which they shed a portion of their charge. By some still

Figure 1. Schematic illustration of the instrumental arrangement for CE/MS using an electrospray interface. The sheath syringe pump and SF6 gas flow are used to assist the electrospray operation in some designs.

Interfacing CE and MS The instrumental arrangement typi cally used for C E / M S is shown in Figure 1. A crucial feature of any CE/MS interface is the method used to establish the electrical connection at the CE capillary terminus, which serves to define the electric field gra dient along the CE capillary. In the first CE/MS interface (7), the electrical connection was made by silver metal deposition onto the fused-silica capillary terminus (Fig-

Figure 2. Schematic illustration of CE/electrospray interfaces. (a) The original design utilizing a metallized capillary terminus, (b) sheath flow (coaxial) interface used for CE/MS, (c) ESI based on a liquid junction, and (d) a sheathless interface design.

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REPORT not well-defined process(es), involv ing t h e emission or d e s o r p t i o n of charged species and continued evap oration of residual solvent, charged species in solution are transferred to the gas phase w i t h very high effi ciency (6). R e m a r k a b l e a s p e c t s of E S I a r e that ionization efficiency can be very high (> 20-50%) and that very large nonvolatile molecular species are amenable to it (6). In 1987 Fenn and co-workers d e m o n s t r a t e d t h a t the ESI of large polymers produced ions with a net charge (z) t h a t increases with molecular size; therefore, con v e n t i o n a l m a s s - t o - c h a r g e (m/z) range i n s t r u m e n t s could be used to detect species with molecular weights many times greater than the maximum m/z of the instrument (8). The next year Fenn and co-work ers demonstrated that precise molec ular weights could also be readily de t e r m i n e d for p r o t e i n s from t h e d i s t r i b u t i o n of c h a r g e s t a t e s ob served across the mass (or more cor rectly, m/z) spectrum (9). Since then the range of applications of ESIMS has grown very rapidly and is facili tated by the c u r r e n t availability of instrumentation from nearly all MS vendors. T h e c o m b i n a t i o n of LC w i t h ESIMS is becoming widely accepted because of the broader use of LC and arguably greater ease of interfacing. As the use of CE grows and inevita bly displaces LC in some applica tions, we anticipate t h a t the role of C E / M S will also grow. In some areas, particularly in biological and biomedical research where sample size is i n h e r e n t l y l i m i t e d or i n creased only at great expense and ef fort, the high resolution and sensi t i v i t y of C E / M S c o m p a r e d w i t h L C / M S will e n s u r e a s i g n i f i c a n t analytical role. Realization of the full potential of CE/MS hinges on inter face performance and MS detection sensitivity, which we now discuss in greater detail.

result of electrochemical processes. Initial studies nevertheless demon strated CE/MS detection limits for quaternary ammonium salts that ex tended to subfemtomole levels (10). The ease of interfacing was d r a matically improved in 1988 by intro duction of t h e s h e a t h flow design, which removed many of these limita tions (11). In this design, a small co axial flow ( 1 - 5 μΕ/πιίη) of liquid serves to establish the electrical con tact with the CE effluent and to fa cilitate the electrospraying of buffers t h a t could not be directly electrosprayed with earlier interfaces. Fig ure 2b shows a schematic illustration of a more recent implementation of the sheath flow design that is distin guished by an etched conical tip of the CE capillary. This design serves to enhance the electric field gradient at the capillary terminus, minimize the effective mixing volume between the sheath liquid and the CE efflu ent, and increase the stability of the electrospray process. Another variation on the CE/MS interface, introduced by Henion and co-workers (12), uses a liquid junc tion to establish electrical contact with the analytical capillary and to provide an additional makeup flow of buffer (Figure 2c). The relative ad vantages of these designs have been c o m p a r e d (13), a n d t h e coaxial s h e a t h flow i n t e r f a c e a p p e a r s to

have several a d v a n t a g e s . A disad v a n t a g e of both a p p r o a c h e s , how ever, is that they depend on an addi tional flow of liquid that incorporates charge-carrying species (e.g., buffer or solvent impurities) and invariably degrades detection sensitivity. There continues to be a n interest in the development of a more versa tile and reliable design that does not depend on an additional liquid flow. We are pursuing several sheathless designs for this purpose, such as that i l l u s t r a t e d in F i g u r e 2d, a n d we show some initial results for one de sign later. Jorgenson and co-workers are pursuing a somewhat similar mi crospray approach for LC/MS inter facing (14) in which the ion source operates under a vacuum in a man ner similar to electrohydrodynamic ion sources for MS. Early generations of commercial CE instrumentation made MS inter facing a difficult t a s k at best. The design of future commercial instru m e n t s will d e t e r m i n e t h e r a t e a t which C E / M S becomes r o u t i n e l y available. Although CE/MS capabili ties exist in a n u m b e r of laborato ries, greater sensitivity (or the gen erally r e l a t e d issues of b e t t e r MS scan speed and resolution) is desired. Sensitivity The small sample sizes associated with CE create demands for the best

Interface methods and performance The initial performance of C E / M S with the metallized capillary termi nus design was limited because flow r a t e s were below o p t i m u m for t h e electrospray design used at the time (7) and because of frequent spray in stabilities. Imposed restrictions on the types of CE buffers effectively precluded t h e use of aqueous a n d higher conductivity buffers. In addi tion, the lifetime of t h e metallized capillary was limited to only a few days of operation, presumably as a

