Anal. Chem. 1995,67,4343-4348
Rapid Tryptic Mapping Using Enzymatically Active Mass Spectrometer Probe Tips David Dogruel, Peter Williams, and Randall W. Nelson* Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-1604
A method has been developed for rapid, sensitive, and accurate tryptic mapping of polypeptides using matrixassisted laser desorptiodionization time-of-flightmass analysis. The technique utilizes mass spectrometer probe tips which have been activated through the covalent immobilization of trypsin. The enzymatically active probe tips were used for the tryptic mapping of chicken egg lysozyme and the results compared with those obtained using either free trypsin or agarose-immobilizedtrypsin. A significant increase in the overall sensitivity of the process was observed using the active probe tips, as well as the production of more characteristic proteolytic fragments and the elimination of background signals due to the autolysis of the trypsin. Further, probe tip digestions were found to be rapid and convenient. Since the advent of matrix-assisted laser desorption/ionizatiop (TvMLDI) time-of-flightmass spectrometry' and, more specifically, the introduction of the cinnamic acid derivatives as mat rice^,^,^ direct mass spectrometric analysis of complex polypeptide mixtures has become a matter of routine. Indeed, direct mixture analysis has proven to be one of the strongest attributes of MALDI and is in fact critical for the successful use of the technique in many applications, e.g., high mass accuracy using internal reference standards? protein ladder sequencing? quantitative analysis,6 direct analysis of biological mixture^,^ and mass spectrometric immunoassay.6 However, the greatest impact on the analysis and characterization of polypeptides results when MALDI mass spectrometry is combined with conventional chemical and/or enzymatic treatments of proteins. While a number of examples of such modifications followed by direct MALDI analysis exist?-14 the protocols used for these reactions were typically those (1) b a s , M.; Hillenkamp, F. Anal. Chem. 1988,60, 2299-2301. (2) Beavis, R. C.; Chait, B. T. Rapid Commun.Mass Spectrom. 1989,3, 432435. (3) Beavis, R. C.; Chaudhary, T.; Chait, B. T. 0%.Mass Spectrom. 1992,27, 156-158. (4) Beavis, R C.; Chait, B. T. Anal. Chem. 1990,62, 1836-1840. (5) Chait. B. T.; Wang, R.; Beavis, R C.; Kent, S. B. H. Science 1993,262, 89-92. (6) Nelson, R W.; McLem, M. A,; Hutchens, T. W. Anal. Chem. 1994,66, 1408-1415. (7) Beavis, R C.; Chait, B. T. Proc. Nutl. Acad. Sci. U S A . 1990,87, 1-5. (8) Nelson, R W.; Krone, J. R; Bieber, A L.; Williams, P. Anal. Chem. 1995, 67, 1153-1158. (9) Juhasz, P.; Papayannopoulos, I. A; Zeng, Ch.; Papov, V.; Biemann, K. Proceedings of the 40th ASMS Conference of Mass Spectrometry and Allied Topics, Washington, DC, May 31-June 5, 1992, pp 1913-1914. (10) Billeci, T. M.; Stults, J. T. Anal. Chem. 1993,65, 1709-1716. (11) Bartlet-Jones, M.; Jeffery, W. A; Hansen, H. F.; Pappin, D. J. C. Rapid Comm. Mass Spectrom. 1994,8, 737-742. (12) Knierman, M. D.; Coligan, J. E.; Parker, K C. Rapid Commun. Mass Spectrom. 1994,8, 1007-1010.
