Anal. Chem. 1994,66, 3423-3430
Matrix-Assisted Laser Desorption/Ionization Using an Active Perfluorosulfonated I onomer Film Substrate Jian Bai, Yan-Hui Liu, Teresa C. Cain, and David M. Lubman' Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48 109- 1055
The use of an active Nafion substrate is shown to enhance the performance of MALDI MS. The use of a Nafion substrate with certain matrices can significantly enhance the signals obtained over those observed with a stainless steel probe substrate. Analytes can often be observed with the use of the Nafion substrate that cannot be easily observed with the standard MALDI procedure, and usually a much wider range of peaks can be observed using MALDI from the Nafion substrate than with any single matrix on a stainless steel substrate. This enhancement of signal from the Nafion substrate is observed only with the sequential deposition of the sample and the matrix onto the Nafion film. If the analyteand matrix are premixed, then the effect is not observed. The use of the Nafion substrate has been shown to be particularly effective in analyzing real biological mixtures without prepurification. This has been demonstrated for various samples including the analysis of the products of chemical digests of proteins, protein profiling in milk and egg white samples, cell lysate analysis, and oligonucleotide detection. Matrix-assisted laser desorption/ionization (MALDI)1.2 has become a powerful tool in biological mass spectrometry. In particular, MALDI has been shown to be a valuable tool for characterizing proteins and peptides by mass spectrometry based upon the high achievable mass range where proteins of molecular mass greater than 200 000 u have been d e t e ~ t e d . ~ Other advantages of MALDI MS analysis include high sensitivity, which may be in the femtomole range, the simplicity of the sample preparation, and the capability to analyze mixtures,& often in the presence of biological interferences. Although initially developed mainly for peptide and protein analysis, various modifications of the technique have allowed its successful extension to other biopolymers such as oligosaccharides/~arbohydrates,~-~~ gangliosides,12 oligonucleo(1) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (2) Tanaka, K.; Waki, H.; Ido Y.; Yoshida Y.; Yoshida, T.Rapid Commun.Mass Specfrom. 1988, 2, 151-153. (3) Beavis, R. C.; Chait, B. T. Rapid Commun.Mass Specfrom. 1989, 3,432435. (4) Chevrier, M.; Cotter, R. J. Rapid Commun. Mass Specfrom. 1991,12,611618. (5) Billeci, T. M.; Stults, J. T. Anal. Chem. 1993, 65. 1709-1716. (6) Perkins, J. R.; Smith, B.; Gallagher, R. T.; Jones, D. S.; Davis, S. C.; Hoffman, A. D. J. Am. Soc. Mass Spectrom. 1993, 4, 670-684. (7) Mock, K. K.; Davey, M.; Cottrell, J. S . Eiochem. Biophys. Res. Commun.
1991, 177,644-651. ( 8 ) Spcngler, B.; Dole, J. W.; Cotter, R. J. Anal. Chem. 1990, 62, 1731-1737. (9) Harvey, D. J. Rapid Commun. Mass Spectrom. 1993, 7, 614-619. (10) Siege], M. M.; Hollander, I. J.; Hamann, P. R.; James, J. P.; Hinmann, L.;
tides13-19 and synthetic organic polymers.20 Initial results obtained with smaller oligonucleotides have also encouraged research on DNA screening and sequencing by MALDI MS.'"l9 The success of MALDI experiments depends heavily on the choice of the UV-absorbing matrix material. Over 30 different m a t r i c e ~ , ~ . ~ Jhave ~ ~ ~been O - ~identified, ~ mainly by trial and error. Although the matrices presently available often meet the need for protein analysis, characterization of suitable matrices still continues for other classes of molecules such as oligonucleotides and oligosaccharides. The development of new matrices, such as 3-hydroxypicolinic acid14 or binary mixtures of common matrices with s ~ g a r s ,has ~ improved MALDI performance in these areas significantly. Despite the successes achieved by MALDI, the diversity of impurities in real biological samples presents real challenges for accurate analysis. Even analyzing mixtures containing several components in relatively pure form can be difficult due to the selectivity of certain matrices for producing ions for different analytes in the MALDI process. In such mixture analysis, often several MALDI experiments with different matrices are required to detect all the components.6 In addition, the presence of contaminants may result in total loss of the ion signal, reduced signal intensity, or poor resolution, depending on the type and concentration of the contaminants and the properties of the analytes. The mechanisms by which different contaminants affect the MALDI experiment are not clearly understood. Water-soluble ionic surfactants appear to have the most harmful effect on the signal.24 Sodium and potassium can easily form cationic attachment products with analytes during MALDI and act as a major peak-broadening source in the mass spectrum. Impurities such as glycerol tend to disturb the crystallization process of the matrix/analyte (13) Currie, G. J.; Yates, J. R., 111. J. Am. SOC.MassSpectrom. 1993,4,955-963. (14) Wu, K. L.; Steding, A.; Becker, C. H. Rapid Commun.Mass Spectrom. 1993, 7, 142-146. (15) Schneider, K.; Chait, B. T. Org. Mass Spectrom. 1993, 28, 1353-1361. (16) Tang,K.;Allman,S.L.;Chen,C.H.;Chang,L. Y.;Schell,M.RapidCommun. Mass Spectrom. 1993, 8, 183-186. (17) Fitzgerald, M. C.; Zhu, L.; Smith, L. M. Rapid Commun.Mass Spectrom. 1993, 7, 895-897. (18) Tang, K.; Taranenko, N. I.; Allman, S.L.; Chang, L.Y.; Chen, C. H. Rapid Commun.Mass Spectrom., in press. (19) Bai, J.; Liu, Y.; Siemieniak, D.; Lubman, D. M. Rapid Commun. Mass Spectrom., in press. (20) Beavis, R. C.; Chaudhary, T.;Chait, B. T.J. Org. MassSpectrom. 1991.27,
156-158.
