Anal. Chem. 1990, 62, 2391-2394
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Recognition of Trypsin Autolysis Products by High-Performance Liquid Chromatography and Mass Spectrometry M a r t h a M.Vestling,*J Constance M. M u r p h y , and C a t h e r i n e Fenselau Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21228
Potential artifactual contributions are assessed in high-pressure liquid chromatograms and fast atom bombardment mass spectra from autolysis of different preparations of the widely used protease trypsin. Both commercially supplied and iaboratory-purified samples were examined. Bovine pancreatic trypsin (1 mg/mL) was found to be completely destroyed in 2 h at pH 8.5, degraded to a complex mixture of small peptides which were characterized by their molecular weights. Some identtfications were supported by sequencing by tandem mass spectrometry or by mass spectrometric analysis of the mixture resulting from a single Edman degradation. Autolysis of porcine pancreatic trypsin produced a completely different set of peptides. Five Snes of hydrolysis at asparagine residues in bovine trypsin were also identified.
INTRODUCTION The serine protease trypsin, also called P-trypsin, is widely used to catalyze the hydrolysis of proteins to produce mixtures of peptides suitable for Edman sequencing and mapping by high-pressure liquid chromatography (HPLC) and mass spectrometry. Pancreatic bovine trypsin is a single chain of 223 amino acids with six disulfide bonds. Its structure has been firmly established by X-ray crystallography (I), and its primary structure is shown in Figure 1. Trypsin functions optimally a t p H 8-9 and shows high specificity for catalyzing the hydrolysis of amide bonds whose carbonyls are part of lysine or arginine (2). Since trypsin’s own structure contains 14 lysines and 2 arginines, it is a substrate for its own action, and this autolysis has long been recognized as a potential source of artifacts in structure studies (3). This paper reports a determination of the autolysis products that are preferentially detected by fast atom bombardment mass spectrometry when the product mixture is analyzed directly and when it is fractionated by HPLC. The autolysis products from trypsin from two different suppliers and from freshly purified material were characterized both by HPLC and mass spectrometry. EXPERIMENTAL SECTION Bovine pancreatic, TPCK treated trypsin, phenyl isothiocyanate, and thioglycerol were obtained from Sigma Chemical Co., St. Louis, MO. Two different lots of trypsin were used. The trypsin, labeled sequence grade, bovine (lots 11594921-02(Nov 89) and 12058920-03 (Nov go)), obtained from Boehringer Mannheim Biochemicals, Indianapolis, IN, was actually porcine trypsin. See Results and Discussion below. Sigma trypsin was used in the studies reported, unless otherwise specified. Trifluoroacetic acid (TFA) was obtained from Aldrich Chemical Co., Milwaukee, WI. High-Performance Liquid Chromatography. HPLC was performed on a Shimadzu dual pump system (Model LC-6A pumps) with a variable wavelength UV detector (Model SPD-6A) equipped with a Brownlee Aquapore RP300 (C8) (4.6 x 250 mm) column: mobile phase A, 0.1% TFA/water; mobile phase B, 0.1% *To whom correspondence should be addressed. On sabbatical leave from the Department of Chemistry, State University of New York College at Brockport, Brockport, NY 14420 0003-2700/90/0362-239 1 $02.50/0