0003-2700/95/0367-4343$9.00/0 0 1995 American Chemical Society
employed for separation methods (e.g., HPLC, SDS-PAGE, CZE). As a result, the speed, sensitivity, and specificity of direct mixture analysis using MALDI were not fully utilized. It is routine to obtain a highquality MALDI mass spectrum of a protein sample withii a few minutes of receiving a picomole quantity of the analyte. Ideally, a method of peptide characterization which incorporates chemical and/or enzymatic modifications followed by mass spectrometry should require little more time, or sample, than that required for a typical MALDI analysis. For example, an enzymatic map/MALDI analysis should be obtainable using -1 pmol of analyte and require only minutes to perform. Further, the mapping analysis should be as easy to perform as a MALDI analysis, requiring little other than application of a few microliters of analyte and matrix solutions to a probe. Finally, the analysis should be free of artifacts due to the reaction (in this case, autolysis products of the enzyme). The above scenario can be accomplished using an approach in which the analyte is applied directly to a mass spectrometric probe tip which actively performs the enzymatic degradation; i.e., the probe tip cam'es the enzymatic reagent. The overall sensitivity of the analysis can be increased by direct application of the analyte to the probe tip, thereby minimizing the number of sample transfers and consequent sample loss. Optimally, the enzymes should be covalently attached to the probe tips, allowing high effective reagent concentrations to be used without the adverse effect of interference signals due to autolysis. A consequence of the high enzyme densities is the ability to perform the digests on a time scale roughly equivalent to that of a typical MALDI analysis (a few minutes). We report here the development and use of such enzymatically active mass spectrometer probe tips. A graphic depiction of the process is given in Figure 1. Enzymatically active probe tips are prepared through a two-step surface activation/enzyme-linkage process. The probe tips are then used for enzymatic degradation of analytes and as devices for sample introduction into the mass spectrometer. Given are examples of the use of trypsin-activated gold (Au/trypsin) probe tips for the peptide mapping of hen egg lysozyme. For comparison, the results obtained using both free trypsin and agarose-immobilized trypsin obtained under identical experimental conditions are given. EXPERIMENTAL SECTION
Chemicals and Reagents. Tosyl-L-phenylalanine chloromethyl ketone- (TPCK-) treated trypsin (from bovine pancreas) (13) Andrews. P. C.; Allen, M. H.; Vestal, M. L.; Nelson, R W. In Techniques in Protein C h e m i s t y U Angeletti, R H.. Ed.; Academic Press, Inc.: New York, 1992; pp 515-523. (14) Hutchens, T. W.; Yip, T. T.; Nelson, R W. In Techniques in Protein Chemistry IK Angeletti, R H., Ed.; Academic Press, Inc.: New York, 1993; pp 33-40.
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OSPnropmpanoi 15 mi".
\ -
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(? NH(.
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0 0 0
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Figure i. Schematic illustrating the general approach of the enzymatically active probe tip/MALDI mass spectrometric method for protein analysis. Probe tips are first prepared, in bulk, by covalent attachment of enzymes to the surface of the probe tip (in this case through DSP activation of a gold surface followed by enzyme linkage via a primary amine). The probe tips can then be used, at will, for protein characterization by application of the protein in an appropriate buffer and allowing time for digestion (digestions are usually performed at elevated temperature). The digestions are stopped with the addition of a MALDI matrix. and the reaction productlmatrix mixture aiiowed to dry (for the case of solidphase matrices). The probe tips are then inserted directly into the mass spectrometer and MALDI-analyzed.