(21) Beavis, R. C.; Chait, B. T. Rapid Commun. Mass Spectrom. 1989, 3, 233-
237.
(22) Juhasz, P.;Costello, C. E.; Biemann, K. J. J. Am. Soc. MassSpecfrom. 1993, 4, 399-409. (23) Cornett, D. S.; Duncan, M. A.; Amster, I. J. Anal. Chem. 1993, 65, 2608-
Smith,B.J.;Famsworth,A.P.H.;Phipps,A.;King,D.J.;Karas,M.;Ingendolf, A.; Hillenkamp, F. Anal. Chem. 1991, 63, 2470-2481. (11) Hurberty, M. C.; Vath, J. E.; Yu, W.; Martin, S. A. Anal. Chem. 1993,65, 2791-2800. (12) Juhasz, P.; Costello, C. E. J. Am. Soc. Mass Spectrom. 1992, 3, 785-796 0003-2700/94/0366-3423$04.50/0
0 1994 Amerlcan Chemical Society
2613.
(24) Ling, V.;Guzzetta, A. W.; Canova-Davis, E.;Stults, J. T.; Hancock, W. S.; Covey, T. R.; Shushan, B. I. Anal. Chem. 1991,63, 2909-2915.
Analytical Chemistry, Vol. 66,No. 20, October 15, 1994 3423
mixture, which is considered to be an important aspect of the sample preparation. Other contaminants seem to destroy the effectiveness of the matrix, either by dilution or by altering its p h o t ~ a c t i v i t y .Even ~ ~ the cleanliness of the substrate or probe surfaces can affect the performance of MALDI. A number of schemes have been used to enhance the performance of the MALDI process through sample preparation or the use of selective adsorption methods. A preliminary purification scheme often involves washing the final sample preparation with ice-cold water as described by Beavis and Chait.26 This procedure has generally been limited to less water solublematrices such as sinapinic acid to aid in removing more water soluble ionic contaminants. Rinsing the sample dried onto the probe tip before adding matrix allowed some hydrophobic analytes to be dete~ted.2~Immobilization/ blotting of samples on poly(viny1idene difluoride) (PVDF) membrane and Nyl0n~8-~O has been used for the cleanup of more hydrophilic proteins and for interfacing MALDI MS with gel electrophoresis. Such membranes are routinely used for enhancing ion production in plasma desorption mass s p e ~ t r o m e t r y . ~More ~ recently, Cottrell and co-workers3' described immobilization protocols using nitrocellulose as a substrate for MALDI. Using this protocol it was shown that spectra could be obtained from low picomole quantities of protein in the presence of contaminants which did not inhibit the binding of the protein to the substrate. Other methods for improving the performance of MALDI developed by Hillenkamp and co-workers include an on-probe purification of DNA samples by ion exchange32 and a novel sample preparation method using fine fibrous paper as the substrate for a liquid matrix.33 More recently, Hutchens and Yip presented two new desorption s t r a t e g i e ~ surface-enhanced :~~ neat desorption (SEND), in which energy-absorbingmolecules are chemically bound to the polymeric layer of the sample presenting surface, and surface-enhanced affinity capture (SEAC), where the probe tip was chemically activated to selectively adsorb the analyte of interest. An important advantage of these schemesincludes the elimination of external matrix and the resulting intensive signal of the matrix ions. In addition, the new probe surface can be exploited as a solidphase reaction site enabling low-affinitymolecular recognition events to be detected directly in situ and can be used for structural analysis through enzymatic modificationof adsorbed analytes free from matrix interference. In the present work, we have applied a Nafion film as an active substrate for MALDI MS. Nafion is a perfluorinated cation exchanger with sulfonic acid groups as the active sites (25) Ehring, H.; Karas, M.; Hillenkamp, F. Org. Mass Sepctrom. 1992, 27,472480. (26) Beavis, R. C.; Chait, B. T.Anal. Chem. 1990, 62, 1836-1840. (27) Hefta, S. A.; Stahl, D. C.; Mahrenholz, A. M.; Rutherford, S. M.; Lee, T. D. Proceedings of the 39th Conference on Mass Spectrometry and Allied Topics; Nashville, TN, ASMS, East Lansing, MI, 1992; p 1416. (28) Vestling, M. M.; Fenselau, C. Anal. Chem. 1994, 66, 471477. (29) Strupat, K.; Karas. M.; Hillenkamp, F.; Eckerskorn, C.; Lottspeich, F. Anal. Chem. 1994, 66, 464-470. (30) Lacey, M. P.; Keough, T. Anal. Chem. 1991, 63, 1482-1487. (31) Mock, K. K.; Sutton, C. W.; Cottrell, J. S. Rapid Commun. MassSpecrrom. 1992,6, 233-238. (32) Nordhoff, E.; Ingendoh, A,; Cramer, R.; Overberg, A,; Stahl, B.; Karas, M.; Hillenkamp, F.; Crain, B. Rapid Commun. Mass Specrrom. 1993, 6, 771776 . . _. (33) Zhao, S.; Somayajula, K. V.; Sharkey, A. G . ;Hercules, D. M.; Hillenkamp, F.; Karas, M.; Ingendoh, A. Anal. Chem. 1991.63, 4 5 W 5 3 . (34) Hutchens, T. W.; Yip, T.-T. Rapid Commun. Mass Spectrom. 1993,7,576580.
