Anal. Chem. 19@2,64, 957-960 (17) Spinner, E. J . Chem. Soc. 1082, 3119. (18) Tape, J.; Tsuruya, T.; Sato, T.; Yoneda, Y. Bull. Chem. Soc.Jpn. 1072, 45, 3609. (19) Shimatla. H.; Ohtoh, T.; Nibu, Y. Fukuoka Unlv. Sci. Rep. 1088, 18 (2). 123. (20) Batts, B. D.; Spinner, E. Aust. J . Chem. 1080, 22, 2595. (21) Bajdor, K.; Nishimura, Y.; Peticoias, W. L. J . Am. Chem. Soc. 1087, 109, 3514.
957
(22) Jones, C. M.; DeVlto, V. L.; Harman, P. A.; Asher, S. A. Appl. Spec-. trosc. 1087, 4 1 , 1268. (23) Hair, M. L.; Hertl, W. J . B y s . Chem. 1070, 7 4 , 91. (24) Dutta, P. K.; Turbeville, W. J . phvs. Chem. 1001, 95, 4087.
RECEIVED
for review September 16, 1991. Accepted J a n W
27, 1992.
Tandem Mass Spectrometric Analysis of Peptides at the Femtomole Level Peter T. M. Kenny and Ron Orlando* Suntory Institute for Bioorganic Research, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618, Japan
I n the sequentlal analysls of peptldes by fast atom bombardment (FAB) tandem m a r spectrometry (MS/MS), the prlnclpal obstacle to decreaslng sample quantltles was determlned to be the rlgnal-to-background ratio of the ionlzatlon/decrorptlon process. By decresdng the background Ion current, contlnuow-flow FAB allows complete analyds (both MS and MS/MS) to be performed on 25-75% l e r sample than requlred for a conventlonal FABMSIMS experlment alone. The comblnatlon of CF-FAB wlth array detection permltted sequential analysls of several peptider (900-2000 Da) at the 900 hnol to 5.8 pmol level, wlthout Interference from the background. These levels do not produce a molecular ton specks easlly dlscernlble above the background In conventlonal FAB.
INTRODUCTION Over the past several years, tandem mass spectrometry (MS/MS)'s2 has played an increasing role in the structural analysis of complex bimole~ules.~ In the case of sequential analysis of peptides, MS/MS offers several advantages over traditional Edman degradations, including the abilities to sequence peptides present in mixtures, identify modified amino acids, and sequence peptides with blocked N termini.3 These advantages often offset the larger sample quantities traditionally required for analysis by MS/MS. Although numerous studies have been performed on reducing the quantity of peptide required for sequencing by MS/MS, probably the most promising new technique is array detection. With the ability to simultaneously detect entire portions of the mass spectrum, array detection has been credited with increasing the sensitivity of MS/MS analysis by a fador of 50-100 over conventional point detection? This development has decreased the sample requirements needed by four-sector MS/MS instruments to the point where they now rival, if not exceed, that of Edman degradations.- With the development of detectors which can simultaneouslyrecord larger portions of the spectrum, further reductions in sample requirements are expected. Although not commonly encountered, artifact peaks have been reported in MS/MS spectra arising from fragmentation of the coselected FAB background ion."" When the sample size is reduced, the intensity of the analyte ion decreases relative to the background leading to increased interference. Eventually, these artifacts dominate the MS/MS spectrum and can obscure all structural information.lOJ1Consequently, the ultimate sensitivity of MS/MS analysis may be imposed 0003-2700/92/0364-0957$03,00/0
by the signal-to-background ratio produced by the ionization process." With the limitation imposed by the isobaric matrix ion, an obvious strategy for reducing sample requirements is to increase the precursor ion intensity relative to the background, which is the focus of this report. By reducing the amount of matrix, continuous-flow FAB (CF-FAB) has been demonstrated to provide a 50-100-fold improvement in the signalto-background ratio obtained in the analysis of peptides at the low-picomole level.12 Similarly, the use of CF-FAB as the ionization process for MS/MS analysis is shown here to yield a 25-75-fold decrease in analyte consumed compared to conventional FABMS/MS. The combination of CF-FAB and array detection decreases the amount of a peptide required for sequence analysis to the femtomole range, a level that is well below that of the stepwise E d " method. Additionally, this strategy allows sequence determination from peptide quantities that do not produce noticeable ions by conventional FAB. EXPERIMENTAL SECTION All