was purchased from Sigma (St. Louis, MO) and used without further purification. Trypsin solutions were prepared at a concentration of 1mg/mL in a buffer of 20 mM sodium dihydrogen phosphate at pH 8.1 (Phos) and used for probe tip preparation. Solutions for free trypsin digestion were prepared at 500 nM (-0.012 mg/mL) in Phos. Agaroseimmobilized TPCK-trypsin was purchased from Pierce (Rockford, K.) and prepared for use through repetitive rinsing (3 x equal volume rinses) with Phos followed by suspension in an equal volume of the same buffer. The activities of the free trypsin and agarose-immobilized trypsin were 135 and 45 units/mL, respectively (as stated from supplier). Gold foil (99.9% purity, 0.01 mm thickness) was purchased from Johnson Matthey Ward Hill, MA) and used as supplied. Dithiobis(succinimidy1propionate) @SP) was used without purification (Pierce) and dissolved in %propanol to saturation (-10 mM). Chicken egg lysozyme was purchased from Sigma and used with no further purification. Lysozyme solutions were prepared in F'hos which contained dithiothreitol (Sigma) at a concentration of 20 nM. Alysozyme concentration of 2.5 pM was used for application to the active probe tips, where a concentration of 500 nM was use for the comparative studies. The MALDI matrix, aqanc-4hydroxycinnamic acid (ACCA) was prepared as a saturated solution in a solvent mixture of 21, 1.3%aqueous triiluoroacetic acid/acetonitrile. Preparation of Trypsin-ActivatedGold Probe Tips ( A d Trypsin). Gold foil circles were cut from the bulk foil to the diameter of the probe tips used in the ASU mass spectrometer (2.5 mm) and placed in a polyethylene microcentrifuge tube. The 4344 Analytical Chemisiry, Vol. 67, No. 23, December 1, 1995
gold circles were activated according to the method of Katd5by treatment with DSP/Z-propanol solution for -15 min. The crosslinking solution was decanted, and the gold circles were rinsed repetitivelywith 2-propanol, followed by rinses with ethanol. The gold circles were then vacuum dried and transferred to a new microcentrifuge tube. The circles were incubated with 1 mg/ mL solution of trypsin (-15 pL/circle), overnight at 4 "C. Following incubation, the gold foil circles were rinsed vigorously with Phos and subsequently with a solution of 0.1%Triton-Xl00 (Aldrich). The gold foil circles were then vacuumdried prior to physical attachment to the stainless steel tips using a small amount of adhesive. The enzymeactivated probe tips were then used for protein digestion as described. In practice, active prohe tips were prepared in bulk and stored at room temperature until needed. ProtemD i g d o d S a m p l e Preparation. AdTypsin Probe Tips. Digestions were performed with the Adtrypsin active probes by the application of aliquots (either 4 pL or 400 nL) of 2.5 pM lysozyme solution directly to the probe tips. The tips were allowed to stand in a humid enclosure maintained at 40 "C. The probe tip solution volume was monitored, and additional aliquots of 1-2 pL of phosphate buffer were added if the volume of liquid on the probe tip appeared to be less than -2 pL After 10 min, the probe tips were removed from the humid environment and 2 pL of ACCA was applied. The mixture was allowed to air-dry prior to insertion into the mass spectrometer. Free TMsin. A 1:1 mole ratio of trypsin to lysozyme was prepared by addition of 2 pL of 500 nM lysozyme to 2 pL of 500 nM trypsin. The combined volume was placed in a 600 pL (15) Katq
E.Y.J Efectmnnl. Ckrm. 1990.291.257-260.
9
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4000
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m/z Figure 2. Results of a 10 min (40 "C)AultrypsinlMALDI map of chicken egg lysozyme. Lysozyme (10 pmol) was applied to the probe tip in the presence of the reducing agent DTT. Thirty-three of a possible 67 digest fragments are observed in the mlz range of -1000-6000 Da (signals below mlz -1000 Da were generally not considered because of possible interferences from the matrix). Signal intensity and quality was such as to allow laser desorption conditions capable of resolving tryptic fragments at mlz 1424.8, 1429.6, and 1435.5 Da (inset). The peaks are marked numerically to correspond with the proteolytic fragments listed in Table 1 and shown in Figure 3.
