Continuous-flow fast atom bombardment mass spectrometry

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Continuous-flow fast atom bombardment mass spectrometry Richard M. Caprioli Houston, TX, U.S.A.

The development and widespread use of fast atom bombardment mass spectrometry (FAB-MS) has led to research and methodology development in fields which require the mass spectrometric analysis of polar compounds’,2. Many of these investigations deal with compounds which were not previously amenable to mass spectrometric analysis without the use of special preparation and derivatization steps. Although there are a number of new ‘soft’ ionization techniques which have been described over the past decade, the general acceptance of FAB is probably the result of it being a convenient techni ue for analyzing samples contained in solutions 3% ’ . This has been of enormous benefit in analytical determinations involving samples derived from biochemical and molecular biological systems where polar and charged molecules are enerally prepared or isolated in aqueous solutions ? . The analysis of a sample by FAB-MS typically involves mixing 0.5-l ,ul of sample solution with 2-3~1 of glycerol, thioglycerol or some other suitable liquid organic compound. This liquid matrix serves several important roles; first, its high viscosity allows the sample to survive the high vacuum introduction process and, second, it keeps the sample surface fluid during the analysis so that the surface is constantly refreshed with new material. Unfortunately, the use of this organic matrix also creates several significant problems. The FAB mass spectrum is often dominated with matrix cluster ions, i.e., for glycerol, ions at m/z 92n + 1, where n is an integer generally from 1 to about 20, and fragment ions of these cluster8. In addition, low level ion intensities are produced at every mass from the radiation damage inflicted on the organic matrix by the atom beam, significantly limiting the detection limit of the technique. This is not surprising since one may typically analyze 200 pm01 of sample contained in 2 ~1 of glycerol, i.e., there is’ about a lo5 molar excess of glycerol over sample. Finally, in many mixtures, ions from certain compounds are not observed and are suppressed by the presence of other compounds in the mixture. This has been rationalized on the basis of the hydrophobicity/hydrophilicity index of the compounds and their interaction with the matrix7’*. 01659936/887$03.00,

Continuous-flow FAB (CF-FAB) was developed to alleviate the problems encountered with the use of excessive amounts of organic matrix and to provide a means for the direct introduction and analysis of aqueous sample solutions”” by mass spectrometry. Since water provides the major source of fluidity for the sample using this technique, the limit of detection of compounds is appreciably lower since much of the background and chemical noise derived from the gl cerol is substantially decreased or eliminated’ x. In addition, ion suppression effects are greatly diminished”. These points are addressed in more detail below. CF-FAB has been shown to be of utility in providing a means for the on-line analysis of biochemical reactions and other processes occurring in aqueous solution5’12. This has included its use as a microbore spectrometry liquid chromate raphy-mass (LC-MS) interface’ B-15 and the potential for utilizing it to couple MS to other separations systems such as electrophoresis devices. A similar technique, termed frit-FAB, has been used for the LC-MS analysis of biological samples16. A schematic diagram of the probe for CF-FAB is shown in Fig. 1. A flow of approximately 5-10 yllmin of liquid supplied from either a mechanical pump or driven by atmospheric pressure enters the source through a fused-silica capillary which extends to the probe tip. The fused-silica capillary is necessary to isolate the high voltage present at the probe tip when the probe is inserted into the ion source of a magnetic mass spectrometer. The flow of liquid is balanced by its removal from the surface of the probe by evaporation or by other means which physically removes the excess liquid. As the liquid

vacuum

stainless

steel

.

sample stage

Fig. I. Schematic

sample load

waste

diagram

of the continuous-flow 0 Elsevier

I pump

FAB probe.

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emerges from the capillary tip, it is bombarded by energetic atoms, usually 6-8 keV xenon atoms, producing ions which are subsequently analyzed by the spectrometer. For convenience, one end of the capillary is attached to an injection valve which permits the rapid introduction of samples. We have found that under optimal conditions, 0.5~1 samples may be injected as often as every 90-120 s without giving any significant memory effect.

