1594
Anel. Chem. 1992, 64, 1594-1600
Matrix-Assisted Laser Desorption Mass Spectrometry of Proteins Isolated by Capillary Zone Electrophoresis Thomas Keough,’ Ray Takigiku, Martin P. Lacey, and Michael Purdon The Procter & Gamble Company, Miami Valley Laboratories, P.O. Box 398707, Cincinnati, Ohio 45239-8707
stabilize the electrospray process.9 Some provision must also be made to minimize wall adsorption processes which can reduce sensitivity and separation efficiency. Use of CZE buffers at high pH is one approach to the problem. If the buffer pH is above the isoelectric point of the protein, then both the capillary wall and the protein are negatively charged and repel each other. Unfortunately,high pH’s favor negative ion formation and result in decreased positive ion sensitivity by over 1 order of magnitude compared with resulta obtained from acidic buffer^.^ Postcolumn manipulation of pH, with a suitable sheath liquid, is one way to minimize this difficulty. However, care must be taken to avoid electrical breakdown, which c a w s serious fluctuations in electrospray ion current.10 Another approachto minimize CZE wall adsorptionis to carry out protein separations at low pH using coated separation columns11J2 as recently demonstrated by Thibault and coworkers.13 This latter approach has the advantage of providing preformed cations for detection by positive ion mace spectrometry. Using this method, on-line CZE/MS has been demonstrated for injections of subpicomole quantitiea of various proteins having molecular massea ranging up to 78 OOO Da (bovine serum apotransferrin). Another important consideration is that electroepray ionization is not u n i v e d y Mass spectrometry (MS) is an ideal detector for capillary applicable to proteins. Peptide and protein analytss must zone electrophoresis (CZE) because it is universal, sensitive, have enough charged residues so that the mass-to-charge ratio and selective. As a result, various methods have been explored (mlz) of the analyte falla within the range accessible to the for on-line coupling of CZE and MS. They include coaxial particular mass spectrometer. A substantial number of continuous-flow fast atom bombardment (CF-FAB),1-3 eleccharges are required if large proteins are to be analyzed with trospray into an atmospheric pressure ion (AF’I) S O U T C ~ , ~ ~ ~ quadrupole mass fiiters. This latter requirement can be and pneumatically assisted electrospray, also into an API problematic for positive ion analysis of some proteins euch ~ource.~JThe CF-FAB approach offers the important as pepsin (M, 34 OOO), which has only four basic reeidues. advantage of being directly compatible with conventional We’ve also encountered difficulties in the analysis of some magnetic sector mass spectrometers. However, CF-FAB small protein fragments produced by specific enzymatic or requires introduction of a viscous liquid matrix and minichemical cleavage. Chait and co-worker8 report that some mization of the preeaure gradient acrossthe CZE/MS interface proteins show large variations in electroepray sensitivities; and is limited by the sensitivity and mass range of the FAB glycoproteins, for example, are not readily analyzed by elecionization process.8 Electrospray methods overcome many trospray ionization even if they contain enough acidic or basic of the problems associated with CF-FAB and offer high residues.14J5 Finally, electrospray ionization, whether inionization efficiencies, subpicomole sensitivity (in favorable terfacedwith CZE or not, produces ionized proteins containing cases), and a demonstrated mass range >1oOOOO Da (offmore than a single charge state. A large protein such as bovine line). serum albumin (BSA)has a charge distribution that may A number of factors must be considered when CZE/eleccontain more than 20 individual charge states. The BSA trospray MS is used for protein characterization. For example, dimer has a charge distribution that is more than 40 charge low CZE flow rates require the use of a make-up flow to A simple method for the off-line coupllng of laser desorption mass spectrometry (LDMS) and capillary zone eiectrophora sk (CZE) k dercrlbed. Representative ma# spectra of subplcomolequantnbsofprotdnkolatedfromCZEarepresented and discussed. The current detectton llmlt for bovlne a-lactaibumin Is 100 hnok injected onto the CZE column. H o w heart myoglobin was demonstratedto be dabk in CHES/KCi, a CZE Mer,for at lead 1month, that sotno isolates can be safely stored for long time perlods prlor to LDYS analysis. Protein stabiilty In 0.1 % aqueous trMuoroacetic ackl (TFA), a common solvent for LDMS, mwt a h be considered. I n the special case d porclne pepsinogen, slgnlficant (>50%) degradation was observed wlthln 5 mln in F A . I n favorable cases, mass measurement accuracks of f0.02 % wore obtainedfor protein kolates. Factors“nlng mass measurement accuracy are presented. Finally, the poesiblltty of identlfylng protein isolates, by combining Nterminal sequencing, molecular mass measurements, and selective peptide "mapping" procedures, is dlscwud.