TFA/acetonitrile. Fractions collected every 3 min in iced polypropylene tubes were frozen and lyophilized. Mass Spectrometry. Conventional magnetic scans were made on the first two sectors (EB) of a JOEL HXllO/HX110 mass spectrometer (Tokyo, Japan) using either a JOEL FAB gun or a JOEL cesium ion gun and the JOEL DA5000 data system. The FAB (fast atom bombardment) gun was operated at 6 kV and the Cs ion gun at 23 kV. Resolution was 3000. Tandem measurements were obtained by utilizing all four sectors (EBEB) on the JOEL HXllO/HX110 instrument. Linked B/E scans were made on MS-2, following mass selection of precursor ions by MS-1. In these experiments,helium was used as the collision gas between the two mass spectrometers, at pressure sufficient to attenuate the precursor ion beam by 80%. The accelerating voltage was 10 kV, and the collision cell was floated at 4 kV. Resolution in both MS-1 and MS-2 was 1000. For both scanning modes, the peptides dissolved in 0.1% trifluoroacetic acid were measured from thioglycerol. The computer program, RESIDUES,written by David Heller (Middle Atlantic Mass Spectrometry Center, Johns Hopkins University, Baltimore, MD) was used to assist interpretation of tandem mass spectra while MacProMass (Terry D. Lee and Sunil Vemuri, Beckman Research Institute, City of Hope, Duarte, CA) and PROCOMP (P. C. Andrews, Department of Biochemistry, University of Michigan, Ann Arbor, MI) supported interpretation of the conventional magnetic scans. Trypsin Autolysis. Trypsin (1 mg) was placed in a 1-dram vial and 1 mL of a solution that was 0.1 M NH4HC03and 0.1 mM CaCl, (pH 8.5) was added. The mixture was allowed to stand at room temperature for 2 h before it was frozen and lyophilized. The reaction was monitored by HPLC: gradient, 10-25% B over 10 min then 25% B for 5 min then 25-40% B for 5 min then 40% B for 5 min then 4040% B for 5 min; flow rate, 1.0 mL/min. Typsin Purification. TPCK treated trypsin from Sigma Chemical Co. (3-5 mg) was dissolved in 0.1 mM CaCl, (6OC-1000 pL) and immediately iced. Trypsin was separated from residual chymotrypsin and autolysis products by reverse-phase HPLC (4): gradient, 10-35% B over 5 min, 35-45% B over 10 min, 45-70% B over 5 min, 70% B for 5 min making a 25-min run; flow rate, 1.0 mL/min; 215 nm; 200 pL sample injected. Edman Degradation. Trypsin autolysis and Edman degradation were carried out sequentially in one 1-dram vial with a Teflon-lined cap. A modified version of the Edman method of Bradley et al. (5) was used. R E S U L T S A N D DISCUSSION Purified and unpurified trypsin dissolved in buffer suitable for incubation with substrate proteins, i.e. p H 8-9, undergo rapid autolysis. In 0.1 M NH4HC03 (pH 8.5) (1 mg/mL) trypsin is completely autolyzed after 2 h a t room temperature, based on HPLC analysis. The same HPLC pattern was obtained after 30 min with a higher trypsin concentration (4 mg/mL). Trypsin dissolved in 0.1 mM CaC1, (pH 6) or in 0.1 7’0 trifluoroacetic acid (pH 2) and kept on ice for several hours showed no change in its HPLC pattern. Comparison of the HPLC traces of autolysis products from purified and unpurified bovine pancreatic trypsin (Figure 2a,b) shows more similarities than differences. The higher molecular weight peptides elute a t the longer retention times and are not fully separated by the gradient shown. The sample from unpurified trypsin has a few more components. This is consistent with the mass spectrometric data, and attributable to the presence and action of chymotrypsin and previous autolysis ( 4 ) . 0 1990 American Chemical Society
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Table I. Observed Autolysis Products from Bovine Pancreatic Trypsin
total hydrolysate purified trypsin"
HPLC fractions unpurified
mlz
trypsino m / z
residues
3210.5 3140.8 2611.8 2552.5 2304.8 2272.5 2192.9 2163.0 1900.9 1775.1 1578.7 1433.8 1375.9 1174.9
[ 140-1491-[150-1701
3209.51
[ 201-2231 [ 10G125jd [ 70-891" [ 70-89Id [ 150-1 701 [ 50-69Id
2613.35 2552.25 2305.16 2273.16 2193.01 2163.06
[ 1-141-[137-1391
1774.86
[ 187-200Id
1433.72 1375.73 1174.65 1174.54 1153.57 1117.53 1111.56 1037.49 1019.52 916.45 906.50 867.42 805.42 802.41 659.38 633.32 562.28 560.30
2612.9 2273.5 2193.0 1901.1 1775.0
1174.8
assignment
[78-891 [80-891 [ 160-1701 [126-136Id [70-791 [209-2 171 [ 150-1591 [140-149Id [70-771 [ 201-208Id [ 15-22] [92-99Id [70-761 [44-49Id [218-223Id [92-971 [ 15-19]
1153.8 1117.6 1111.8 1037.6 1020.7 916.4 906.6 867.7 805.5 802.7 659.5 633.4 562.3 560.4