mass spectra were acquired with a JEOL (Tokyo, Japan) HX/HXllOA tandem four-sectormass spectrometer, which was operated at 10-kV accelerating potential. Spectra acquired by MS 1are averaged profile data of 12 scans as recorded by a JEOL complement data system. These spectra were acquired from m / z 1000 to 1500 in 6 s at a rate that would scan from m/z 1to 6000 in 1min. A fitering rate of 300 Hz and an approximate resolution of lo00 were used in acquiring these spectra Ions were produced by fast atom bombardment with xenon using a JEOL FAB gun operated at 6 kV, with either a conventional FAB or a FRIT-FAB ion source. In the case of conventional FAB, the samples were diluted in a 1%aqueous solution of trifluoroacetic acid. Aliquota of the sample, 1 pL, were mixed with 1 pL of thioglycerol, the FAB matrix, on the probe tip. For the FRIT-FAB spectra, the samples were dissolved in a 1:l mixture of water and methanol. This solution also contained 10% trifluoroacetic acid and 4 % thioglycerol. The concentrations of peptides were 370 fmol/pL substance P, 580 fmol/pL porcine renin substrate tetradecapeptide, 90 fmol/pL for the synthetic peptide ASHIPRFV-NH,, and 300 fmol/pL for the synthetic peptide SDYEGRLIQNSL. Samples were introduced to the FRIT-FAB source by a 60-pm-i.d. fused-silica capillary tube that was 37 cm in length, which was inserted into the 10-pL micropipet that contained the sample. This combination of capillary size and length was found to introduce the sample at a constant rate of 2 pL/min, without the need for a syringe pump. Approximately 10 pL of the sample solution was sufficient to fill the transfer line, ensure constant flow, tune the mass spectrometer, and acquire both MS and MS/MS spectra. Further decreases in sample volume were not investigated, as this appeared to be the minimum volume needed to wash the sides of the sample tubes, ensuring complete use of 0 1992 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 8, APRIL 15, 1992
300
550
800
1050
m/z
Flgure 2. Conventional FABMS/MS spectrum acquired on 740 fmol of substance P (RPKPQQFFGLM-NH,). Ions produced by loss(es)of neutral thbglvcerol rolecules: (*) [M ~thkglY),l+;(+I [M (tMogly), - la]+; (X) [M - (thiogly), - 361'. Protonated thioglycerol clusters: (0) [(thl~ly),+ HI+; (0)[(thIOgly), + H - 1 8 1 ' .
-
1
I!
J
1300
1350
1400
m/z Flgun 1. Conventional FAB spectrum acquired with (a) 7.4 pmol and (b) 740 fmol and (c) the CF-FAB spectrum acqulred with 740 fmol of substance P (RPKPQQFFGLM-NH,).
the sample. Samples of substance P and porcine renin substrate tetradecapeptide were obtained from the Sigma Chemical Co., St.Louis,MO, end were used as received. The synthetic peptides ASHIPRFV-NH2and SDYEGRLIQNSL were synthesized by solid-phase methods on an automated (Applied Biosystems 430A) synthesizer using standard methodology. The crude peptide was deprotected in anhydrous HF and then purified by trituration in ether and reversed-phase high-performance liquid chromatography. Collisionally induced dissociation (CID)was performed in the third field-free region, using helium as the collision gas. The helium pressure was sufficient to attenuate the primary ion beam by 75%. The collision cell was floated at 8 kV. Fragment ions were detected by a JEOL MS-ADS11 variable dispersion array detector, which has been previously described.* A fragment ion dispersion of 1-1.2 (20%)was used. A dead time of 0.1 s was used between magnetic and electric field steps to allow stabilization of the magnetic field. The CID spectrum of substance P was acquired from m/z 75 to 1325in 16 steps, with each segment being recorded for four 65ms periods. The MS/MS spectrum of renin substrate tetradecapeptidewas acquired from m/z 80 to 1735 in 17 steps,with each segment being acquired for eight 65msperiods. Similarly, 15 steps were used to acquire the MS/MS spectrum of ASHIPRFV-NH2( m / z 60-905) with each segment averaged 10 times, and 16 steps to record the m / z range from 75 to 1365 in the tandem spectrum of SDYEGRLIQNSL. In this experiment each segment was averaged four times. The totalscan times were 16 s for substance P, 23 s for renin substrate tetradecapeptide, 22 s for ASHIPRFV-NH2,and 16 s for SDYEGRLIQNSL. The difference between the calculated time (sum of the steps, exposures, and dead times) and the actual "scan" time results from the time required to transfer the data from the photodiode array to the computer.