(1)Moseley, M. A.; Deterding,
L.J.; Tomer, K. B.; Jorgenson, J. W.
Rapid Commun.Moss Spectrom. 1989,3, 87-93. (2) Moeeley, M. A.; Deterding, L.J.; Tomer, K. B.; Jorgenson, J. W. J. Chromatogr. 1989,480,197-209. (3) Moeeley, M. A.; Deterding, L.J.; Tomer, K. B.; Jorgenson, J. W. Anal. Chem. 1991,63,109-114. (4) Smith,R.D.; Loo,J. A.; Barinaga, C. J.; Edmonds, C. G.; Udeeth, H. R.J. Chromatogr. 1989,480, 211-232. ( 5 ) Smith, R.D.; Loo, J. A.; Edmonds, C. G.; Barinaga, C. J.; Udseth, H. R. Aml. Chem. 1990,62,882-899. (6) Lee, E. D.; Muck, W.; Henion, J. D.; Covey, T. R. J. Chromatogr. 1989,458, 313-322.
(7) Lee,E. D.; Muck, W.; Henion, J. D.; Covey,T. R.Biomed. Enoiron. Mass Spectrom. 1989,18, 253-267. (8) Barber, M.; Green, B. N. Rapid Commun. Mass Spectrom. 1987, 5,80-84. 0003-2700/92/0364-1594$03.00/0
(9) Parker, C. E.; Perkins, J. R.; Tomer, K. B. Proceedings of the 39th ASMS Conference on Moss Spectrometry and Allied Topics, Nashville, TN, M a y 19-24, 1991. (10) Shabanowitz,J.; Moseley, M. A.; McCormack,A.; Michel, H.; Martino, P.; Hunt, D. F.; Tomer, K. B.; dorgemon, J. W. Proceedings of the 38thASMS Conference onMassSpectrometryand Allied Topics,Tucaon, AZ,June 3-8,1990. (11) McCormick, R. M. Anal. Chem. 1988,60, 2322-2328. (12) Cobb,K. A.; Dolnik, V.;Novotny, M. Anal. Chem. 1990,62,2478-
2483. (13) Thibault, P.; Paris, C.; Pleasance, S. Rapid Commun. Moss Spectrom. 1991,5, 484-490.
(14) Chait,B.T.;Chowdhury,S.K.;Katta,V.;Beavie,R.C.Proceedings
of the 38th ASMS Conference on Mass Spectrometry and Allied Topics, Tuacon, AZ,June 3-8,1990. (16) Chowdhury, S. K.; Katta, V.; Chait, B. T. Rapid Commun.Mass Spectrom. 1990, 4, 81-87. 0 1002 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 64, NO. 14, JULY 15, 1992
states wide. For large proteins such as these, the electrospray ion current is distributed over a number of mlz values, decreasing absolute sensitivity at any particular mlz. Smith and co-workers have specifically investigated the dependence of maximum electrospray signal intensity as a function of protein molecular mass.16 Given the impressive attributes of CZE and the significant recent results obtained by on-line coupling of CZE with MS, we sought to develop methods to isolate proteins from CZE for analysis with the desorption mass spectrometry methods available in our laboratory. Desorption MS offers an independent and complementary method to on-line CZE/electrospray MS. Initially, we demonstrated a method to collect fractions from capillary zone electrophoresis (CZE) in a manner similar to that used with liquid ~hromatography.'~ The resulting peptide and protein fractions were analyzed with plasma desorption mass spectrometry (PDMS).laJgOffline CZE/PDMS was evaluated with simple mixtures such as derivatives of bradykinin, and for small peptides, detection limits of about 250 fmol were obtained. Detection limits for larger proteins such as a-lactalbumin were considerably higher, on the order of 5 pmols injected. PDMS mass measurement accuraciestypically average 0.1-0.2 76 .a While CZEIPDMS does not appear to be as convenient as current on-line methods, it does offer the opportunity to independently optimize the p H s of the CZE buffer and the analyte prior to MS analysis. Matrix-amisted laser desorption mass spectrometry (LDMS) has recently emerged as a powerful new method for the characterization of proteins.21-27 In some instances, it offers advantages relative to both PDMS and electrospray ionization. LDMS appears to be universally applicable to proteins and glycoproteins. It is applicableto large proteins in addition to small peptides. So far, the highest mass ion measured by LDMS is the dimer of glucose isomerase, having a mass of 344800 Da.28 LDMS detection limits for proteins can be several orders of magnitude lower than for PDMS. A detection limit of 1 fmol has been demonstrated for small proteins like cytochrome C.29 Mass measurement accuracies of &0.01% have also been demonstrated for proteins having molecular masses up to about 30 OOO Da.30 This accuracy is comparable to that obtained with quadrupole electrospray ionization20 and represents 1 order of magnitude improvement over typical PDMS results. Finally, unlike electrospray ionization, LD mass spectra typically do not exhibit broad distributions of charge states, and high sensitivity has been