Figure 3. Dependence of MS signal intensity on analyte mass flow rate to the electrospray source for relative analyte/background electrolyte flow rates of A, A/10, and A/100. Behavior predicted using Equation 1 ; it is expected to be qualitatively correct when the CE current exceeds the ESI current.


possible detection sensitivity. Gener ally, CE is not well suited for trace analyses and is probably inappropri ate for such applications when large amounts of sample are available and LC separations are adequate. CE is a t i t s b e s t , h o w e v e r , in s a m p l e limited situations and may consti tute the only practical approach in some cases (e.g., sampling from sin gle biological cells). Recent work has demonstrated that CE/MS is capable of providing exceptional sensitivity in certain situations (15). It is essen tial, however, t h a t consideration of CE/MS application involve a practi cal understanding of ESIMS perfor mance and sensitivity issues. The response characteristics of a particular detector are referred to as either "concentration sensitive" or "mass flow sensitive." For example, a UV detector functions as a concen t r a t i o n - s e n s i t i v e detector. Higher flow rates will decrease peak widths but will not increase peak heights; consequently, the peak a r e a is in versely related to the flow rate. Con versely, t h e conventional electron ionization method used for GC/MS functions as a mass flow-sensitive detector. Higher flow rates will de crease peak widths but also increase peak heights; t h u s , peak a r e a s are independent of flow rate.

P o p u l a r d i s c u s s i o n s of E S I M S h a v e l a b e l e d it a c o n c e n t r a t i o n sensitive detection method. At the typical sample flow r a t e s used for ESIMS, and thus for LC/MS, this is generally correct. However, because of the small sample size a n d flow r a t e s associated with C E / M S , it is possible for ESIMS to function in ei ther a concentration-sensitive or a m a s s f l o w - s e n s i t i v e m a n n e r . The distinction between these two detec tion regimes is essential for under standing the CE/MS sensitivity gains t h a t arise when capillary di a m e t e r s or electromigration r a t e s are reduced. U n d e r most conditions used for ESIMS, the number of charged spe cies in solution delivered to the electrospray source is much greater than the number the electrospray process can transfer to the gas phase as de tectable ions. This situation applies for typical CE capillary d i a m e t e r s and buffers in which CE c u r r e n t s r a n g e from ~ 5 to 100 μΑ. In con trast, ESI currents for aqueous solu tions range from 0.1 to 0.5 μΑ. Thus, even if the entire electrospray cur rent could be converted to desolvated ions in the gas phase, the overall ef ficiency of t h e i o n i z a t i o n process would be only on the order of 1%. Details of the ion formation pro

cess and any discrimination in ion i z a t i o n efficiency b e t w e e n buffer c o m p o n e n t s (i.e., t h e b a c k g r o u n d electrolyte that substantially exceeds the analyte in concentration) and analyte species would obviously affect the overall efficiency. The important point, however, and one that is qual itatively consistent with the current phenomenological understanding of ESIMS (5, 6), is that discrimination processes do not substantially alter the expectation t h a t the ionization efficiency will be low u n d e r such conditions. The qualitative features relevant to sensitivity can be described with a simple model. In an a d a p t a t i o n of the approach of T a n g and Kebarle (16), the a n a l y t e signal i n t e n s i t y , 7(A+), can be expressed as a function of the mass flow rate of the analyte, 7 m (A + ), and background electrolyte, ^ m (B + ), as

where / is the total electrospray cur rent, Ρ is a variable related to the s a m p l i n g efficiency of t h e ESIMS system (assumed constant), and / is a constant representing the fraction of droplet charge converted into gasp h a s e ions. For simplicity, we as-

Figure 4. Comparison of normal constant field strength (top) and reduced elution speed (bottom) CE/ESIMS analysis of a tryptic digest of bovine serum albumin. The reduced elution speed CE/ESIMS analysis was conducted at 300 V/cm until 1 min before elution of the first analyte, when the electric field strength was reduced to 60 V/cm. Single-ion electropherograms corresponding to several of the tryptic (polypeptide) fragments are shown on the right (designated by use of the single-letter code for amino acid residues). (Adapted with permission from Reference 17.)


REPORT sume no discrimination in the ion ization efficiency for A + and B + . Most relevant to CE is the limit at which the background electrolyte (buffer) mass flow rate is assumed constant and much greater t h a n the analyte mass flow rate. Here Equation 1 re duces to

The analyte signal intensity in this high-background electrolyte concen tration regime is predicted to be di rectly proportional to t h e a n a l y t e concentration. Moreover, the analyte s e n s i t i v i t y is p r e d i c t e d to be i n versely p r o p o r t i o n a l to t h e b a c k ground electrolyte m a s s flow r a t e . Analyte sensitivity can thus be im proved by decreasing the background electrolyte mass flow rate. The importance of this observation to detection sensitivity is illustrated in Figure 3, where the relative ana lyte signal intensity is shown as a function of t h e a n a l y t e m a s s flow rate. For curve A, the maximum and minimum a n a l y t e m a s s flow r a t e s cover a range from 3 orders of mag nitude less t h a t of the background m a s s flow r a t e up to a m a s s flow rate equivalent to t h a t of the back g r o u n d e l e c t r o l y t e . According to Equations 1 or 2, an increase in ana lyte sensitivity is predicted when the m a s s flow r a t e of t h e b a c k g r o u n d electrolyte is d e c r e a s e d . T h i s i n crease in sensitivity is illustrated in Figure 3 where the middle curve (A/ 10) represents the signal intensity predicted when both the analyte and background mass flow rates are re duced by a factor of 10 relative to curve A, and the upper curve (A/100) c o r r e s p o n d s to r e d u c t i o n of m a s s flow rates by a factor of 100 for both flow rates. At low analyte concentrations, the analyte signal intensity is predicted to be 2 orders of magnitude greater when both the analyte and the back ground ions are reduced by 2 orders of magnitude compared with a de crease only in the analyte mass flow rate (curve A). Thus, a large increase in sensitivity is predicted. This in crease arises directly from the de crease in background electrolyte and is derived from the greater fraction of the a n a l y t e converted into g a s phase ions. The situation described by Equa t i o n s 1 or 2 clearly fails a t suffi ciently low-background electrolyte flows. Equation 2 is not realistic; it predicts that the ion signal intensity will remain constant as the analyte