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Analytical Chemistiy, Vol. 66, No. 20, October 15, 1994
and has been used as a substrate for PDMS.35136Thin and homogeneous Nafion films can be easily prepared from liquid solutions. Nafion possesses a high surface activity and can selectively and effectively bind small cations as well as large organic cations, as demonstrated by PDMS studies.35~36 Another unique property of the Nafion is that it has very limited solubility after drying when exposed to aqueous media. This makes it compatible with MALDI and other desorption techniques since water-soluble ionophores can have a deleterious effect on the desorption/ionization process. In this work, Nafion was used as an active substrate for MALDI detection of various macromolecules including mixtures such as a CNBr digest of cytochrome c, commercial milk, chicken egg white, Escherichia coli cell extract, and oligonucleotide samples. The improvement in detection and resolution using the Nafion substrate compared to an inert stainless steel probe tip is demonstrated.
EXPERIMENTAL SECTION The TOF MS used in these studies was a modified WileyMcLaren d e ~ i g n with ~ , ~ ~the capability for high-voltage acceleration up to f20 kV (manufactured by R. M. Jordan Co., Grass Valley, CA). The laser source was a DCR 1 1 Nd:YAG laser system (Spectraphysics, Mt. View, CA) which provided 266 and 355 nm radiation for desorption. The laser beam was focused onto the probe tip at a 45" angle to the probe surface with a single 12.5 in. focal length quartz lens. The detector was initially a dual microchannel plate (MCP), which is sufficient for detection of ions of less than m/z 10 000. For the detection of heavier ions, a triple MCP detector with a Cu/Be conversion dynode, designed for postacceleration capability up to f 15kV, was used. The postacceleration stage enhances the efficiency for detection of heavy species, but at some expense to the resolution. Data was recorded using a Lecroy 9400 digital oscilloscope and were subsequently transferred to a 386 IBM PC /AT for processing. Some commonly used matrix materials, including 2,5dihydroxybenzoic acid (DHB), caffeic acid (CA), sinapinic acid (SPA), 3-hydroxypicolinic acid (HPA), and a-cyano4-hydroxycinnamicacid (CHCA), were obtained from Aldrich Chemical Co. (Milwaukee, WI) and used without further purification. Near-saturated matrix solutions were prepared with 50% deionized water and 50% acetonitrile as solvents; 4-6 p L was applied to the probe tip. Two commonly used sample preparation methods were used, where one involves premixing the matrix with the sample and the second is the sequential application of the sample and matrix solutions. All sample deposits were air-dried. Dairy milk and chicken egg were obtained from a local grocery store. The 5-mer ss-DNA oligomer was synthesized by the core facility of The University of Michigan Medical Center. Cyanogen bromide digest of cytochrome c (horse heart) and ss-DNA oligomers of 18-, 42-, 53-, and 69-mer were provided by Dr. Rachel Ogorzalek Loo and Mr. David Siemieniak of The University of Michigan Medical Center, respectively. Escherichia coli obtained from Carolina Bio(35) Jordan, E. A.; Macfarlane, R. D.; Martin, C. R.; McNeal, C. J. rnr. J . Mass Spectrom. Ion Phys. 1983, 53, 345-348. (36) McNeal, C. J.; Macfarlane, R. D. J . Am. Chem. SOC.1986,108,2132-2139. (37) Wiley, W. C.; McLaren, I. H. Reu. Sci. Imtrum. 1955, 26, 1150-1157.