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80 70

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60 50 40

2 GLYC

30

Advantages of CF-FAB A significantly lower limit of detection observed with CF-FAB, as compared to standard FAB, is the result of the removal of most of the organic matrix and also an increase in the sputter yield of ions produced from the more aqueous sample”. This is exemplified by comparing the analysis of the peptide substance P [molecular weight, (MW) 13471 using standard FAB, i.e., with sample containing 90% glycerol, and CF-FAB where the sample contains only about 5% glycerol. At a concentration of 100 fmol, the [M+H]+ ion for this peptide is not discernible above background in the case of standard FAB but is observed with a signal-to-noise ratio of about 5 with CF-FAB. It should be noted that the ion intensity at m/z 1348 for the measurement obtained using standard FAB is 8495 counts while that for CF-FAB is 454 counts, i.e., in this case, about 95% of the ion current in standard FAB is derived from the glycerol matrix. Of course, the magnitude of the improvement in the limit of detection achieved with CF-FAB will vary with the nature of the sample and is dependent upon the concentration of sample. The ion suppression effect which has often been observed with standard FAB for mixtures of compounds usually occurs when these compounds differ markedly in hydrophobicity7. Generally, more hydrophobic compounds are capable of occupying the surface of the droplet in disproportion to their concentration and suppress ion formation of more hydrophilic compounds. Using CF-FAB, it has been observed that this effect is greatly diminished and even eliminated in many cases”. This effect is attributed to the dynamic state of the liquid on the probe tip and the extensive mixing which occurs. Fig. 2 shows this for the analysis of a mixture of six peptides at a concentration of 300 pmol each using both CFFAB and standard FAB. Table I lists these compounds together with their hydrophobic indices. Clearly it can be seen that the data obtained for the sample introduced with the CF-FAB shows more intense [M+H]+ signals for the peptides at m/z 777, 735, and 720 relative to that obtained for standard FAB. These data also show that the suppression effect is complex and that other factors in addition to

4

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J

0lk 780

760

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720

700

680

640

620

600

580

M/Z

4

100

6 90

I

CONTINUOUS-FLOWFAB

a0 70 60 50 40 30 20

10 0

780 760 7&O 720

700

680

660

640

620

600

580

M/Z Fig. 2. FAB mass spectra of a mixture of heptapeptides obtained from (top) standard FAB, and (bottom) CF-FAB techniques. The peptides used in the mixture are listed in Table I. The peaks derived from glycerol are noted as GLYC.

TABLE

I. Synthetic

heptapeptides

No. Sequence Ala-Phe-Lys-Lys-Ile-Asn-Gly Ala-Phe-Asp-Asp-Ile-Asn-Gly Ala-Phe-Lys-Ala-Lys-Asn-Gly Ala-Phe-Lys-Ala-Ile-Asn-Gly Ala-Phe-Asp-Ala-Ile-Asn-Gly Ala-Phe-Ala-Ala-Ile-Asn-Gly

analyzed

by FAB-MS

(m/z>

Hydrophobic indexa

777.4 751.3 735.4 720.4 707.3 663.3

37 80 331 59 80 80

[M+H]+

a Hvdrophobicihydrophilic index as described in ref. 7. from data 0; H. B. Bull and K. Breese, Arch. Biochem. Biiphys., 161(1974)665.

the hydrophobic and hydrophilic nature of peptides can play major roles in determining the surface composition of mixtures17. These may include the charge state in solution, secondary structure, interaction with the matrix, ion pairing, etc. Nevertheless, the overriding determinant remains the ‘ability of the

330

peptide to occupy the surface layers of the liquid sample. One of the major advantages obtained with the use of CF-FAB is its ability to provide quantitative measurements of high accuracy from sample to sample without the need for internal standards. For reaction monitoring, this is of great importance in either off-line or on-line operations. In contrast, for standard FAB, it is generally very difficult to load the probe tip and place it into the spectrometer in exactly the same manner without getting enormous variation in ion currents. Since the CF-FAB probe is not moved for sample injections and the liquid flow is held constant, a series of samples can be quantitatively related without using internal standards. Under stable operating conditions, replicate injections of the same sample routinely show a standard deviation of the area under the peaks of approximately & 10%) or better. Disadvantages of CF-FAB The dynamic nature of the surface of the liquid on the probe tip creates some difficulties. Because the surface is constantly in flux, the bombardment process gives rise to ion signals that may fluctuate more from moment to moment than it does in standard FAB. In this case, averaging spectra over an injection peak is of considerable value. Also, a period of 5-15 min may be required to establish stable operation after insertion of the probe. This period will vary depending on the nature of the solvents and the balance of temperature and flow-rate used. Water flowing into the ion source chamber at a rate of 5-10 pllmin will produce a source ion gauge pressure of about 2.10-4 Torr with the pumping capacity available on most commercial instruments. For magnetic instruments, care should be taken to insure that proper electrical grounds have been installed so that high voltage arcs do not cause damage to the electronics in the instrument. We have operated the CF-FAB probe with a Kratos MS50 at 8 keV of accelerating potential without difficulty. Finally, one must keep in mind the problems encountered with capillary bore tubing, nanoliter injection valves, and low-dead-volume plumbing connections. Cleanliness is essential and it is recommended that solvents and sample solutions be filtered to prevent blockage of small bore devices. Attention to eliminating dead volumes in connections cannot be overemphasized in order to minimize tailing of injections and memory effects. As a practical estimate, a 0.54 sample of a compound injected into a carrier solvent flowing at 5 ,&min should produce a peak which is approximately 30 s in duration at 10% peak height when using the CF-FAB probe.