and background electrolyte mass flow rates are decreased in an indef inite m a n n e r . Consequently, a sec ond d e t e c t i o n r e g i m e m u s t occur when charge-carrying species are no longer supplied to the ESI source at a rate sufficient to sustain the maxi mum electrospray current (i.e., when the CE current becomes comparable to or less than the maximum electrospray current), and the ionization ef ficiency for this regime is

Both Equations 2 and 3 predict that the observed MS signal will be pro portional to analyte concentration; however, one i m p o r t a n t and some w h a t subtle distinction exists. For Equation 2, a change in background electrolyte mass flow rate will affect a n a l y t e signal i n t e n s i t y . In the c o u r s e of a given CE s e p a r a t i o n , Km(B+) is generally constant, and the signal intensity will directly reflect electrolyte mass flow rate. In this re gime the ESIMS detector appears to function as a m a s s flow-sensitive detector. The transition between the regimes described by E q u a t i o n s 2 and 3 is evident when further de creases in the background electrolyte do not lead to additional gain in ana lyte sensitivity and is predicted to occur when the CE c u r r e n t is less than the normal ESI current (17-19). These considerations indicate that a n a l y t e s e n s i t i v i t y in C E / E S I M S m a y be increased by r e d u c i n g t h e m a s s flow r a t e of t h e b a c k g r o u n d components. Separations by CE are generally performed u n d e r condi tions in which the analyte concentra tion is m u c h less t h a n t h e buffer concentration. The lower limits to CE current with larger capillary di a m e t e r s a r e g e n e r a l l y defined by trace ionic contaminants; low-con ductivity liquids are thus generally impractical for CE. However, t h e m a s s flow r a t e of electrolyte from the analytical capil lary can be reduced in several other ways, including reduction of the CE electric field strength or migration r a t e of electrolyte into the electrospray source, or the use of small i.d. capillaries, which reduces the mass flow rate of both the analyte and the background ions. Experimental stud ies have recently demonstrated that both approaches are viable and lead to t h e a n t i c i p a t e d a d v a n t a g e s for CE/MS detection (17-19). Reduced elution speed CE/MS The small solute quantities used in CE and the low signal i n t e n s i t i e s

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generally produced by ESIMS typi cally result in maximum analyte ion MS detection rates no greater t h a n 1 0 5 - 1 0 6 ion counts per second with current quadrupole instrumentation (5). Statistical considerations, there fore, effectively limit the maximum practical scan speeds with quadru pole m a s s s p e c t r o m e t e r s . In addi tion, MS resolution is often sacri ficed because it generally comes at the expense of sensitivity. Thus, de pending on the m/z range to be ex amined, solute concentrations, and other factors related to the nature of t h e solute a n d buffer species, t h e maximum practical MS scan speeds are often insufficient to exploit the speed or h i g h - q u a l i t y s e p a r a t i o n s feasible with CE. The reduced elution speed (RES) method for CE/MS circumvents, to some extent, both the sensitivity and t h e speed l i m i t a t i o n s of s c a n n i n g mass spectrometers (17). Involving only step changes in the CE electric field strength, the technique is sim ple and readily implemented. Before elution of the first analyte of interest into the ESI source, the electrophoretic voltage is decreased a n d electromigration r a t e s are slowed,

Figure 5. Reconstructed ion current electropherograms obtained for the peptide mixture containing tryptophan (204 Da), leucine-enkephalin (555 Da), and melittin (2845 Da), using capillaries of 100-, 50-, 20-, and 10- μιη i.d., and displayed on the same absolute intensity scale. The relative amounts of sample injected are proportional to the capillary cross-sectional area (i.e., the amount injected for the ΙΟ-μπι-i.d. capillary is 2 orders of magnitude smaller than for the ΙΟΟ-μπη-i.d. capillary). The gains in sensitivity for the ΙΟ-μΓη-i.d. capillary correspond to factors of 25 to 50.

allowing more or longer MS scans to be a c q u i r e d . U n d e r c o n d i t i o n s in which the amount of solute entering the ESI source per unit of time ex ceeds its ionization capacity, the ear lier discussion suggests t h a t no sub s t a n t i a l decrease in m a x i m u m ion intensity will result. The RES method is illustrated in Figure 4, which c o m p a r e s n o r m a l and RES total ion electropherograms from CE/MS analyses of a tryptic di gest of bovine serum albumin (a pro tein with a mass of ~ 66,430 Da). In each separation a total of 40 fmol of protein was used, producing a sepa ration t h a t yielded more t h a n 100 overlapping peaks for various pep tide digestion products. W h e n t h e RES separation method was used to slow the elution by a factor of 5, only a 20% decrease resulted in maximum ion intensities. Figure 4 shows four of the more than 100 peaks attributable to tryp tic polypeptides observed during the RES C E / M S experiment. The RES technique allows more components to be detected and effectively reduces