I
100
90 80 70
40
3
30
20
30 10 0
20
b s
0
3000
10
1 2
B~
6000
9000
12000
15000
MI2 IDaltonl
0 0
3000
6000
9000
12000
15000
MI2 IDalton)
Figure 1. Positive ion MALDI mass spectra of the CNBr digest of cytochrome c with (A) CA matrix and (E) DHB matrix using 355 nm irradiation. The spectrum consists of 200 laser pulses averaged. Acceleration voltage, 18 kV.
Figure 2. Positive ion MALDI mass spectrum of the CNBr digest of cytochrome con the Nafion substrate with CA as the matrix using 355 nm irradiation. The spectrum consists of 200 laser pulses averaged. Acceleration voltage, 18 kV.
logical Supply Co. (Burlington, NC) was harvested at midlog phase (12 h growth) from trypticase soy broth by centrifugation and suspended in one-tenth the culture volume in 10 mM Tris buffer (pH 7.8). A cell extract was obtained by sonicating the cell suspension and centrifuging to remove any unbroken cells or cell fragments. Gramicidin S,Nafion, and polystyrene (MW 1800) were all obtained from Aldrich Chemical Co. and were used as provided. All samples were dissolved with deionized water. The sample probe tip was inserted through a vacuum lock and into the acceleration region such that the front end stainless steel surface was flush with the first acceleration plate. Nafion Film and Polystyrene Film Preparation. Nafion is available in liquid form at a concentration of 5% in a mixture of 10% water and lower aliphatic alcohols. The pH of this solution was estimated with pH paper to be -3. Approximately 5 pL of this solution was applied to the stainless steel probe tip (i.d. 5.6 mm) and air-dried, which generally produced a homogeneous film of 12"*in size. Polystyrene film was prepared by applying a 5% solution in ethyl acetate to the stainless steel probe tip and air-drying. This solution tends to spread over the stainless steel probe tip during application, resulting in a larger and somewhat nonhomogeneous glassy deposit. Intensive heating of the deposit generated a more homogeneous yellowish layer and was used as an alternative for the preparation. Both forms of polystyrene film were tested and compared with Nafion films.
CA as the matrix when the sample and matrix were mixed before application to the stainless steel probe tip. In all the spectra, expected cleavage products at methionine are obtained along with other minor peaks. Table 1 summarizes the possible cleavage products at methionine and tryptophan. The variations of the relative intensity of the different peaks reflect the enhancement or suppression effects of the matrices toward different analytes. A method where the sample was deposited directly onto the stainless steel probe tip before applying the matrix was also tested for this cytochrome c digest using several matrices, but produced basically the same result. In order to enhance the detection of analytes by MALDI, Nafion, and polystyrene films were also tested as substrates for the MALDI analysis of cytochrome c digest. The various matrices used in these experiments included DHB, CA, and SPA. In the first set of experiments, the matrix and analyte were premixed and then applied to the probe surface coated with Nafion. This procedure produced mass spectra similar to those obtained in the MALDI experiment from the stainless steel probe as in Figure 1. However, a second set of experiments involved the sequential application of the sample and matrix to the Nafion film. A significant difference was obtained when the sample was deposited on the Nafion film and allowed to dry before adding CA as matrix. The mass spectrum of the CNBr digest of cytochrome c using this procedure is shown in Figure 2. It is apparent that a more homogeneous desorption of all the major components was obtained with the use of the Nafion surface and that the selectivity seen by the individual matrices is eliminated. Polystyrene surfaces were used instead of Nafion, but showed no improvement over the stainless steel probe. DHB on the Nafion surface showed minimal improvement. SPA was found to be unsuitable as a matrix when used with the Nafion film, as serious deterioration or total loss of the analyte signal was experienced. A visual inspection of the sample preparation revealed possible polymerization of the sinapinic acid, where the sample deposit turned into a foamy film during laser irradiation. The molecular weight of the fragments detected from the CNBr digest compares favorably with the calculated values
-
RESULTS AND DISCUSSION CNBr Digest of Cytochrome c Analysis. In this work we demonstrate the capabilities of a Nafion surface for enhancing detection of components in mixtures. In particular, an important problem we examine is the analysis of the products of the CNBr digest of cytochrome c. Cytochrome c is a wellcharacterized protein with two methionine residues. Conventional methods such as electrophoretic analysis and HPLC with amino acid composition analysis have allowed the three major fragments and partial cleavage products to be identified after CNBr digestion. Figure 1 shows the MALDI mass spectra of the CNBr digest mixture obtained with DHB and
AnalyticalChemistty, Vol. 66,No. 20, October 15, 1994
3425
Table 1. CNBr Digest Fragments of Cytochrome c and the Calculated and Measured Molecular Weight MW residues cleaved calcb,c frag. no.' at C-terminal fragment length
1 2
3
4 5 6
7
8 9 10
G(l)-W(59) G( 1)-HS(65) C(1)-HSL(65) G( 1)-M(65)-HS(80) G( 1)-M(65)-HSL(80) G( 1)-HS(65)-HS( 80) G( 1)-HS(65)-HSL(80) G( 1)-E( 104) G(60)-HS(65) G(60)-HSL(65) G(60)-M(65)-HS(80) G(60)-M(65)-HSL(80) G(60)-HS(65)-HS(80) G(60)-HS(65)-HSL(80) G(60)-M(65)-M(80)-E( 104) G(6O)-HS(65)-M(8O)-E( 104) G(60)-M(65)-HS( 80)-E( 104) G(6O)-HS(65)-HS(8O)-E( 104) E(66)-HS(80) E(66)-HSL(80) E(66)-M(80)-E( 104) E(66)-HS(80)-E( 104) P(8 1)-E(104)
7142 7713 1755 9567 9549 9537 9519 12360 649 63 1 2442 2424 2412 2394 5235 5205 5205 5175 1781 1761 4574 4544 2781
meas
error 7%
7138
0.06
7754
0.01
9528 ndd
0.09
621
0.63
2400
0.25
ndd 1754
0.40
4550 2788
0.13 0.25
a Fragment number identical to the peak number in the mass spectra. Cleavage at methionine produces C-terminal homoserine (HS) or homoserine lactone (HSL). Side reaction at methionine (not cleaved) results in HS. nd, not detected.