1505
observed even for proteins having molecular masses of 150 OOO Da and beyond. This paper describes our first results obtained using LDMS as an off-line detector for CZE. Representative LD mass spectra of subpicomole quantities of proteins isolated from CZE are discussed. The sensitivityof the method was a s a d by CZEILDMS analysis of a-lactalbumin. Usable mass spectra were obtained with as little as 100 fmols injected oncolumn. These results will be directly compared with results from our previous PDMS study.17 Myoglobin was found to be stable when stored in CHEWKC1 (CZE buffer consisting of (cyc1ohexylamino)ethanesulfonicacid/KCl 10mW20 mM, pH = 9.0) at -70, -15, and 25 "C for periods of up to 1month, suggestingthat some protein isolates can be safely stored for long periods of time prior to LDMS analysis. Not suprisingly, porcine pepsinogen, an acid-labile zymogen of pepsin, decomposes rapidly in a common LDMS solvent system (0.1% aqueous TFA). The intact molecule completely decomposes to pepsin in less than 1 h in TFA (pH -2). The maas measurement accuracy obtained with CZE/LDMS was determined by analysis of a series of small model proteins. With current equipment, accurate mass measurements require use of an internal mass standard, and the quantity of standard added to any particular isolate must be optimized. In some cases, mass measurement accuracy can be adversely affeded by the low mass resolution inherent in the linear time-offlight analyzer employedon our present instrument. Finally, one of the principal advantages of off-line techniques in general is that they make protein available for analysis with important complementary biochemical methods such as automated Edman sequencing. Here we demonstrate the combined use of molecular mass determination, N-terminal sequence analysis, and CNBr peptide "mapping" to identify proteins isolated by CZE.
EXPERIMENTAL SECTION Capillary Electrophoresis and the Procedure for Eluant Collection. All separations were carried out on a home-built CZE unit as previously described." Samples were introduced using electromigration, and the quantity of analyte injected was calculatedas previously reported.31The cathodic end of the CZE column was fitted with a porous glass used to complete the electrical circuit and enable fraction collection in a fashion similar to that used with HPLC. On-column W detection was accomplished with a Linear detector (Model Uvis 203, Reno, NV). The migration times of analytes from the injector to the UV detector were used to calculateoverallmigration times to the outlet of the separation column. These calculations established the times at which fractions were collected.17 (16) Smith, R. D.; Loo, J. A.;Edmonds, C. G.; Barinaga, C. J.; Udseth, Two proteins were collected onto PVDF membranes (ProH. R. J. Chromatogr. 1990,516, 157-165. (17) Takiaiku, R.; Keough, T.; Lacey, M. P.; Schneider, R. E. Rapid Blott, Applied Biosystems, Foster City, CA) for subsequent Commun. M a s Spectrom.-1990,4, 24:29. analysisby automated Edman degradation. a-Lactalbumin and (18) Hakansson, P.; Kamensky, I.; Sundqvist, B.; Fohlman, J.; Petersubtilisin BPN' were collected directly from the outlet of the son, P.; McNeal, C. J.; Macfarlane, R. D. J. Am. Chem. SOC.1982,104, separationcolumn onto 1-cm2piecesofprewetted (MeOH) PVDF 2948-2949. membrane. In the fiit experimentwith a-lactalbumin,the PVDF (19) Sundqvist, B.; Macfarlane, R. D. Mass Spectrom. Reu. 1985,4, 421-460. membrane was not washed prior to insertion into the sequencer (20) Loo, J. A.; Edmonds, C. G.; Smith, R. D.; Lacey, M. P.; Keough, (ABI,Model 475A, Foster City, CA). Unfortunately, this T. Biomed. Enuiron. Mass Spectrom. 