t h e complexity of i n d i v i d u a l m a s s spectra or the likelihood of missing some c o m p o n e n t s (a complication that arises because analyte zones for conventional s e p a r a t i o n s are often s h o r t e r or c o m p a r a b l e to t h e MS scan time). Similar advantages have also been demonstrated for protein m i x t u r e s for w h i c h h i g h - q u a l i t y m a s s spectra can be obtained with injections of only 60 fmol of each pro tein (17). RES CE/MS provides an increase in the effectiveness of mass spectrometric scanning compared with con ventional CE/MS methods. The method does not increase solute con sumption; does not degrade detection sensitivities for peptide and protein a n a l y s e s e x t e n d i n g into t h e lowfemtomole regime; and incurs very little loss in ion intensity, which is p a r t i c u l a r l y i m p o r t a n t for t a n d e m MS methods and their potential ap plication to peptide sequencing. Per haps more important, RES illus trates a flexibility available with CE; separations may be slowed or even stopped instantaneously with little

Figure 6. Comparison of CE/MS separations for a mixture of pentapeptides in which the amount of sample injected was varied by more than 2 orders of magnitude. The separations used a bare fused 20^m-i.d. capillary and a 10-mM acetic acid buffer with a sheath flow interface.

loss of separation efficiency (18)—a feature not readily achieved with LC. It is anticipated t h a t this capability could be particularly useful in con j u n c t i o n w i t h t h e a d v a n c e d MS methods that we will discuss later in this article. CE/MS with small-diameter capillaries When greater sensitivity is desired or sample volume is extremely lim ited, smaller capillary diameters are advantageous. For a given buffer and CE electric field s t r e n g t h , t h e CE current has a quadratic dependence on the capillary diameter. The sim ple model for E S I s e n s i t i v i t y d e scribed earlier leads one to expect t h a t the best sensitivity will be ob tained when the CE c u r r e n t is less t h a n the ESI current. Even with low-conductivity 10-mM acetic acid buffer s y s t e m s often used for C E / MS, this happens only for capillary diameters of < 3 0 - 4 0 μπι, somewhat smaller t h a n those typically used for CE. Because of complexities i n t r o duced by the sheath flow, it was un certain whether the predicted sensi tivity gains could be realized. To examine this question, we com pared CE/MS separations for capil laries ranging from 100- to 5-μπι i.d. (19). Figure 5 shows CE/MS selected ion electropherograms for a simple mixture plotted on the same absolute intensity scale obtained using capil laries of 100-, 50-, 20-, and 10-μπι diameters. The electrokinetic injec tions result in sample sizes propor tional to the capillary cross-sectional area, so t h a t 2 orders of magnitude more sample was injected into t h e 100 -μπι capillary (~ 800 fmol per component) t h a n into the 10-μπι cap illary (- 8 fmol per component). The absolute signal intensities were found to decrease by factors of only 2 - 4 , r a t h e r t h a n t h e 2 o r d e r s of m a g n i t u d e t h a t might be expected, which corresponds to a sensitivity increase by factors of 2 5 - 5 0 for the smallest capillary diameter. Similar experiments with protein mixtures, in which MS signal inten sities are generally lower, showed that useful mass spectra could be ob tained from subfemtomole injections u s i n g 5 ^ m - i . d . capillaries (15). A detailed study of the role of sample concentration and capillary diameter on CE/MS sensitivity has been pub lished (19). Applications Currently at least a dozen laborato ries are actively involved in develop ing and applying CE/MS techniques


REPORT and m e t h o d s , b a s e d on p u b l i s h e d work. In addition to those from our laboratory, numerous contributions and applications have been demon s t r a t e d by Henion and co-workers (20-23); Moseley, Tomer, Jorgenson, a n d t h e i r colleagues {24, 25); a n d Thibault et al. (26-28). A l t h o u g h o u r focus h e r e is o n CE/MS using ESI interfaces, other methods have been reported. In par ticular, interfaces based on continu ous-flow fast a t o m b o m b a r d m e n t have been developed by Tomer, Caprioli, Reinhold, and their respective co-workers (29-32), as well as oth ers. Wilkins et al. (33) recently dem

onstrated the off-line combination of CE w i t h h i g h - r e s o l u t i o n F o u r i e r t r a n s f o r m ion cyclotron resonance (FTICR) MS b a s e d on m a t r i x - a s sisted l a s e r desorption ionization. Currently, the ESIMS interface ap pears to provide the best approach to on-line C E / M S , although t h e s e al ternatives may have advantages for specific applications. The range of potential CE/MS ap plications is far too broad to cover in this article, and it substantially mir rors that for CE. Applications to date include t h e a n a l y s i s of oligonucle otides, DNA adducts, peptides, pro teins, carbohydrates, pesticides, var

ious metabolites, and small organic a n d inorganic ions. The e m p h a s i s here is on free-solution-zone electro phoresis, the most widely used CE format. Other CE formats have been inter faced to M S . C a p i l l a r y i s o t a chophoresis/MS (CITP/MS), devel o p e d by U d s e t h e t a l . i n o u r l a b o r a t o r y (34), can be a powerful tool in some applications because the background electrolyte is effectively eliminated during analyte detection. As a result, greater signal intensities and improved detection limits can be obtained w i t h use of C I T P / M S . In a d d i t i o n , H e n i o n a n d G a r c i a (35) d e m o n s t r a t e d t h e c o m b i n a t i o n of c a p i l l a r y gel e l e c t r o p h o r e s i s w i t h pneumatically assisted ESIMS. These developments emphasize the f l e x i b i l i t y of E S I M S i n t e r f a c i n g methods for CE. Toward uitrahigh-sensitivity CE/MS

Figure 7. Comparison of three total ion electropherograms obtained for CE/MS separations of tryptic digests of cytochrome c species. The separation used a ΙΟ-μπι-Î.d. capillary and a new sheathless CE/MS interface. Mass spectra are shown for representative regions and generally indicate elution of more than one component.