in Table 1. The mass calibration for the CNBr digest was done using internal standards including gramicidin S (m/z 1141) and substance P (m/z 1348). The m/z values obtained experimentally compared to those calculated are also compared in Table 1, where the percent difference may range from 0.0 1% to 0.63%. There is no significant difference between the mass values measured on the stainless steel probe tip and on the Nafion surface. The typical resolution of the peaks in these spectra is 40-50. The expected cleavage products of the CNBr digest of cytochrome c dominate the spectrum; however, products due to incomplete cleavage at the two methionine amino acids were also detected and labeled as peaks 3 and 9. Peak 9 is one of the two components whose sensitivity was clearly improved on the Nafion surface. The other peak labeled as 1 is produced due to oxidative cleavage at the tryptophan site, which should produce two fragments of molecular weights 7142 and -5200. A peak was not detected at molecular weight 5200 using MALDI on the stainless steel probe tip or with the Nafion surface. This would appear to indicate that this peak results from the cleavage of fragments 2 and/or 3 during sample handling/storage. Such cleavage should produce two additional products with molecular weights around 630 and 2400. The former peak was observed at 627 (peak 5 ) and the latter can be identified around 2400 (peak 6) in the MALDI spectrum obtained with the DHB matrix. In addition, a broad peak around 6100 was detected in all cases (except with the DHB matrix) which appeared as a doublet in the CA/Nafion spectra with molecular weights of 6050 and 6200. These two peaks could be the cleavage products at another site since their sum is close to the molecular weight of intact cytochrome c. In addition, the peak around 3840 in the CA MALDI spectrum may be the doubly charged species of peak 2. CNBr digestion is a specific and valuable chemical cleavage method in protein characterization. MALDI MS in com3426
Analytical Chemistry, Vol. 66, No. 20, October 15, 1994
bination with the use of a Nafion substrate has shown that side products in addition to expected cleavage products are observed. The use of a Nafion substrate allows more uniform detection of the peaks present in the mixture than with the use of MALDI MS on a stainless steel probe. The identification of such peaks may provide additional structural information on the protein or on the chemical digestion reaction or may help identify the presence of impurity peaks. MALDI MS on a Nafion substrate provides a rapid means of screening such digestion reactions with identification of species not easily detected by the usual MALDI MS method. Dairy Milk Analysis. The use of matrices more tolerant toward contaminants such as sinapinic acid has allowed direct analysis of complicated mixtures such as dairy milk with the identification of several proteins.26 In this experiment, a 2% fat commercial milk sample was diluted 50-fold with deionized water. Mixing this diluted milk with DHB and SPA allowed us to obtain useful mass spectra under normal MALDI operation. The spectrum obtained with DHB is presented in Figure 3. Lactalbumin, lactoglobulin, and casein are tentatively identified and are labeled in all the mass spectra. Caffeic acid appears to be a less effective matrix for producing ions from this mixture and only one major peak was detected and identified as lactoglobulin. Application of sample onto the Nafion film substrate before adding matrices was also tested. Figure 4 shows the spectra obtained with CA as the matrix and with CA and sample deposited on the Nafion film. A pronounced improvement was achieved for caffeic acid using the Nafion substrate. The peaks detected with other matrices were present, along with the appearance of higher molecular weight species. A similar improvement was also observed for DHB used with the Nafion film. The higher molecular weight peaks could simply be the cluster ions or other higher molecular weight species in the milk, which are difficult to detect using MALDI with conventional matrices. SPA with Nafion film again failed to produce any useful spectra as before.