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vol. 7, no. 9, 1988

Applications Operating modes

The CF-FAB probe may be operated in several modes depending on the application and the specific nature of the information required. We have generally used it in one of two modes, constant-flow and flow-injection. In the constant flow mode shown in Fig. 3A, the aqueous reaction solution is allowed to continuously flow into the ion source, with the 5-10% glycerol required added to the reaction solution or added in a make-up tee prior to entering the probe capillary18. This type of operation is useful where rapid changes in concentrations are expected in the reaction and where reaction conditions are compatible with bombardment conditions in the source. The driving force for liquid flow can be a syringe or other low flow-rate pump or atmospheric pressure itself as shown in Fig. 3A. Although the latter is simpler, it has the disadvantage of being a pressure-regulated rather than a flow-regulated device, making it more susceptible to particulate matter which can cause partial blockage of the capillary and hence give uneven liquid flow. The other mode of operation, flow injection, is shown in Fig. 3B and is used in cases where slower changes of concentrations of compounds are anticipated and where reaction conditions are not favorable to give maximum ion yields. An example of this would be the monitoring of the tryptic cleavage of a peptide at pH 8.5, but where greatest sensitivity for detection would re-

A. Constant Flow

Fig. 3. Use of the CF-FAB flow-injection applications.

probe for (A) constant-flow, See text for details.

and (B)

trends

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TABLE

II. Composition

Peptide

Renin Substrate Neurotensin a-MSH Bombesin Angiotensin I Physalaemin Angiotensin II Angiotensin III Met-Enkephalin-RF Gly Octapeptide Kemptide

of peptide mixture used for on-line monitoring of trypsin digestion Sequence

[M+H]* (m/z)

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Asn pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu Ac-Ser-Tyr-Ser-Met-Glu-Phe-His-Arg-Trp-Gly-Lys-Pro-Val(NH~) pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met(NH,) Asp-Arg-Val-Tyr-Be-His-Pro-Phe-His-Leu pGlu-Ala-Asp-Pro-Asn-Lys-Phe-Tyr-Gly-Leu-Met(NH~) Asp-Arg-Val-Tyr-Ile-His-Pro-Phe Arg-Val-Tyr-Ile-His-Pro-Phe Tyr-Gly-Gly-Phe-Met-Arg-Phe Gly-Arg-Gly-Leu-Ser-Leu-Ser-Arg Leu-Arg-Arg-Ala-Ser-Leu-Gly

quire pH 1 on the probe tip. In this case, the carrier liquid contains sufficient acid to maintain pH 1 at the probe tip while still accepting small volume (0.5 ~1) injections of the reaction solution itself. This mode also lends itself to automated control for on-line process monitoring over long periods of time.

Reactant

Products

1759 1672 1665 1620 1296 1265 1046 931 877 845 773

1488, 1030, 1098, 1224, 1025, 629, 775, 775, 730, 632, 503,

290 661 585 414 290 584 290 290 290 232 288

-f

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1265

Reaction monitoring

In an investigation aimed at studying the catalytic properties of trypsin, it was desirable to present the enzyme with a substrate ‘cocktail’ consisting of mixtures of peptides. CF-FAB was used for reaction monitoring of the digestion of this mixture of eleven peptides (see Table II). The relative rates of cleavage of these peptides were of interest as specific reaction conditions were altered. In one example, the reaction solution contained 100 pmol of each peptide, 50 mm Tris (pH 8.4), with a substrate-totrypsin ratio of 2OO:l (w/w). The flow-injection mode was employed with data acquired with a Kratos MSSORF high-field mass spectrometer. A Brownlee Microgradient pump was used to deliver the liquid and control the automated process which allowed 0.5 ,~l sample aliquots to be injected into the carrier flow (95% water, 5% glycerol, 0.1% trifluoroacetic acid) every 5 min. Individual molecular species were followed to monitor both the decrease in the reactants and increase in products during the reaction over a period of about 50 min. Fig. 4 shows the selected ion chromatograms for the [M+H]+ ions of physalaemin at m/z 1265 and its tryptic fragment at m/z 629. The relative rate curves for the hydrolysis of this peptide as well as several others are shown in Fig. 5.