580 A • ANALYTICAL CHEMISTRY, VOL. 65, NO.13,JULY 1, 1993

An obvious approach to improving CE/MS detection limits is to modify the concentration during the injec tion step. Such techniques have been demonstrated for CE/MS; a particu larly useful approach involving isotachophoretic sample preconcentration has recently been reported by Tinke et al. (36) a n d by K a r g e r a n d co workers (37). However, when sample size is limited or such techniques are impractical because of degradation of separation quality, detector sensitiv ity becomes the crucial issue. Our understanding of ESIMS sug gests t h a t s e n s i t i v i t y can be o p t i mized by using small-diameter cap illaries (< 20 μπι). ESIMS interfaces allow a wide range of CE buffers to be electrosprayed successfully, pro viding considerable flexibility d e spite some constraints (e.g., high salt and surfactant concentrations re main problematic). Aqueous and mixed aqueous/organic buffers of at least 0.1 M can be used for CE/MS when dilution occurs by the sheath liquid or liquid-junction buffer. Im proved electrospray source designs and the coaxial flow of electron scav engers (e.g., SF 6 ) also serve to extend the range of compatible liquids. Because of p r a c t i c a l s e n s i t i v i t y constraints, however, CE buffer con centrations are generally minimized. For the low-conductivity buffers and sheath liquids generally used for CE/ MS, 10- to 20-μπι capillaries provide good sensitivity while avoiding most of t h e difficulties associated w i t h smaller capillary diameters, such as low permeability (which makes sur face t r e a t m e n t and column flushing

difficult) and greater susceptibility to plugging. The major considerations relevant to MS detection are the n a t u r e and complexity of the particular sample and pragmatic compromises between detection sensitivity, resolution, and scan speed. F o r q u a d r u p o l e m a s s spectrometers, selected ion monitor ing (SIM) yields significantly e n hanced detection limits compared with scanning MS operation because of the greater dwell time for signal a c q u i s i t i o n a t e a c h s e l e c t e d m/z value. For samples in which analyte molecular w e i g h t s a r e k n o w n a n d m/z values can be predicted, SIM de tection is an obvious choice. If suffi cient sample is available, direct infu sion can be used to produce a mass spectrum of t h e u n s e p a r a t e d mix ture, and the results can be used to guide selection of specific m/z values for SIM detection. The sensitivity gain of SIM detec tion versus scanning detection with conventional quadrupole MS instru ments can be s u b s t a n t i a l . Figure 6

s h o w s t h r e e selected ion e l e c t r o pherograms for injections from serial d i l u t i o n of a simple p e n t a p e p t i d e mixture. For the 5-10-fmol injection (top), SIM detection provides excel lent signal-to-noise ratios (S/N), and the CE performance and solute zone profile is captured by the MS detector. For injection sizes corre sponding to 0 . 5 - 1 . 0 fmol (middle), increased chemical noise attributable to buffer impurities is evident. Injec t i o n s of 4 0 - 7 5 amol (bottom) still give good response, with estimated d e t e c t i o n l i m i t s of 10 a m o l . T h e chemical noise background for this s e p a r a t i o n r e s u l t s from " r e a l " sig n a l s a r i s i n g from solution compo n e n t s . T h u s , higher p u r i t y buffers may provide a basis for an extension to subattomole detection limits. An a d v a n t a g e of MS r e l a t i v e to other detectors is its high specificity. Obtaining the maximum number of theoretical plates possible with C E / M S is r a r e l y r e q u i r e d u n l e s s closely related mixture components have similar molecular weights. Ana lyte m i g r a t i o n t i m e s can shift be tween runs because of (sometimes un avoidable) capillary surface modi fication and degradation. With less specific detectors, great care is gener ally necessary to establish reproduc ible m i g r a t i o n t i m e s to identify eluents; with C E / M S , approximate migration times are usually sufficient, and one is generally more concerned with MS sensitivity and resolution. A sheathless CE/MS interface Although the coaxial sheath flow in terface h a s facilitated progress in

C E / M S , it does h a v e some d r a w backs. It contributes electrolyte to t h e E S I source t h a t can d e c r e a s e sensitivity (but less t h a n might be e x p e c t e d , p r e s u m a b l y b e c a u s e of poor mixing w i t h t h e CE effluent) and gives rise to chemical noise. Our laboratory h a s revisited the initial sheathless CE/MS interface ap proach (7) with use of a metallized contoured capillary t e r m i n u s (38), such as that illustrated in Figure 2d. F i g u r e 7 s h o w s s e p a r a t i o n s of tryptic digests obtained for three cy tochrome c proteins (bovine, Candida krusei, a n d equine, with molecular masses of - 12 kDa). Approximately 30 fmol of the s t a r t i n g protein (be fore d i g e s t i o n ) w a s c o n s u m e d for each s e p a r a t i o n . T h e s e p a r a t i o n s were conducted in a 50-cm χ 10-μπιi.d. capillary with its inner surface chemically modified with 3-aminopropyltrimethoxysilane, using a 10mM a q u e o u s a m m o n i u m a c e t a t e / acetic acid buffer at pH 4.4. By scan ning rapidly (0.6 s per scan), m a s s spectra could be acquired for each separation in < 6 min, allowing each of the tryptic fragments, and other mixture components attributable to incomplete digestion, to be identified (38). The routine implementation of such capabilities can have enormous impact for more rapid and sensitive protein mapping and, when used in conjunction w i t h t a n d e m M S , for protein sequencing. The future Recent work with CE/MS using quadrupole mass spectrometers has d e m o n s t r a t e d the potential for ex-