100
80
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,
in
1
loo
Ovalbumin
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5 60 ,i50 5
-d
40
30
20 10 0
1 0
0
10000
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0
50000
20000
40000
Flgure 9. Positive ion MALDI mass spectrum of commercial milk with DHBas the matrix on the stainless steel probe using 355 nm irradlation. The spectrum consists of 200 laser pulses averaged. Acceleration voltage, 18 kV. 100
80000
100000
120000
140000
Flgure 5. Posltive ion MALDI mass spectrum of chicken egg whlte on Nafion film using CA as the matrix with 355 nm kradiatlon. The sDectrum consistsof 200 laser Dulses averaaed. Acceleration voltage, 20 kV.
-
loo
90
60000
MI2 IDaltonI
MI2 (Dalton1
7[
Locmglobulin
h
10
0
0
10000
20000
30000
40000
50000
MI2 IDaltonI
0
20000
40000
60000 BO000 MI2 (Dalton1
100000
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140000
Flgure 4. Posltive mass spectra of commercial milk with CA matrix on (A) the stainless steel probe and (B) on the Nafion film using 355 nm irradiation. The spectrum consists of 200 shots averaged. Acceleration voltage, 18 kV.
Flgure 6. Positiveion mass spectrum of the aqueous layer from chicken egg white extract using CA as the matrix with 355 nm irradiation.The spectrum consistsof 200 laser pulses averaged. Acceleration voltage, 20 kV.
Chicken Egg White Analysis. Chicken egg white, taken from a whole egg without disturbing the egg yolk, was diluted with deionized water and sonicated for 10 min. The final working mixture contains -20 mg of egg white/mL. An attempt to analyze this mixture by MALDI using DHB, SPA, and CA matrices on the stainless steel probe by either premixing or sequential application of the sample and matrix did not generate any significant signal. However, excellent results for this sample were readily obtained with CA on the Nafion film. Massive components as large as 100 000 u were detected, as shown in Figure 5. Two of the peaks are tentatively identified as egg white lysozyme and ovalbumin. The components of the egg white sample were further studied by use of a simple extraction procedure. Approximately 0.5 mL of 200 mg of egg white/mL was extracted into 1 mL of chloroform. The extract was sonicated for 7 min and allowed to settle for 20 min. Three separate layers were obtained from this procedure. After separation, the aqueous layer was analyzed by MALDI using DHB, SPA, and CA matrices without the Nafion substrate. As shown in Figure 6 , the aqueous layer contains the unidentified component with the broad peak between mlz 25 000 and 30 000. The aqueous
layer shows a slightly reduced level of lysozyme and ovalbumin. The precipitate obtained from the extraction procedure was washed with deionized water and was dissolved with 3 mL of a water/acetone/methanol (v/v/v 1:l:l) solution. The components of this mixture could also be detected using the standard MALDI procedure with DHB and SPA matrices. The Nafion substrate was unnecessary for detection in this case. The result shown in Figure 7 is basically the same spectrum obtained from the egg white sample on the Nafion substrate shown in Figure 5 , with the broad peak at m/z -25 000, which was extracted into the aqueous layer, considerably reduced. The key point observed in the egg white extract experiment is that a simple procedure, Le., using a Nafion film, enabled the major components in egg white to be detected with a standard MALDI procedure using a stainless steel probe, indicating that impurities or components of a real mixture may be a potential source of interferences in MALDI experiments. These impurities did not affect the MALDI mass spectrum obtained on a Nafion surface. Escherichia coLiExtract Analysis. An important problem in which mass spectrometry can be particularly useful is the AnalyticalChemjstry, Vol. 66,No. 20, October 15, 1994
3427
100
I
80
70
10
0 0
20000
40000
60000
80000 Mi2 lDaltonl
100000
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Figure 7. Positive ion mass spectrum of chicken egg white precipitate usingDHB as the matrix with 355 nm Irradiation. The spectrum consists of 200 laser pulses averaged. Acceleration voltage, 20 kV. 100 1
80
0
2000
4000
6000
8000
10000 12000 14000 16000 18000
Mi2 (Dalton1
Figure 8. MALDI mass spectra of an E. coli extract with CA matrix (A) on the stainless steel probe and (8) on the Nafion substrate using 355 nm irradiationaveragedfor 250 laser pulses. Acceleration voltage, 20 kV.