RESULTS AND DISCUSSION In the analysis of peptides at the low-picomole level, the analyte signal observed in a conventional FAB mass spectrum
-
is only slightly larger than the background, as demonstrated by the spectrum of 7.4 pmol of substance P (Figure la). Although the protonated molecule is easily recognized, the signal-to-background ratio implies that the sample accounts for only half of the ions present in this peak. As the sample quantity is further reduced, the signal-to-background ratio decreases and eventually the protonated molecule becomes indistinguishable from the background, as seen when the amount of sample is reduced to 740 fmol (Figure lb). In this case, prior knowledge of the sample is eseential, i.e., molecular weight, since the precursor ion cannot be distinguished from the background. Additionally, the low relative intensity of the analyte ion suggests that matrix ions account for the vast majority of the precursor selected for MS/MS analysis. With these handicaps, it is not surprising that the MS/MS analysis performed on 740 fmol of substance P (Figure 2) is dominated by peaks produced by fragmentation of the coselected matrix ion. Although the identity of this background ion is unknown, the fragment ions observed in this spectrum consist of two series: (1) losses of neutral thioglycerol molecules from the precursor; (2) protonated thioglycerol clusters. Both of these series contain satellite peaks corresponding to the loss(es) of water molecules. These ions account for virtually all the peaks observed in Figure 2 and conceal all structural information pertinent to the analyte. The inability to identify any of the sequence ions present in this spectrum suggests that the ultimate sensitivity of FABMS/MS analysis is primarily limited by the chemical background. Further support for this theory is obtained from comparison of the limits of analysis described in two recent studies. In one report, 15 pmol of substance P was required for CID analysis using a four-sector tandem maas spectrometer with a conventional point detector.'3 In a different study using array detection, the analysis of 10 pmol provided complete sequence information, but only half of the these fragments were observed when the sample size was reduced to 1pmol." The similarity in sample requirements between these two studies, combined with the lack of structural information in Figure 2, suggests that the obstacle limiting sample quantity reduction is the interference from the chemical background, and not detector sensitivity. Consequently, simultaneous detection of larger percentages of a FABMS/MS spectrum will not lead directly to reduction in sample quantities. By reduction of the amount of matrix, CF-FAB provides significantly better signal-to-background ratios than conventional FAE3.l2 This can easily be seen by comparing Figure IC(CF-FAB) with Figure I b (conventional FAB). Although
ANALYTICAL CHEMISTRY, VOL. 64, NO. 8, APRIL 15, 1992 I
Flguro 3. CF+ABMS/MS spectrum acquired with 200 fmoi of substance P (RPKPQQFFGLWNH,).
OS0 I
Flgurr 5. CF-FABMS/MS spectrum acquired with 70 fmol of the synthetic peptide ASHIPRFV-NH,.
-Y '2
E
f
PI
9 C P
a
100
600
1100
1600
L , 300
550
m/z
Flguro 4. CF-FABMS/MS spectrum acquired with 440 fmol of renln substance tetradecapeptide (DRVYIHPFHUVYS).
Ftgurr 6. CF-FABMSIMS spectrum acquired with 160 fmoi of the synthetic peptide SDYEQRLIQNSL.