Table 1. Performance characteristics for mass spectrometers with potential for CE/MS Mass spectrometer

Figure 8. Application of CE/MS to the study of ribonuclease S, a noncovalent complex consisting of an A-protein and a smaller A-peptide. The CE separation in an acetic acid buffer (pH 3.4) gives two peaks with mass spectra corresponding to the dissociated A-protein and A-peptide species. The separation in an ammonium bicarbonate buffer (pH 8.0) gives one broad peak. The mass spectrum obtained during elution of this peak is dominated by the intact ribonuclease Α-complex, although a smaller contribution attributable to the A-protein formed by dissociation of the complex in the ESI interface is also observed.

Quadrupole" Orthogonal time-offlight6 ITMS° FTICR

Ion utilization Spectra/s efficiency8

Resolution

MS"

Commercial availability'

Cost ($K)»

0.03-0.8

1 104

103 200-1000

n=2 n=1

Now 1993 (?)

200-400 150-200

0.1-0.5 0.03-0.3 c

1-5 0.05-0.5°

103" 10 4 -10 5

η=·\"

1994 (?) 1993 (?)

200-300 400-500

ίο- 3 -1er 4

n>2

3

Estimate based on scanning operation or duty cycle. Based on the efficiency with which a con tinuous ion current contributes to useful signal but does not consider trapping efficiency of ion trap mass spectrometry (ITMS) or FTICR instruments. b Estimate range of performance arises because of tradeoffs between resolution and scan speed or m/z range. ° Projected based on initial performance demonstrated with instrumentation at the authors' labora tory (48, 49). Higher resolution requires somewhat slower speeds. d Higher resolution can be obtained, but currently requires slow scanning (ion ejection) operation that is not generally compatible with CE/MS demands. 8 A value of n> 2 is possible, but the ITMS uses destructive ion detection and true multiple gen eration spectra require restarting from new precursor ion populations for each step. 'Based on estimated availability of ESI interfaces, but implementation may suffer from low ion sampling or ion use efficiency and other limitations that hinder CE/MS operation (indicated by "?"). ο Approximate range, but will vary widely with commercial implementation and options.


REPORT traordinary sensitivity and the capa b i l i t y to a c c o m m o d a t e e x t r e m e l y small s a m p l e sizes. For e x a m p l e , sample volumes w i t h 5- to 10-μπιi.d. capillaries are generally on the order of 10 pL. H a n d l i n g of femtomole-sized samples is generally im practical, and integrated or on-line methods of sample handling are es sential. Microcolumn methods for protein digestion (using immobilized en zymes), combined with other frontend sample workup strategies, must be optimized for specific a p p l i c a tions. It is anticipated t h a t such CE methods a n d ancillary "picoscale" manipulation techniques will provide an essential toolbox for biochemical analysis on the femtomole and subfemtomole levels. The opportunities for biochemical research are enormous but extremely demanding. For example, the capa bility for analysis at the single-cell level could provide insights into cel lular chemical processes without the necessity of averaging over large cell

populations. Novel studies of DNA d a m a g e p r o d u c t s , protein produc tion, a n d i m p o r t a n t p o s t - t r a n s l a tional protein modifications would become possible. Such studies will r e q u i r e MS detection s e n s i t i v i t i e s extending to the zeptomole range. The demands imposed by m a t e r i als such as glycoproteins are essen tially o p e n - e n d e d because of t h e i r possible structural complexity (i.e., heterogeneity arising primarily from the structural complexity of carbohy drates and further convoluted by the number of possible association sites). Comparable challenges exist for the c h a r a c t e r i z a t i o n of l a r g e biopolymers, in which higher order tandem MS methods (e.g., MS", where η > 2) may offer the possibility of obtaining complete sequence information a s well as the identity and location of structural modifications. The gentle n a t u r e of ESI has also created new opportunities to directly probe noncovalent associations in so lution (39-43) and even qualitative aspects of higher order structure. Al

Figure 9. An initial demonstration of on-line CE/FT-ICRMS. Shown on the left is the total ion electropherogram for separation of a polypeptide and five proteins, in which the elution rate was reduced by half prior to elution of the first solute to the electrospray source. Partial mass spectra obtained during this separation are shown on the right for three of the proteins (ubiquitin, 8,565 Da; carbonic anhydrase, 28,802 Da; and myoglobin [minus the heme moiety], 16,951 Da). Inserts to the mass spectra show that resolution (30,000-50,000) sufficient to resolve the 1 -Da spacing attributable to isotopic contributions is obtained for each of the proteins.