microcentrifuge tube and incubated at 40 "C for 10 min, at which point the reaction was halted by the addition of 2 pL of ACCk The total volume of the digest/matrix mixture was placed on an inert mass spectrometer probe tip and allowed to air-dry prior to mass spectrometry. Agarose-Immobilized Tmsin. A 1:l volume ratio of lysozyme to agarose/trypsin was prepared by addition of 2 pL of 500 nM lysozyme to 2 pL of slurried agarose/trypsin. It was estimated that the slurried agarose contained -1 pL of beaded reagent. The combined volume was placed in a 600 pL microcentrifuge tube and incubated at 40 "C for 10 min. The reaction was halted by the addition of 2 pL of ACCA, and the total volume (with beaded reagent) of the digest/matrix mixture was placed on an inert mass spectrometer probe tip. The mixture was allowed to air-dry prior to mass spectrometry. Time-of-FlightMass Spectrometry. Mass spectrometry was performed using a linear timeof-night mass spectrometer that has been previously described.8 All mass spectra were obtained in the positive-ion mode and calibrated using an external calibration equation generated from the singly- and doubly-charged ion signals of bovine insulin (MW = 5733.5 Da). Masses of digest fragments were calculated using the known hen egg lysozyme
amino acid sequence16 and the peptide data manipulation routine PROCOMP.17 RESULTS Figure 2 shows a Au/trypsin/MALDI map of 10 pmol of lysozyme resulting from a 10 min digestion at 40 "C. Ion signals representing 40 distinct species are observed, 33 corresponding to digest products of the lysozyme. Table 1shows the correlation between the masses calculated for the enzymatically cleaved fragments and those observed in the mass spectrum. The average mass error over the entire series is less than 1 Da, with the greatest contribution arising from signals of weak intensity (due presumably to a less precise peak-centroid determination). No unexplained signals are observed in the mass spectrum, and there are no background signals due to autolysis of the trypsin. The quantity and quality of the ion signal per laser shot was such to allow laser irradiances only slightly above threshold without (16)Imoto, T.; Johnson, L. N.; North, A C. T.; Phillips, D. C.; Rupley, J. A In The Enzymes, 3rd ed.; Boyer, P. D., Ed.; Academic Press, Inc.: New York, 1972; pp 666-868. (17) Andrews, P. C. PROCOMP, University of Michigan Medical School, Ann Arbor, MI.
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32
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KVFGRCEWMKRHGLDNYRGYSLGNW V C M K F E S N F N T ~ T N R M O G S T D Y G i L ~ l N S R ~ C N ~ R T P ~ R N L C N l P C ~ L L S S D l T ~ V N C A K K l V S ~ N ~ N A ~ A W R N R C K G T D V ~ W i R G C R L
10
20
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40
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Figure 3. Coverage map of hen egg lysozyme digested using Au/trypsin mass spectrometer probe tips. The 33 proteolytic fragments result in the mapping of the entire protein with at least a 3-fold redundancy. The fragments are labeled to correlate with Table 1 and Figure 2.
sacrifice to the overall signal-to-noise ratio (S/N) of the mass spectrum. As a result, the mass resolution was adequate to resolve the three tryptic species in the mid-1400 Da range. This is shown in the inset of Figure 2. Three species are observed at m / z values of 1424.8, 1429.6, and 1435.5 Da. The full width at half-maximum resolution for these species is 300-400, roughly half that dictated by the isotopic envelope (a peak width of -2.5 Da is expected for the isotopic envelopes). While this resolution is below that expected (the linear time-of-flight instrument used for these studies is capable of achieving resolutions of -500 in this mass range), it is notable considering that these signals were acquired simultaneously with the signals of 30 other tryptic species (Le., a certain minimum laser irradiance is required to register ion signals over the entire mass range-in this case the irradiance was not optimal for the 1400 Da species). Figure 3 gives an iqdication of the coverage observed during the mapping. The entire protein was mapped with a 3-%fold redundancy during the experiment; Le., each amino acid of the protein was included in at least three and up to eight of the proteolytic fragments. Such oversampling of the primary structure of the protein is invaluable in the isolation of sequence modiiications,I8 and had a sequence error occurred in the case presented here, the region of the molecule containing the error could (theoretically) be localized to within 23 residues (fragment 22; the longest stretch of the protein not overlapped by multiple proteolytic fragments). Using mass spectrometer probe tips activated with yet other proteolytic enzymes, the error region might be further localized with the more complete mapping of the protein. We have pursued such methods for the sequence verification of recombinant proteins (and isolation of sequence defects)Ig and found the enzymatically active probe tips highly useful and, considering the expense of the analytes, economical. Reducing the amount of lysozyme applied to the tip by a factor of 10 (1 pmol) resulted in the Au/trypsin/MALDI map shown in Figure 4. As expected, a sacrifice in S/N is observed. Additionally, a decrease in resolution is observed as a slightly higher laser irradiance was used to acquire the spectrum (although not so low as to completely eliminate resolution of the 1400 Da species; see Figure 4 inset). Nevertheless, approximately the same fragmentation pattern as before is observed with roughly the same mass (18) Biemann, K. In Protein Sequencing, a Practical Approach: Findlay, J. B. C., Geisow, M. J., Eds.: IRL Press: Cary, NC, 1989: pp 101-106. (19) Krone, J. R.; Dogruel, D.; Williams, P.; Nelson, R. W., in preparation.