both of these spectra were obtained on identical quantities of substance P (740 fmol), only the CF-FAB spectrum displays a protonated molecule observable above the FAB background. Additionally, the signal-to-background ratio obtained by CF-FAB (Figure IC)is clearly superior to that of the conventional FAB spectra obtained by using 10 times the amount of sample (Figure la). Although the protonated molecule produced by CF-FAB (Figure IC),allows easy identification and selection in MS 1, the low intensity of this ion requires the increased sensitivity of array detection for CID analysis. The CF-FABMS/MS spectrum obtained by consuming 200 fmol of substance P (Figure 3) contains two prominent series of N-terminal ions (a and d which confirm the amino acid sequence. Numerous low m / z immonium ions, which are useful for determining the amino acid composition of the peptide, are also present in this spectrum. Figure 3 is very similar to the numerous high-energy CID spectra of substance P present in the literature."J3-" The reduction in artifacts from the coselected background ion provided by CF-FAB, is easily wen in the MS/MS spectra by comparing Figure 3 (CF-FAB) to Figure 2 (conventional FAB). Even though only onefourth of the material was used, the CF-FABMS/MS spectrum does not contain any of the interfering peaks that dominate the spectrum obtained with conventional FAB. Therefore, by enhancing the percentage of analyte ions in the precursor, CF-FAB decreases the background interference, allowing reduced sample quantities. To further demonstrate the sensitivity of this strategy, Figure 4 shows the MS/MS spectrum acquired with 440 fmol of renin substrate tetradecapeptide. This spectrum contains both immonium ions and peptide sequence ions that provide
conformation of this peptide's sequence, without interference from the background ion. Also,the spectrum shown in Figure 4 is good agreement with those previously reported for highenergy CID of this peptide using larger sample sizes.4J4 The combination of CF-FABMS/MS with array detection has been used for the sequential analysis of several synthetic peptides. For example, the MS/MS spectrum acquired by consuming 70 fmol of ASHIPRFV-NH2is shown in Figure 5. This spectrum is dominated by C-terminal fragment ions, as predicted by the arginine at position 6, and allows the entire peptide to be sequenced. Additionally, half of the amino acid composition of this peptide is also obtained from the immonium ions. The MS/MS spectrum of a second synthetic peptide, SDYEGRLIQNSL, allowed sequence confirmation by consuming 160 fmol (Figure 6). In both of these cases, no interferences are observed from the coselected background ion. The complete mass spectral analysis, molecular weight determination and tandem spectra, used a total of 900 fmol (ASHIPRFV-NH2)and 3 pmol (SDYEGRLIQNSL). The decreased interference provided by CF-FAB combined with the increased sensitivity of array detection yields dramatic reductions in sample quantities consumed during the experiment compared to FAB MS/MS. For example in the analysis of substance P, this strategy provided a 25-75-fold decrease in sample quantity compared to the previous limits of analysis with FAB MS/MS."J3 Similarly, the amount of renin substrate tetradecapeptide used in this study is 22 times less than that reported previously for MS/MS analysis with array d e t e ~ t i o n . ~ Although this report e m p h a s h the advantages of CF-FAB, it also suggests a general strategy for reducing sample quantities for MS/MS analysis. As demonstrated, the current
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Ct”. 1902, 64. 960-964
barrier to decreasing sample sizes is the intensity of the precursor ion relative to the chemical background. Therefore, any technique that increases this ratio is expected to provided a similar gain in overall sensitivity, and several different possibilities can be suggested. Increasing the resolution of MS 1 may allow separation of the sample from the background, but with the reduced precursor ion intensity MS/MS analysis may not be possible. Alternatively, choice of the proper gas may allow reaction-induced dissociations’* (RID) to fragment the analyte ion selectively while neutralizing the interfering FAB background. Another approach would be the use of an ionization technique that provides a higher signal-to-background ratio than FAB, as demonstrated in this study. Regardless of the methodology, reducing the background interference yields a significant decrease in sample requirements, and permits more extensive use of the increased sensitivity provided by array detection.
ACKNOWLEDGMENT We thank J. A. Hill, J. E. Biller, and K. Biemann for providing information from their manuscript before publication and R. K. Boyd for helpful comments with this work.