rapid screening of cell contents. FAB MS has been used for the profiling of bacteria toward the development of a rapid method for the identification of b a ~ t e r i a . 3In ~ ~this ~ ~work, a crude protein extract was obtained from the cell extract from disintegrated E. coli by precipitation with cold acetone. The precipitate was dispersed in deionized water for MALDI MS analysis using DHB, CA, SPA, and CHCA on the stainless steel probe tip and the Nafion substrate. CHCA gave the best result on the stainless steel, allowing the observation of several rather broad peaks. CA matrix on the stainless steel probe tip also allowed the detection of two components with m/z above 2000. Figure 8 shows the mass spectra of E . coli extract on the stainless steel and the Nafion substrate using CA matrix. As can be seen from Figure 8, the largest number of peaks were detected on the Nafion substrate with excellent sensitivity and signal-to-noise ratio. An interesting observation in this experiment is that, upon mixing CA with the sample and application of the mixture onto the stainless steel probe, (38) Heller, D. N.; Fenselau, C.; Cotter, R. J.; Demirev,P.; Olthoff, J. K.; Honovich J.; Uy, M.; Tanaka, T.; KishimotoY.Biochem. Biophys. Res. Commun. 1987, 142, 194-199. (39) Heller, D. N.; Cotter, R. J.; Fenselau, C.; Uy, 0. M. Anal. Chem. 1987, 59, 2806-2809.
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i
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+ iIl II
U & iL 0
11
1
IN
J-L+\ 500
1000
1500
2000
Mi2 lDaltonl
Figure 9. Negative ion mass spectra of a 5-mer ss-DNA oligomer (5'-ClT AA-3') obtained on (A) a stainless steel probe and (b) Naflon film with CA as the matrix with 355 nm irradiation; -20 pmol of sample loaded. The spectra consist of 100 laser pulses averaged. Acceleration voltage, -15 kV.
the sample deposit developed a black tint, indicating possible reactions which might interfere with the MALDI experiment. No such changes were observed when the sample and CA matrix were sequentially applied onto the Nafion substrate. Mass spectra from the MALDI process from the Nafion film using CHCA as the matrix showed an enhanced signal-tonoise ratio, but gave basically the same spectra as with CHCA on the stainless steel substrate. DHB did not generate any significant signals on either the stainless steel or the Nafion substrate. Oligonucleotide Analysis. The analysis of oligonucleotides has proven to be particularly difficult because the MALDI process has been found to be very dependent on the matrix used. Various matrices have led to MALDI mass spectra which are base composition dependent15 or result in fragmentation15 or cation exchange.32 Finding matrices of improved performance still constitutes a potential breakthrough in the area. The use of 3-HPA as a matrix has allowed oligonucleotide strands as long as 90 bases to be observed in MALDI MS,14,16and the use of a 3-HPA and picolinic acid matrix mixture has recently allowed the detection of DNA strands up to 500 base pairs long.ls Purification procedures3* and other sample preparation alternatives13 have been shown to enhance the ability to obtain MALDI mass spectra of oligonucleotides. In Figure 9 is shown the negative mass spectra of a singlestranded 5-mer obtained with CA matrix from the stainless steel probe and using a Nafion film substrate. The MH- peak obtained using a CA matrix on a stainless steel probe shows several adduct peaks as is also observed for the HPA, DHB, and SPA matrices. These adduct peaks result from cation attachment. The intensity of these adducts depends on the matrix used and the concentration of the impurities present. These adduct peaks were effectively eliminated using the Nafion substrate. It appears that the Nafion effectively binds the inorganic cations to the anionic sites and eliminates these interferences during the MALDI process. DNA oligomers including an 18-, 42-, 53-, and 69-mer were also analyzed using the Nafion film. The observed mass spectra showed some improvement in sensitivity and signals that lasted over
an extended period of time. The improvement in signal for these relatively pure samples was found to be limited though, Le., a factor of -2. However, in more recent work,lg DNA strands exceeding 200 base pairs in length obtained from restriction enzyme digests of plasmid DNA have been greatly enhanced by the Nafion surface. In this case though, the Nafion assists MALDI of a relatively impure biological sample. Factors Affecting MALDI from a Nafion Film Substrate. In MALDI, as well as in other desorption processes which produce gas-phase ions from solid phases, impurities and strong interactions among large polar analytes are factors that affect the performance of the method. The introduction of matrix in MALDI partially relieves these strong interacti~ns.~O However, the presence of an active substrate can also effectively reduce the presence of impurities and their interactions with the analyte and/or matrix. The selective adsorption of impurities by active surfaces may result in the in situ purification of the sample. In the case of the Nafion film, small inorganic cations are adsorbed onto the surface and strongly bound, so that they are excluded from the analyte/ matrix interactions. The procedure used in this work for applying the analyte and matrix to the surface may further enhance this effect. The sample is applied directly to the Nafion first, where it is in direct contact with the active polymer surface and the impurities can easily access the anionic sites in the Nafion and bind. The matrix is then applied to the sample, and sufficient analyte appears to interact with the matrix for MALDI to enhance the signal without involvement of the potential contaminants. If the matrix and sample are first mixed and then applied to the Nafion, then little effect is observed. It appears that the analyte and impurities do not easily access the active sites due to the large amount of matrix present under these conditions. The use of active surfaces such as Nafion can allow removal of contaminants by selective adsorption provided they are either strongly or loosely adsorbed. If the contaminants are loosely adsorbed, then they can be washed away with water or other solvents. Such washing was performed with no obvious effect on the results. This leads us to believe that the impurities are strongly bound and do not interfere with the MALDI process. The use of an active surface in the MALDI process may also involve other factors besides selective adsorption of contaminants. The analytes too may become strongly adsorbed on the surface and thus reduce the effectiveness of MALDI. This may be influenced by such factors as the counterions present in the Nafion surface, the solvent used, and the affinity of the matrix toward the analyte. The latter may explain the selectivity of a matrix toward enhancing the MALDI signal and explain the variation of the improvement observed in these experiments with different matrices with the use of the Nafion film. The crystallization process in MALDI may also be affected by the active surface. In these experiments, the presence of a small amount of liquid Nafion in DHB can totally destroy the effectiveness of this matrix. Thus, any solvent that dissolves dried Nafion films should be avoided. In addition, an important factor to be considered is the capacity of the Nafion and the amount of salt impurity that (40) Karas, M.; Bahr, U.; Giessmann, U. Reu. MassSpectrom. 1991,10,335-357.