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Table 1. Residues of Lysozyme Identified Following I O min Digestion of 10 pmol on an Au/Trypsin Probe Tip
no.a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
mass(obs)b 936.6 1046 1277.4 1424.8 1429.6 1435.5 1475.8 1548 1581 1677.1 1707.2 1755 1805.4 1947.5 1977.8 2076.1 2126 2281.7 2306.9 2339.2 2437.1 2467.4 2680.8 3172.4 3334.8 3537.2 3689.9 3754.4 4125.5 4418.4 5098 5273 5488.3
mass(cald
Ad
residues
937.02 1045.54 1276.64 1424.78 1428.64 1434.63 1474.75 1546.78 1580.96 1676.90 1707.04 1754.86 1805.08 1947.19 1977.33 2075.37 2125.38 2281.57 2304.69 2336.72 2436.88 2464.90 2679.94 3171.41 3333.84 3535.68 3692.05 3751.26 4122.78 4415.79 5097.81 5271.70 5487.10
-0.42 0.46 0.76 0.02 0.96 0.87 1.05 1.22 0.04 0.20 0.16 0.14 0.32 0.31 0.47 0.73 0.62 0.13 2.21 2.48 0.22 2.50 0.86 0.99 0.96 1.52 -2.15 3.14 2.72 2.61 0.19 1.30 1.20
62-68 117-125 22-33 1-13 34-45 62-73 117-129 113-125 1-14 98-112 115-129 46-61 97-112 98-114 113-129 97-114 15-33 14-33 97-116 74-96 1-21 74-97 22-45 46-73 97-125 15-45 14-45 62-96 74-112 74-114 1-45 15-61 46-96
Refer to Figure 3. * Observed m l z values in daltons. Average chemical masses, in daltons, for protonated ([M + HI+) tryptic fragments. Difference, in daltons, between observed and calculated masses.
accuracy. Slight differences are also observed among the relative signal intensities, possibly due to the effective increase in the enzyme-to-substrate ratio. For comparison, 1 pmol of lysozyme (2 p L of a 500 nM solution) was digested using either an equal mole ratio of free trypsin or an equal volume of agarose-immobilized trypsin. Results using free trypsin are shown in Figure 5. Observed are a number of low-mass signals identifiable as trypsin-generated fragments (see Table 2). Several signals are also observed for
Table 2. Residues of Lysozyme Following 10 min Free Trypsin, or Digestion of 1 pmol with A-rypsin, Agarose-Immobilized Trypsin
Au/trypsin 935.5 1045.1 1276.5 1347.9 1423.3 1428.5 1434.2 1474.8 2000
4000 m/2
Figure 4. Same as Figure 2 except with the application of 1 pmol of lysozyme. A fingerprint equivalent to that in Figure 2 is observed with an expected decrease in the overall signal-to-noise ratio. Acquisition conditions were such as to not completely eliminate the resolution of the mid-1400 Da components (inset).
1546.7 1579.9 1675.9 1705.2 1753.4 1804.1 1946.2 2074.6 2125.0
1
2305.8 2336.8 2464.6 2656.6 3170.4 1 5
ey
mass(obsP free trypsin agarose trypsin 935.3 1046.1 1176.7 1277.4
residues
936.5 1046.1
62-68 117-125
1276.9
115-125
1435.6 1458.9 1476.9 1497.9 1548.8 1582.4 1678.2 1709.4 1756.7 1806.4
1434.9
1-13 34-45 62 -73
2166 2190.6 2274
2164.4
X
2274.8
X X
X
X
117-129 X
1581.8 1677.6 1755.5 1805.8
13-125 1-14 98-112 115-129 46-61 97-112 98-114 97-114 15-33
97-116 74-96 74-97
2471.5 3168 3227.9 3372.5
46-73 X
3371.1
X
15-45 14-45
3534.8 3689.7 4305.9 4820.4 5096.4 0
autolysis
1-45
Observed m/z values in daltons.