t h o u g h t h e gentle interface condi tions necessary to preserve such in teractions by ESIMS can result in a substantial decrease in sensitivity, C E / M S is still feasible, as demon strated in Figure 8. Shown are separa tions of ribonuclease S (RNase S) with use of two different pH buffers (44). RNase S is a noncovalent complex consisting of a 13.7-kDa A-protein and a 2166-Da Α-peptide. The com plex dissociates under acidic buffer conditions, resulting in two peaks in the CE separation. The mass spec t r u m in Figure 8 was obtained from a C E / M S s e p a r a t i o n u s i n g an a m m o n i u m b i c a r b o n a t e buffer. T h e m a s s s p e c t r u m is d o m i n a t e d by peaks a r i s i n g from intact RNase S (i.e., the A-peptide-A-protein com plex). The ability to use C E / M S to probe n o n c o v a l e n t D N A - p r o t e i n , protein-ligand, DNA-drug, and other solution interactions is an ex citing prospect. The practicality of such applications will benefit greatly from improved MS performance. The prospects for improved perfor mance are excellent. The sensitivity of the ESIMS detector can be attrib uted to two factors: the overall ionsampling and transmission efficiency of t h e E S I M S i n t e r f a c e (typically only 10~ 3 -10~ 5 with present instru mentation) and the ion utilization ef ficiency, which derives from the type of m a s s spectrometer used and its mode of operation. For example, a quadrupole m a s s spectrometer with a n ion-sampling efficiency of 10~ 3 operated in a scan ning mode would typically have a n ion utilization efficiency of - 10 3 , w h e r e a s in SIM mode it m i g h t be 10" 1 or better. If 100 ions m u s t be detected to give an S/N of 2 during elution of an analyte zone, the best achievable sensitivity would corre spond to - 0.6 and 60 amol d u r i n g scanning and SIM operation, respec tively—even if ESI efficiency were 100%. In fact, our best results are close to these levels of performance. Progress continues in improving ESIMS sampling and t r a n s m i s s i o n efficiency, but efficient utilization of the ions created at atmospheric pres sure remains elusive. More promis ing in the short term are approaches t h a t address mass spectrometric ion utilization efficiency. High-performance CE/MS instru mentation, in our opinion, will most likely be based on either orthogonal time-of-flight (TOF) or ion trapping (quadrupole ion t r a p FT-ICR) m a s s spectrometers. Table I compares ex isting quadrupole MS i n s t r u m e n t a tion w i t h t h e e s t i m a t e d or a n t i c i -

pated characteristics of three of the most promising c o n t e n d e r s among high-performance m a s s spectrome t e r s for C E / M S . O u r l a b o r a t o r y is currently investigating both the or thogonal TOF and FT-ICR ap proaches. The rationale behind this selection is t h a t the TOF offers the g r e a t e s t scan speeds a n d possibly t h e best achievable sensitivity (po t e n t i a l l y u s i n g n e a r l y e v e r y ion transmitted into vacuum); the F T - I C R p o t e n t i a l l y offers excep tional performance (high MS resolu tion, precise m a s s m e a s u r e m e n t s , and high sensitivity simultaneously, as well as MS") but at slower spec tral acquisition rates. Thus, the CE/orthogonal TOF ap proach a p p e a r s to be ideally suited for ultrahigh sensitivity and for situ ations in which very high separation speeds are desired (45). Such appli cations may include single-cell anal yses, rapid sampling for the analysis of physiological systems, and twodimensional LC/CE/MS. The C E / F T - I C R combination of fers exciting opportunities for situa t i o n s t h a t require high MS resolu tion, accurate m a s s m e a s u r e m e n t , and high sensitivity. Building on the pioneering work of McLafferty et al. (46, 47), we have recently developed a new FT-ICR i n s t r u m e n t t h a t has been used to obtain ultrahigh-reso lution mass spectra (in excess of 106) a n d good sensitivity (below femtomole detection limits for small pro teins) for off-line ESIMS (48). Figure 9 shows an initial example from o u r l a b o r a t o r y t h a t d e m o n s t r a t e s t h e p r a c t i c a l i t y of o n - l i n e CE/FT-ICRMS (49). The left side of the figure shows the total ion electropherogram obtained for a separation of a polypeptide a n d five p r o t e i n s . The duty cycle (ion injection time di vided by the total time between spec tra) obtained in these experiments is low (2-10%), and a m a s s spectrum could be a c q u i r e d only every 6 s because of data storage limitations. However, excellent quality spectra w i t h r e s o l u t i o n s of 3 0 , 0 0 0 - 5 0 , 0 0 0 were obtained from 500-fmol injec tions of individual components. Figure 9 also shows p a r t i a l m a s s spectra for three of the proteins. As shown by the inserts, the FT-ICR in s t r u m e n t provides resolution of the 1-Da spacing of the isotopic envelope associated with each protein molecu lar ion charge s t a t e . Significantly, s u c h h i g h MS r e s o l u t i o n w a s o b t a i n e d even for t h e largest protein examined (carbonic anhydrase, 28,802 Da) for which t h e isotopic peaks for the 25+ charge s t a t e are