1153.8 1117.7 1037.7 1018.8 906.7 805.6 659.8 633.7 560.7
cutsb
calculated monoisotopic MH+
additional informationc
E
E S, ES S, E S, ES
ES
S S, E S, E S, E S S
Monoisotopic masses were measured (6). *Residues that contributed the carbonyls from each hydrolyzed bond. S, sequenced by tandem mass spectrometry; E, N-terminus residue confirmed by Edman degradation; ES, peptide product from one Edman degradation sequenced by tandem mass spectrometry. Predicted. e Methionine sulfone. Trypsin [bovine] 1 I 1 I I IVGGYTCGANTVPYQVSLNSGYHFCGGSLINSQYWSAAHCYKSGIQVRL
-,
-I/OD
I I I I I GEDNINWEGNEQFISASKSIVHPSYNSNTLNNDIMLIKLKSAASLNSRV 110
>40
I
I
I
I
:
I
ASISLPTSCASAGTQCLISGWGNTKSSGTSYPDVLKCLKAPILSNSSCKS *a0
,IO
------
I I I I I AYPGQITSNMFCAGYLQGGKDSCQGDSGGPWCSGKLQGIVSWGSGCAQK '0
2
I I 1 NKPGVYTKVCNYVSWIKQTIASN
,---
Dlsvlflde bonds 7 137 25 41 109 210 116 183 148 162 173 187
Flgure 1. Primary structure of bovine pancreatlc trypsin from ref 1 See Table I for the protonated monoisotopic weights of the underlined peptides
Table I summarizes the mass spectra of the autolysis products of bovine trypsin. Determinations are reported for an unfractionated mixture from autolysis of purified trypsin and of seven fractions collected at 3-min intervals during the chromatographic separation of the autolysis mixture from unpurified bovine trypsin. Only ions with a signal to noise ratio greater than 3 are reported and only ions that appeared in determinations using both lots of Sigma trypsin. Additional ions were detected in the composite of the HPLC fractions, compared to the unfractionated sample, due both to the chymotryptic contamination and to the preferential desorption of peptides in mixtures that has been previously demonstrated (7). All the peaks detected in the mixture were also found in the fractions.
Formation of 16 peptides is predicted, based on the specificity for lysine and arginine, and assuming that all the disulfide bonds are reduced. Lysine at position 200 is regarded as not hydrolyzable because of the proline at position 201. Although these assumptions are qualified to some extent by the peptides identified in Table I, nonetheless, 12 of the predicted peptides are observed (starred). In five cases these assignments are confirmed by sequencing by tandem mass spectrometry (8) and/or by a single cycle of Edman degradation. The predicted autolysis fragments that are not observed include the heaviest, >4000 u and the three lightest, e400 u. Two disulfide bonded products were identified, [ 140-149]-[ 150-170] and [ 1-1414 137-1391, and five peptides were assigned as having been released from disulfide bonds, perhaps by reduction in the thioglycerol matrix used for fast atom bombardment mass spectrometry. Control of pH and/or a nonreducing matrix might preserve disulfide bonds and shift some of the potential autolysis background to a higher mass range, albeit with potentially altered solubility and sensitivity. Some peaks were assigned to peptides formed by incomplete hydrolysis, such as m/z 2612 to peptide [201-2231. Note also that m / z 2163 supports glutamic acid not glutamine at positions 52 and 62 in agreement with the X-ray data of Bode and Schwager ( I b ) . A number of ions were observed that could not be explained by cleaving only disulfide linkages and bonds containing lysine and arginine carbonyl groups. T o assign structures to these ions, the primary structure of trypsin was searched for peptides of appropriate masses. In most cases this generated several candidates. In seven cases the final assignment is based on sequencing by tandem mass spectrometry and/or analysis of the products after one Edman degradation cycle (Table I). Edman degradation indicated that the m / z 1175
ANALYTICAL CHEMISTRY, VOL. 62, NO. 21, NOVEMBER 1, 1990
2393
I
MIJ+l
nlz
Mass spectrum obtained by colliiionai activation in a tandem mass spectrometer of QVSLN [15-191. Assignment of sequence ions follows Biemann (8).