(4) Hill, J. A,; Biller, J. E.; Martin, S. A.; Blemann, K.; Yoshldome, K.; Sato, K. Int. J . Mass Spectrom. Ion Processes 1888, 92. 211. (5) Cottrell, J. S.; Evans, S. Anal. Chem. 1987, 59, 1990. (6) Evans, S. In Methods in E~Z~mObgyY; McCloskey, J. A., Ed.; Academ ic Press: San Dbgo, CA 1990; Vol. 193. (7) &oss. M. L. I n M e W s in €nzyfno&gy; McCloskey, J. A,, Ed.; Academic Press: Sen Dlego. CA, 1990 Vol. 193. (8) HIII, J. A.; Biller, J. E.; Biemann, K. Inf. J . Mass Spectrom. Ion Processes, in press. (9) Falick, A. M.; Medzihradszky, K. F.; Walls, F. C. f?apMC”un. Mass Specfrom. 1990, 4 . 318. (10) Bryant, D. K.; Orlando, R. RapidCommun. Mass. Spectrom. 1881, 5 . 124. (11) Wails, F. C.; Baldwin, M. A.; Fallck, A. M.; Glbson, B. W.; Kaur, S.; Maltby. D. A.; GllleceCastro, 8. L.; Medzihradszky, K. F.; Evans, S.; Burlingame, A. L. I n Biological Mass spectrometry; Burlingame, A. L., McCloskey, J. A., Eds.; Elsevler: Amsterdam, 1990. (12) Caprioli, R. M. Bhxhemlstry 1988, 27, 513. (13) Bean, M. F.; C a r , S. A.; Thorne, G. C.; Reilly, M. H.; Qaskell. S. J. Anal. Chem. 1881. 63, 1473. (14) Martin, S. A.; Johnson, R. S.; Costello, C. E.; Blemann, K. I n Analysk Of fepfMes and Proteins; McNeal, C. J., Ed.; Wiley: Chichester, England, 1988. (15) Johnson, R. S.; Martin, S. A.; Blemann, K. Int. J . Mass Spectrom. Ion Processes 1888, 86. 137. (16) Poulter, L.; Taylor, L. C. E. Int. J . Mass Spectrom. Ion ProCeJses 1888. 91, 183. (17) Scoble, H. A.; Martin, S. A.; Biemann, K. Biochem. J . 1987, 245, 821. (18) Orlando, R.; Fenselau, C.; Cotter, R. J. J . Am. Soc. Mass Spectrom. 1881, 2 , 189.
REFERENCES (1) Busch, K. L.; Cooks. R. 0. Anal. Chem. 1883, 55, 38A. (2) Yost, R. A,; Enke. C. 0. J . Am. Chem. Soc.1978. 100, 2274. (3) Blemann. K. Anal. Chem. 1886, 58, 1289A.
RECEIVED for review September 30,1991. Accepted February 13, 1992.
TECHNICAL NOTES Liquid Membrane Electrode for Guanosine Nucleotides Using a Cytosine-Pendant Triamine Host as the Sensory Element Koji Tohda, Masahiro Tange, Kazunori Odashima, and Yoshio Umezawa* Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060, Japan Hiroyuki Furuta and Jonathan L. Sessler Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas 78712 INTRODUCTION The recognition and complexation of target chemical substances by synthetic host molecules and consequent signal transduction involving changes in membrane potential constitute an important approach to chemical sensing. A number of ion-selective electrodes (ISEs) based on polymer matrix liquid membranes have been investigated. Many of these display high selectivity for particular target substances and are now commercially available.’* However, most of the polymer matrix liquid membrane ISEs developed so far have focused on the recognition of alkali and alkaline earth metal cations by the use of natural and synthetic cyclic and acyclic neutral ionophores as sensory elements.’~~ Recently, a number of anion-selectiveelectrodes have been developed by the use of alkyltin compounds? vitamin Blz and metalloporphyrin derivative^,'^'^ diphosphonium16J7 and diammonium cations,ls and macrocyclic polyamines.’*21 Whereas the former two types of electrodes are based on reversible coordination of the anionic guests to the vacant coordination site(s) of the central metal ions, the latter two are based on electrostatic interaction between the anionic guests and the cationic hosts. Of these, the macrocyclic polyamine electrodes are characteristic in that the hosts
function as anion receptors by protonation at the membrane surface.1sa One of the remarkable features of the macrocyclic polyamine electrodes is their ability to discriminate among the adenosine nucleotides as a function of the number of negative charges. As a result, by far the strongest potentiometric response is observed for ATP“ as compared to ADP3and AMP2-.19 However, since these protonated macrocyclic polyamines appear to bind mainly to the phosphate group of nucleotides, it would be difficult to effect discrimination among similarly charged nucleotides bearing different kind of bases. Recently, a cytosine-pendant triamine host (la) was developed as a new type of receptor for the recognition and binding of guanosine 5’-monopho~phate.~~ This host has ditopic recognition sites for guanosine nucleotides, i.e., the cytosine moiety for complementary base pairing with the guanine base and the (protonated) triamine moiety for electrostatic binding with the phosphate group. The formation of a stable complex with the above nucleotide was observed in dimethyl sulfoxide (K,= 2.6 X lo4M-l at 23 “C for the 1:l complex25). In this paper, we report potentiometric response properties for organic and inorganic anions of a polymer matrix liquid membrane electrode using as a sensory element host
0003-2700/92/0364-0960$03.00/00 1992 American Chemical Society