s e p a r a t e d by only 0.040 mlz u n i t s . The combination of sensitivity and M S r e s o l u t i o n d e m o n s t r a t e d by these initial results represents a b r e a k t h r o u g h in C E / M S p e r f o r m a n c e . Work in o u r l a b o r a t o r y is aimed at obtaining further gains in t h e duty cycle, sensitivity, and MS resolution of CE/FT-ICRMS. Although only beginning to be re alized, this combination of capabili ties could ultimately allow the sam pling, separation, and structural determination (e.g., sequencing, lo cation of modifications, or noncovalent associations) from attomole and even subattomole quantities of mate rial. A current goal at our laboratory (50) is t r a p p i n g a n d s e q u e n c i n g a single (i.e., individual) ionized seg m e n t of DNA. A t t a i n m e n t of t h i s ambitious goal will depend on t h e unique capabilities of nondestructive detection, long trapping times, high s e n s i t i v i t y (one ion if sufficiently charged), and MS" methods (where η can be very large) t h a t are currently unique to FT-ICR (51). Because CE provides the capabil ity to slow or completely stop a sepa r a t i o n almost instantaneously, t h e potential exists to fully exploit t h e sensitivity and high-resolution capa bility of FT-ICR in conjunction with MS" experiments for structural char acterization of biopolymers. The r e alization of such advanced C E / M S instrumentation should provide the m e a n s to address a profoundly ex p a n d e d r a n g e of q u e s t i o n s across broad fields of chemical and biologi cal research. This research was supported by internal PNL exploratory research and by the Director, Office of Health and Environmental Research, U.S. Department of Energy. Pacific Northwest Labo ratory is operated by Battelle Memorial Insti tute for the U.S. Department of Energy, through contract DE-AC06-76RLO 1830. We thank C. J. Barinaga, J. E. Bruce, G. A. Ander son, R. T. Kouzes, H. R. Udseth, D. C. Gale, and A. Brown for contributions and helpful discus sions related to the work described. References (1) Yergey, A. E.; Edmonds, C. G.; Lewis, I.A.S.; Vestal M. L. Modern Analytical Chemistry; Plenum Press: New York, 1990. (2) Jorgenson, J. W.; Lukacs, K. D. Sci ence 1983, 222, 266-72. (3) Yamashita, M.; Fenn, J. B. /. Phys. Chem. 1984, 88, 4671-75. (4) Fenn, J. B.; Mann, M.; Meng, C. W.; Wong, S. F. Mass Spectrom. Rev. 1990, 9, 37-70. (5) Smith, R. D.; Loo, J. Α.; Edmonds, C. G.; Barinaga, C. J.; Udseth, H. R. Anal. Chem. 1990, 62, 882-99. (6) Smith, R. D.; Loo, J. Α.; Ogorzalek Loo, R. R.; Busman, M.; Udseth, H. R. Mass Spectrom. Rev. 1991,10, 359-451. (7) Olivares, J. Α.; Nguyen, N. T.; Yon-

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REPORT (37) Thompson, T. J.; Foret, F.; Vouros, P.; Karger, B. L. Anal. Chem., in press. (38) Wahl, J. H.; Smith, R. D., submitted for publication in / Chromatogr. (39) Ganem, B.; Li, Y-T.; Henion, J. D. /. Am. Chem. Soc. 1991, 113, 6294-95. (40) Katta, V.; Chait, B. T. /. Am. Chem. Soc. 1991, 113, 8534-35. (41) Baca, M.; Kent, S.B.H./. Am. Chem. Soc. 1992, 114, 3992-93. (42) Light-Wahl, K. J.; Springer, D. L.; Winger, B. E.; Edmonds, C. G.; Camp, D. G., II; Thrall, B. D.; Smith, R. D. /. Am. Chem. Soc. 1993, 115, 803-04. (43) Goodlett, D. R.; Camp, D. G., II; Hardin, C. C ; Corregan, M.; Smith, R. D. Biol. Mass Spectrom. 1993, 22, 18183. (44) Goodlett, D. R.; Ogorzalek Loo, R. R.; Loo, J. Α.; Wahl, J. H.; Smith, R. D., unpublished work. (45) Sin, C. H.; Lee, E. D.; Lee, M. L. Anal. Chem. 1991, 63, 2897-900. (46) Henry, K. D.; Quinn, J. P.; McLafferty, F. W./. Am. Chem. Soc. 1991, 113, 5447-48. (47) Beu, S. C ; Senko, M. W.; Quinn, J. P.; McLafferty, F. W . / Am. Soc. Mass Spectrom. 1993, 4, 190-92. (48) Winger, B. E.; Hofstadler, S. Α.; Bruce, J. E.; Udseth, H. R.; Smith, R. D. J. Am. Soc. Mass Spectrom., in press. (49) Hofstadler, S. Α.; Wahl, J. H.; Bruce, J. E.; Smith, R. D., unpublished work. (50) Edmonds, C. G.; Smith, R. D. ACS Div. Anal. Chem. Newsletter 1990, 27-34. (51) Marshall, A. G.; Grosshans, P. B. Anal. Chem. 1991, 63, 214 A-229 A.

include combining LC and CE with MS for biochemical applications.

Richard D. Smith (left) is a senior staff scientist and group leader for the Chemi cal Methods and Separations Group in the Chemical Sciences Department at Pa Steven A. Hofstadler (left) received his cific Northwest Laboratory. He obtained B.S. degree in chemistry from the Univer his Ph.D. from the University of Utah and sity of New Mexico and his Ph.D. in chem spent a year as an NRC postdoctoral asso istry from the University of Texas. He is ciate at the Naval Research Laboratory currently a postdoctoral research associ before joining PNL in 1976. His current ate focusing on the development and bioresearch interests include the developmentanalytical applications of ESI-FT-ICRMS. of separation techniques, their combina He will join the PNL staff this year as a se nior research scientist. tion with MS, and new methods for the ultrasensitive structural characterization David R. Goodlett received his M.S. de of large molecules based on MS. gree from Auburn University and his Ph.D. Jon H Wahl received his B.S. degree in in biochemistry from North Carolina State chemistry from Michigan Technological University. He recently completed a post doctoral appointment at PNL and has University and his Ph.D. in analytical chemistry from Michigan State Univer joined the Immunobiology Research Insti tute. His research interests include the rela sity. He is currently a postdoctoral re searcher at PNL involved in the develop tionship between structure and function in ment ofCE/ESIMS. HL· research interests biological macromolecules.

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