Figure 3.
2
SAASLN b3
-
% I
60
40 nlz
25
Figure 4.
Mass spectrum obtained by collisional activation in a tandem
mass spectrometer of SAASLN [92-971.
10 0
Table 11. Observed Autolysis Products from Porcine Pancreatic Trypsin total hydrolysatea
mlz
assignment residues cutsb
calculated monoisotopic MH+
additional informatione
2283.3
K/K 2283.18 S [70-89Id R/K 2211.10 S [50-69Id 1768.9 [108-125Id R/K 1768.80 1737.0 K/1736.84 S 1736.84 1469.9 K/K 1469.73 S [126-139Id 1143.0 K/Y 1143.55 [203-2121 1106.7 [77-861 F/M 1106.48 1045.8 K/R 1045.56 [90-99Id 1006.7 1006.49 S d140-149ld K/K 842.8 842.51 S [100-107Id R/R 515.8 R/R 515.33 S [46-49Id 'Monoisotopic masses were measured (6). Residues that contributed the carbonyls from each hydrolyzed bond. e s, sequenced by tandem mass spectrometry. Predicted, assuming complete hydrolysis at lysine and arginine and reduction of all disulfide bonds. 2211.1
A
8
I2
1'6
IO
24
28
Minutes Figure 2. Reverse-phase HPLC of trypsin autolysis products: (a) trypsin from Sigma, (b) HPLC purified trypsin, and (c) sequence grade trypsin from Boehringer-Mannheim. See text for details.
peak corresponds to two isobaric peptides. T o obtain assignments for the rest of the ions, priority was given to sequences that would be formed with at least one cut at lysine (K) or arginine (R)and, secondarily to peptides formed with the proteolytic selectivity expected from chymotrypsin (9) or $-trypsin (IO), an early trypsin autolysis product with proteolytic activity like chymotrypsin. Four peptides in Table I {[70-761, [70-771, [70-791, and [l-141-[137-13911 should be viewed as suggestions since additional experimental confirmation was not obtained. It is of interest that formation of six of the peptides whose structures are confirmed by additional experiments (Table I) requires cleavage at five amide bonds whose carbonyl group
is donated by asparagine. Spectra of two of these, [15-191 and [92-971 obtained by tandem mass spectrometry are presented in Figures 3 and 4. Fragment ions are identified following the nomenclature of Biemann (8) and support the assignment indicated. This detailed examination identifies 5 of the 16 asparagine residues as hydrolyzable under autolysis conditions. Nonenzymatic degradation of proteins a t asparagine has been previously observed ( I 1). Autolysis of sequence grade trypsin from two different lots provided by Boehringer Mannheim produced a different HPLC pattern (Figure 2c) and mass spectra with no coincident peaks when compared to that from Sigma bovine trypsin. However, the peptide molecular weights observed in Table I1 are consistent with the structure of porcine trypsin (12) shown in Figure 5. Cleavage occurred primarily, but not exclusively a t lysine and arginine. Although 80% or 179 of the amino acids are identical in bovine and porcine trypsins, the remaining 20% or 44 amino acid differences are sufficient
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 21, NOVEMBER 1, 1990
Trypsin, porcine IO
IO
20
10
yi
I I I I I IVGGYTCAANSVPYQVSLNSGSHFCGGSLINSQWVVSAAHCYKSRIQVRL 7 60
-
0
80
111
90
I I I I I GEHNIDVLEGNEQFINAAKIITHPNFNGNTLDNDIMLIKLSSPATLNSRV 110
130
120
.IO
151
I I I I i ATVSLPRSCAAAGTECLISGWGNTKSSGSSYPSLLQCLKAPVLSDSSCKS :Lo
180
170
ZOO
lii
I I I I I SYPGQITGNMICVGFLEGGKDSCQGDSGGPWCNGQLQGIVSWGYGCAQK
210