1990, 19, 286-294. approach prevented the observation of glutamic acid residues (21) Karas, M.; Hillenkamp, F. And. Chem. 1988, 60, 2299-2301. because of an HPLC interference from the CHES/KCl buffer. (22) Karas, M.; Bahr, U.; Hillenkamp, F. Znt. J. Mass Spectrom. Zon In subsequent experimentswith subtilisin BPN', the membrane Processes 1989,92, 231-242. (23) Hillenkamp, F.; Karm, M. Method Enzymol. 1990,193,280-295. was thoroughly washed with deionized water prior to ineertion (24) Bmvi~,R. C.; Chait, B. T. Rapid Commun.Mass Spectrom. 1989, into the automated sequencer. This procedure completely 3, 233-237. eliminated the interference. (25) Benvis, R. C.; Chait, B. T.Rapid Commun.Mass Spectrom. 1989, Sample Preparation Prior to LDMS. All CZE separations 3,432-436. discussed in this paper were carried out in CHEWKC1 buffer (26) Beavis,R. C.; Chait, B. T. Rapid Commun.Mass Spectrom. 1989, (pH = 9.0; (cyclohexy1amino)ethanesulfonicacid/KCl 10 mM/ 3, 436-439. (27) Beavis, R. C.; Chait, B. T. Proc. Natl. Acad. Sci. U.S.A. 1990,87, 20 mM). The isolates were collected in 1-2-pL volumes in poly6873-6677. propylene microcentrifugetubes. In some experiments,they were (28) Karas,M.;Bahr,U.;Ingendoh,A.;Nordhoff,E.;Stahl,B.;Stupat, diluted with small volumes of 0.1% (vol/vol) aqueous trifluoK.; Hillenkamp, F. Anal. Chim. Acta 1990,241, 175-185. (29) Stupat, K.; Karas, M.; Hillenkamp, F. Znt. J . Mass Spectrom. Zon Processes 1991,111, 89-102. (30) Beavis, R. C.; Chait, B. T. Anal. Chem. 1990, 62, 1836-1840.
(31) Rose, D. J., Jr.; Jorgenson, J. W. Anal. Chem. 1988,60,642&. (32) Wallingford,R. A.; Ewing, A. G. Anal. Chem. 1987,59,1762-1766.
1506
ANALYTICAL CHEMISTRY, VOL. 64, NO. 14, JULY 15, 1992
roacetic (TFA) to a final concentration of 51 pmol/pL. One to two microliters of the final isolate solutions were then mixed with an equal volume of a saturated sinapinic acid solution, prepared by dissolving 10 mg of sinapinic acid into 1 mL of aqueous 30 % acetonitrile (vol/vol) containing 0.1 % trifluoroacetic acid, according to the procedure of Beavis and ChaiLZ7 One to two microliters of the resulting sinapinic acid solutions were loaded onto the end of a flat solids probe and allowed to dry at ambient temperature prior to insertion into the mass spectrometer. The CNBr digestion of subtilisin BPN’ isolated by CZE was accomplished as follows: The isolate was diluted to a volume of 200 pL with aqueous 0.1 N HC1. A small (unweighed)crystal of CNBr was added to the reaction vessel which was then sealed, wrapped in aluminum foil, and inserted into a heating block at 37 OC for 2 h. The solution was then evaporated to dryness and redissolved in 0.1% TFA prior to LDMS analysis. Laser Desorption Mass Spectrometry. LD mass spectra were obtained on a Vestec 2000 linear time-of-flight mass spectrometer (Vestec Corp., Houston, TX). Protein isolates, dissolvedin a saturated sinapinicacid matrix,=-%were irradiated with the frequency-tripledoutput of a Q-switched Nd-YAG laser (Lumonics HY-400,355 nm, pulse width 10 ns, repetition rate 10Hz). Desorbed secondary ions were accelerated in two stages to 35 kV. After acceleration, product ions drifted down a 2-m flight tube and were detected with a 20-stageelectron multiplier. Electronicsfor recording maas spectra include a transient recorder (TR8828D) and a CAMAC crate controller (Model 6010) both from LeCroy (Chestnut Ridge, NY). The transient recorder was operated with a time resolution of 5 ns. Typically, spectra produced from 50 laser shots were accumulated in the transient digitizer using LeCroy-suppliedsoftware. Accumulated spectra from the transient recorder were then directly imported into a Compaq 386/33 (Houston, TX) using a converting program written in-house. Spectra were then processed using Lab Calf, a commercially available software package (Galactic Industries Corp., Salem, NH). Spectra were then mass calibrated and peak centroids were measured, again using software developed inhouse. A complete description of our Lab Calf based time-offlight data system is currently in preparati~n.