0 2000
4000
E
m/1
Figure 5. MALDI mass spectrum of 1 pmol of lysozyme digested with an equimole amount of free trypsin (40 "C, 10 min). Thirteen digest fragments are observed accompanied by signals due to the autolysis of the enzyme (marked by *). A decrease in S/N is also observed, relative to Figure 3, and is attributed to loss of sample in transfer and handling.
autolysis products of the trypsin (confirmed by MALDI mass analysis of a trypsin blank data not shown). In the present analysis, the autolysis signals do not significantly interfere with the lysozyme fingerprint (only one potential lysozyme fragment is overlapped by the autolysis signals). However, the autolysis signals do present a significant complication during the analysis of unknowns and should be eliminated if possible. Digests were performed using agarose-immobilized trypsin in an attempt to reduce the autolysis background contribution. The results obtained from the 10 min, 40 "C digestion of 1 pmol of lysozyme are shown in Figure 6. In contrast to both the Au/trypsin probe tips and the free trypsin digestions, few peaks corresponding to digest fragments are observed (see Table 2). Also quite noticeable is the marked decrease in the overall intensity of the ion signals. It is believed that this is due in part to the loss of digest fragments through absorption to the agarose media and to inefficient elution using a small volume of matrix.
0
2000
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"dl
Figure 6. MALDI mass spectrum of 2 pL of a 500 nM solution of lysozyme digested with an equal volume of slurried agaroseimmobilized trypsin (40 "C, 10 min). Few tryptic fragments are observed with an overall poor SIN. The decrease in ion signal intensity is attributed to loss of analyte in transfer and handling and retention by the agarose media.
DISCUSSION
Some features of the enzymatically active probe tip approach are worth noting. The most striking aspects of the active probe tip approach are those of sensitivity, mass spectral quality, and Analytical Chemistry, Vol. 67, No. 23, December 1, 1995
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speed. Conventionally, to minimize the autolysis background peaks, tryptic digests are performed with a large excess of analyte, and the low relative concentration of trypsin can lead to long digestion times (several hours). The short analysis times using the active probe tips can be attributed to a relatively high enzymatic activity-from the digest pattern (degree of total digestion), it is estimated that the activity of the probe tips falls somewhere between that of the agarose-immobilized and free trypsin (-0.05 and 0.15 TAME units, respectively)-and to performing the digests at elevated temperature. The overall sensitivity of any process is dependent on a number of factors including sample loss to the walls of the reaction vessel and loss in transfer. Sensitivity using the active probe tips is improved by the elimination of unnecessary sample transfers. As shown, it is not possible to match the speed or sensitivity of the trypsin-active probe tips using either high concentrations of free trypsin or agarose-immobilized trypsin. In the one case, autolysis products dominate the mass spectrum, while in the other, signiiicant sample losses occur and essentially no useful signals are seen from
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picomole quantities of analyte. It is apparent that the enzymatically active probe tips were capable of providing the greatest quantity (more fragment ion signals) of highest quality data (greater S/N), while having the least interferences from reagentbased signals. A final aspect is that of convenience. While this is a subjective issue, we have found the use of the enzymatically active probe tips quite routine (as applied to dozens of polypep tides), requiring virtually the same protocol as that used for normal MALDI preparation. ACKNOWLEDGMENT This work was supported in part by Department of Energy Grant DEFG02-91ER61127 and Arizona State University Investigator Incentive Funds. Received for review June 13, 1995. Accepted September 12, 1995.@ AC950577C Abstract published in Advance ACS Abstracts, November 1, 1995.