220
.23
I I i NKPGVYTKVCNYVNWIQQTIAAN I
Dlsulhde bonds
7 137
zs 4 1
IW ZIO
1
116 183 148 162 173 197
Figure 5. Primary structure of porcine pancreatic trypsin from ref 11. See Table I I for the protonated monoisotopic weights of the underlined peptides
to completely change the HPLC and mass spectrometric maps. In Table I1 (peaks observed from both lots), the number of peaks observed is smaller than that in column 1 of Table I; however, this may only reflect the slower proteolysis rate of porcine trypsin. The autolysis studies reported here were carried out in the absence of substrate in order to produce the most inclusive set of potential artifactual peptides. The level and also the selection of autolytic peptides encountered will vary with the length of exposure of trypsin at pH 8, in stock solutions as well as in the proteolytic reaction itself. The level and selection of artifactual peaks that might be observed in mass spectra of total or partially fractionated trypsin digests may also vary with the protein substrate, since the peptides produced from different substrates may affect selective desorption
in the FAB source differently and may inhibit autolysis a t different rates. The value of the present experimental determination of autolysis products is confirmed by significant deviations from prediction based on trypsin’s known specificity. Hydrolysis of bovine trypsin at asparagine under standard trypsin digestion conditions was documented.
LITERATURE CITED (a) Huber, R.: Kukla, D.; Bode, W.; Schwager, P.; Bartels, K.; Deisenhofer, J.; Steigeman, W. J. Mol. Biol. 1974, 89,73-101. (b) Bode, W.; Schwager, P. J. Mol. Biol. 1975, 98,893-717. (a) Walsh. K. A. Methods Enzymol. 1970, 19, 41-63. (b) KeiCDlouha, V.; Zylber, N.; Tong, N.-T.; Keil, B. FEBS Lett. 1971, 76, 287-290. (c) Poncz L.; Dearborn, D. G. J. Biol. Chem. 1983, 258, 1844-1850. For example, Andrews, P. C.; Dixon, J. E. Methods Enzymol. 1989, 768,97. Titani, K.; Sasagawa, T.; Reslng, K.; Walsh K. A. In High-Performance Liquid Chromatography of Proteins and Peptides ; Academic Press: New York, 1982; pp 23-27. Bradley, C. V.; Williams, D. H.; Hanley, M. R. Biochem. Siophys. Res. Commun. 1982. 704, 1223-1230. Yergy, J.; Heller, D.; Hansen. G.; Cotter, R. J.; Fenselau, C. Anal. Chem. 1983, 55, 353-357. (a) Hemling, M. E.; Carr, S. A.; Capiau, C.; Petre, J. Biochemistry 1988, 27, 699-705. (b) Gibson, B. W.; Biemann, K. Proc. Narl. Amd. Sci. U . S . A . 1984, 87, 1956-1960. (c)Naylor, S. A,; Findeis, A. F.; Gibson, B. W.; Williams, D. H. J. Am. Chem. Soc. 1986, 108, 6359-6363. Biemann, K. Biomed. Envirffn. Mass Spectrom. 1988, 16, 99-111. (a) Baumann, W. K.; Bizzozero, S. A,; Dutler, H. FEBS Lett. 1970, 8, 257-260. (b) Berezin. I . V.; Martinek, K. FEBS Lett. 1970, 8 , 26 1-262. KelDlouha, V.; Zylber, N.; Imhoff, J.-M.; Tong, N.-T.; Keil, B. FEBS Lett. 1971, 76, 291-295. Geiger, T.; Clarke, S. J. Biol. Chem. 1987, 262, 785-794. Hermodson, M. A.; Ericsson, L. H.; Neurath, H.; Walsh, K. A. Biochemistry 1973, 72, 3146-3153.
RECEIVED for review October 30, 1989. Revised manuscript received June 29, 1990. Accepted July 30, 1990. The work was supported in part by a grant from the National Science Foundation, BB5-8714238, and was presented a t the 37th ASMS Conference on Mass Spectrometry and Allied Topics.