~~ Proteins. All proteins, with the exception of subtilisin BPN’, were purchased from Sigma Chemical Co. (St. Louis, MO). The proteins used and their catalog numbers are as follows: ribonucleaseB (R-5750),bovine a-lactalbumin (L-5385),horse heart cytochrome C (C-2506), bovine trypsinogen (T-1143), porcine pepsinogen (P-4666), horse heart myoglobin (M-1182),bovine serum albumin (A-3675), bovine &lactoglobulin A (L-7880), bovine erythrocytecarbonic anhydrase (C-6653). SubtilisinBPN’ was produced and purified in-Company.
RESULTS AND DISCUSSION Representative LD Mass Spectra of CZE Isolates. Several model proteins were isolated from CZE and analyzed by LD mass spectrometry. Some of the proteins used in this evaluation have been previously characterized with PDMS or electrospray ionization. Some were specifically included in the study because they are problematic for PDMS and/or electrospray ionization and they illustrate areas where the LDMS technique might make significant contributions. Others were included because they point out limitations with the current LDMS method, particularly when using a linear time-of-flight mass analyzer. A representative series of LD mass spectra of proteins isolated from CZE are given in Figure 1. Figure l a shows the spectrum obtained from analysis of 8% of an isolate from a 7-pmol injection of ribonuclease B onto the CZE column (560 fmol loaded onto the LDMS solids probe assuming 100% recovery of the protein following electrophoresis). The spectrum in Figure l a illustrates the presence of an unresolved mixture of both ribonuclease A (33) Lacey, M. P.; Keough, T.;Shaffer, J. D.; Hayworth, M. S.; Nelson, R. W., in preparation.
I
16000
14000
I
10000
sublilisin
I
20000
15000 mir
mil
BPN’ tl
,
$
6
6
O
o
m
1300
P
f
c
6550
200
MOO0
20000 mil
40000
60000 mis
80000
Flgure 1. Representative LD mass spectra of proteins Isolated from CZE: (a) ribonuclease B, (b) lactaibumln, (c) subtilisin BP” and (d) bovine serum albumin.
and B. These proteins have identical primary structures but differ in that ribonuclease B contains a single polysaccharide moiety attached through N-acetylgalactosamine to asparagine 34. The carbohydrate consists of two N-acetylgalactosamine residues, and the remainder is six to eight mannose groups arranged with two 1,3 and 1,6 branch points.34 The spectrum is consistent with the LD mass spectrum of ribonuclease B previously obtained by Beavis and Chait.35 It is noteworthy because it reiterates that LDMS is applicable to both proteins an glycoproteins, an attribute that has been previously reported.28*36,37 Significantly, ribonuclease B was not observed in this sample in an earlier electrospray and plasma desorption mass spectrometry study.20 On the other hand, electrospray mass spectra of ribonuclease A demonstrate the presence of a completely resolved series of impurity ions that are only partially resolved from the ribonuclease A molecular ion envelope in the present experiment. The ribonuclease A molecular ions, the impurity ions (M + X + H)+ and the photochemically generated adduct ions (M + P H)+are shown expanded in the inset of Figure la. The impurity ions have been shown to differ in mass by 98 Da from the molecular ions and their origins have been discussed by Chait and co-worker~.~~ The measured molecular mass of ribonuclease A is 13 682 i 4.2 Da (average of seven determinations from a single sample loading), which agrees well with the value calculated (13 682.4 Da) based on the known primary structure of the molecule.39 The spectrum in Figure l b was obtained by LDMS analysis of 7 % of an isolate from a 9-pmol injection of bovine cy-lactalbumin onto the CZE column. Cytochrome C was added to the isolate as an internal mass standard. A total of 140 fmol of cytochrome C and 630 fmol of lactalbumin were loaded onto the mass spectrometer solids probe for analysis. The measured molecular mass of 14 181 f 1.4 Da (n = 7) agrees well with the expected value (14 179.1Da) and with the value (14 180 Da) recently obtained by on-line CZE/electroepray MS.13