M/Z 629

300

Non-aqueous applications

Although this review has emphasized the analysis of aqueous solutions, it should be pointed out that the CF-FAB probe has been utilized for the intro-

400

500

scan Fig. 4. Selected ion chromatograms for the time-course tryptic hydrolysis of physalaemin (mlz 1265) and a fragment product (mlz 629) using the flow-injection CF-FAB technique.

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%

lOO_ m/z: 916-917 8060. m/z

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1265

4020-

1

I\

1046

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0

4

a

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16 TIME (minutes

20

24

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x-E+07 3.165

28

1

Fig. 5. Relative rates of hydrolysis of a mixture of peptides by trypsin; mlz 1265, physalaemin; mlz 1046, angiotensin II; mlz 1665, bombesin. The flow-injection CF-FAB technique was used to obtain the data without the use of an internal standard.

duction of organic carrier solvents for those samples which are not soluble in water. Barber and co-workers19,*’ have employed acetonitrile containing 5% thioglycerol as the solvent system for the analysis of glycerides such as 1,2- and 1,3-distearin and tripalmitin, chloroform as solvent for the analysis of cholesterol, and toluene with 5% m-nitrobenzyl alcohol as the flow solvent for analysis of air and moisture sensitive compounds such as phosphines.

40

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100 scan

120

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160

180

Fig. 6. Microbore LC-MS analysis of the tryptic digest of ,%lactoglobulin A, using the CF-FAB interface with a 0 to 60% acetonitrile gradient. (top) Selected ion chromatogam of mlz 916 and (bottom) reconstructed total ion chromatogram.

916.6

*E-l

900

910

920

930

940

"I"","'.,"","1000

1500

mlz

2000

2500

Fig. 7. FAB mass spectrum of the chromatographic peak shown in Fig. 6 (top) centered at scan 63.

Microbore HPLC-MS

The CF-FAB probe has been used successfully as an interface for microbore LC-MS for the analysis of peptides and derivatized oligosaccharides’3-‘5. For example, we have used the technique for the analysis of the tryptic digest of ,&lactoglobulin A with both MS and UV detection*l. An Applied Biosystems Model 130A microbore LC system was linked to a Finnigan MAT 90 high-field magnetic mass spectrometer. The LC was fitted with a Brownlee RP300 (50 x 1 mm) column which was eluted at approximately 25 ,&min with a 0 to 60% acetonitrile gradient. The eluent was split 4:l to UV and MS detectors, respectively, to give a 5 ,&min flow into the mass spectrometer. The mass spectrometer was continuously scanned from m/z 600 to 2900 at a rate of approximately 10 s per scan. Fig. 6 shows the reconstructed ion chromatogram (RIC) for the MS analysis of 40 pmol of the digested protein. Individual ion searches were used to locate peptides in the chromatogram, as illustrated in the top panel of Fig. 6 for m/z 916. The mass spectrum obtained for the apex of this chromatographic peak is shown in Fig. 7. In all,

fifteen peptides were identified. Simultaneous UV detection and sample collection of the effluent permits additional sample work-up on mass identified peak fractions. The major advantages of the CF-FAB interface for LC-MS analyses are (1) high sensitivity and high mass capability (> m/z 6000), (2) compatibility with low flow-rate microbore techniques, (3) compatibility with high-voltage magnetic instruments, and (4) a greatly diminished ion suppression effect in comparison to standard FAB. Conclusion CF-FAB has been shown to be an effective means to introduce samples in volatile liquids into a FAB mass spectrometer. It is amenable to on-line operation for analysis of batch reactions and also for the analysis of reactions where a continuous analysis is of value. Perhaps its greatest use will come in the area of biochemistry and molecular biology where

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trends i_nanalytical chemistry, vol. 7, no. 9,198s

the vast majority of compounds are present in aqueous media. It provides a means for the analyst to utilize the mass spectrometer for the molecularly specific determination of compounds directly in water solutions without the need for isolation and derivatization, thereby maintaining the dynamic chemical balance of a reaction during the analysis. Further, it has been shown to be an excellent LC-MS interface for microbore separations. A number of papers has shown the method to be of particular utility in the identification of protease digests of large polypeptides and proteins, especially where product verification and purity are being assessed. Acknowledgements The author thanks William T. tin, Beverly DaGue and Anne help in obtaining data presented Petrie of Applied Biosysytems, the synthetic heptapeptides.