can be tolerated, The capacity of the Nafion is estimated to be 0.9 mequiv/g (based on an equivalent molecular mass of 1 100 Da). Loading of 5 pL of the Nafion (5%) corresponds to a theoretical capacity of -0.2 pequiv. However, since the final form of the Nafion is a thin film, the effective capacity remains unknown and is complicated by factors such as the Nafion film thickness, the proportion of active sites on the surface available for ion exchange, whether ion exchange occurs due to ion transport/diffusion within the film, etc. The experimental evaluation of the actual capacity of the Nafion substrate is also complicated by (1) the MALDI process itself, which can tolerate moderate amounts of salt, and (2) the addition of (in)organic salts to the sample, which results in species due to ion exchange such as HC1, H3P04, or acetic acid, if salts of those acids are added, which may affect the MALDI process. In these experiments, the loading of 0.25 pequiv of NaH2P04 begins to degrade the MALDI spectrum of mixtures obtained on the stainless steel probe tip as compared to a loading of 0.35 pequiv of NaHzP04 to reach a similar degradation using the Nafion substrate. However, further tests indicate that the CNBr digest of cytochrome c, for example, can still be observed upon loading of 1 pequiv of NaH2P04 on the Nafion film. The use of Nafion film for enhancing the observed signal is found to be particularly effective for impure samples. A dramatic enhancement is observed for egg white, E . coli lysate, and milk samples. The enhancement is these cases may be the difference between not observing a useful signal on the stainless steel probe to that of observing a strong signal intensity on the Nafion substrate. The signal enhancement in the case of the E . coli lysate appears to be greater than 1OOX. In the case of relatively pure samples such as the CNBr digest of cytochrome c or oligonucleotides, the enhancement effect is considerably smaller. For the CNBr digest, the Nafion was found to enhance components that could not be observed with specific matrices and provide a more general spectrum than any particular matrix. The overall intensity of the spectrum though was not enhanced significantly. Thesame was observed for synthesized oligonucleotides that were relatively pure, where little enhancement was observed using Nafion in the MALDI process. CONCLUSION This work has shown that active substrates such as Nafion can enhance considerably the performance of MALDI MS. The use of a Nafion substrate with certain matrices can significantly enhance the signals obtained over that observed with a stainless steel probe substrate. In addition, analytes that cannot be easily observed with the standard MALDI procedure can often be observed with the use of the Nafion substrate, and generally a much wider range of peaks can be observed using MALDI from the Nafion substrate than with any single matrix on the stainless steel substrate. The Nafion substrate can thus eliminate some of the matrix dependence of the MALDI process. The MALDI process from the Nafion substrate though is still very matrix dependent and often appears to work best with caffeic acid. In addition, the enhancement of signal from the Nafion substrate is observed only with the sequential application of the sample and the Analytical Chemistty, Vol. 66, No. 20, October 15, 1994
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matrix on the Nafion. If the analyte and matrix are premixed, the effect is not observed. This would seem to indicate that impurities in the sample that would normally inhibit the MALDI process are bound to active sites in the Nafion more tightly than the large analyte molecules. These impurities thus remain strongly bound during desorption and do not interfere with the MALDI process. The use of the Nafion substrate has been shown to be particularly effective in analyzing real biological mixtures without significant prepurification. This has been demonstrated for various samples ranging from chemical digests of proteins to cell lysates. The further development of substrates to enhance the MALDI
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process will no doubt continue to be an important area of investigation. ACKNOWLEDGMENT This work received support from the National Science Foundation under Grant BIR-9223677, the National Institutes of Health under the National Center for Human Genome Research Grant IR21H60068501A2, and the Environmental Protection Agency under Grant R-8 19605-01-0. Received for review March 29, 1994. Accepted July 7, 1994." a
Abstract published in Advance ACS Absfructs. September 1, 1994