Moore, Millie MarBallatore for their here, and Gordon Inc., for preparing

References M. Barber, R. S. Bordoli, R. D. Sedwick and A. N. Tyler, J. Chem. Sot., Chem. Commun., (1981) 325. R. M. Caprioli, in P. A. Lyon (Editor), Desorption Mass Spectrometry-Are SIMS and FAB the Same, American Chemical Society, Washington, DC, 1985, Ch. 12, p. 209. R. M. Caprioli, in M. E. Rose (Editor), A Specialist Periodical Report: Mass Spectrometry, The Royal Society of Chemistry, London, 1985, Vol. 8, Ch. 8, p. 184. R. M. Caprioli, in S. J. Gaskell (Editor), Mass Spectrometry in Biomedical Research, Wiley, New York, 1986, Ch. 4, p. 41. R. M. Caprioli, Biochemistry, 27 (1988) 513. M. Barber, R. S. Bordoli, G. Elliott, R. D. Sedgwick and A. N. Tyler, Anal. Chem., 54 (1982) 645A. S. Naylor, A. F. Findeis, B. W. Gibson and D. H. Williams, J. Am. Chem. Sot., 108 (1986) 6359. W. V. Lignon, Jr., Anal.‘ Chem., 58 (1986) 485.

9 R. M. Caprioli, T. Fan and J. S. Cottrell, Anal. Chem., 58 (1986) 2949. 10 R. M. Caprioli and T. Fan, Biochem. Biophys. Res. Commun., 141 (1986) 1058. 11 R. M. Caprioli, W. T. Moore and T. Fan, Rapid Commun. Mass Spectrom., 1 (1987) 15. Biomed. Environ. Mass Spectrom., 15 12 R. M. Caprioli, (1988). 13 A. E. Ashcroft, Org. Mass Spectrom. Lett., 22 (1987) 753. 14 R. M. Caprioli, B. DaGue, T. Fan and W. T. Moore, Biothem. Biophys. Res. Commun., 146 (1987) 291. 15 R. M. Caprioli, W. T. Moore, B. DaGue and M. Martin, J. Chromatogr. SC., (1988) in press. 16 Y. Ito, T. Takeuchi, D. Ishii and M. Goto, J. Chromatogr., 346 (1985) 161. 17 R. M. Caprioli, W. T. Moore, G. Petrie and K. Wilson, Znt. J. Mass Spectrom. Ion Phys., (1988) in press. M. A. Moseley, K. B. Tomer 18 J. S. deWit, L. J. Deterding, and J. W. Jorgensen, Rapid Comm. Mass Spectrom., 2 (1988) 100. R. S. 19 M. Barber, L. W. Tetler, D. Bell, A. E. Ashcroft, Brown and C. Moore, Org. Mass Spectrom. Lett., 22 (1987) 647. 20 M. Barber, L. W. Tetler, D. Bell, D. B. Gordon, A. E. Ashcroft, R. S. Brown and G. Elliott (Editors), Proceedings of the 35th ASMS Conference on Mass Spectrometry and Allied Topics, Denver, Colorado, May 24-29, 1987. 21 R. M. Caprioli, unpublished data. Richard M. Caprioli is at the Analytical Chemistry Center and Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, TX 77030, U.S.A. He is a Professor of Biochemistry at the University of Texas Health Science Center. He obtained his Ph.D. from the Department of Biochemistry at Columbia University of New York in 1969. After a one year post-doctoral with Prof. John H. Beynon at Purdue in 1970, he was appointed Assistant Professor of Chemistry at Purdue. In 1975, he moved to the University of Texas at Houston and was appointed Professor in 1980. He is also Director of the Analytical Chemistry Center at the University of Texas in Houston. The Center is a research resource which specializes in the application of modern analytical instrumentation and techniques~to biomedical research programs.

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