+
(34) Williams, R. L.; Greene, S. M.; McPherson, A. J. Biol. Chem. 1987,262, 16020-16030. (35) Beavis, R. C.; Aduru, S.; Chait, B. T. Proceedings of the 38th ASMS Conference on Muss Spectrometry and Allied Topics, Tucson, AZ, June 3-8,1990. (36) Karas, M.; Bahr, U. Trends Anal. Chem. 1990,9, 321-325. (37) Karas, M.; Hillenkamp, F. In Muss Spectrometry of Large Znuolatile Molecules for Murine Organic Chemistry;Hilf, E. R., Tsuzynski, W., Eds.; World Scientific: Singapore, 1990. (38) Chowdhury, S. K.; Katta, V.; Beavis, R. C.; Chait, B. T. J . Am. SOC.Mass Spectrom. 1990, I , 382-388. (39) Protein Identification Resource (PIR), National Biomedical Research Foundation, Release 19.0 (3187 December 1988).
ANALYTICAL CHEMISTRY, VOL. 64, NO. 14, JULY 15, 1992
The spectrum in Figure ICwas obtained by analysis of about 7 % of a subtilisin BPN’ isolate following an injection of 3.6 pmol onto the CZE column. A total of 240 fmol were loaded into the mass spectrometer for analysis. Subtilisin BPN’ was chosen as a representative single-chain alkaline serine protease. The chemical propertiesm and some of the s e q ~ e n c e s ~of l - these ~ ~ enzymes have been published. The spectrum exhibits excellent signal to noise and should be contrasted with previously reported PD mass spectra that required data acquisition times of between 7 and 14 h with 500 pmol of material.44 Subtilisin BPN’ and homologous enzymes such as proteinase K have been problematicfor electrospray ionization; subtilisin BPN’ provided an unexpected molecular and proteinase K was not detected.20 In the present experiment, the measured molecular mass of 27 536 f 2.5 Da (n= 7) is in good agreementwith the calculated value (27 533.7 Da). The spectrum in Figure Id was obtained by analysis of about 50% of an isolate from an injection of 1.5 pmol of BSA on column. The measured molecular mass of BSA was 66 795 Da, which is higher than the calculated value (66 432.3 Da) by 0.55% . This level of mass error is consistent with previous measurements in this mass range23and results because of the poor mass resolution inherent with current time-of-flight analyzers. The molecular ion envelope contains unresolved contributions from matrix adducts formed photochemically. The LD mass spectrum of porcine pepsin (not shown) was generated by loading 4% of an isolate obtained from a 2.5pmol injection of porcine pepsinogen. The measured molecular mass of the isolate (34 593.8 f 8.7 Da) is in closer agreementwith the values expectedfor porcine pepsin (34 460 and 34644 Da based on the known heterogeneity for this enzyme39) than with the value calculated for pepsinogen (39 485 Da). The spectrum suggests that either the starting pepsinogen was unstable under the analysis conditions or that the sample had decomposed to pepsin prior to analysis. To address this issue, porcine pepsinogen (not electrophoresed) was prepared in CHES/KCl buffer at a concentration of 5 pmol/pL. To this solution was added an equal volume of sinapinic acid, containing bovine trypsinogen as an internal standard, and the sample was quickly analyzed. The resulting spectrum is given in Figure 2a. It exhibits a molecular mass that is consistent with that calculated for authentic intact pepsinogen. On the other hand, the spectrum obtained after having first dissolved pepsinogen in 0.1 7% aqueous TFA for about 5 min and then mixing with the sinapinic acid matrix is given in Figure 2b. Clearly, significant decompositionof pepsinogen had occurred in the TFA solution within only a few minutes. The decomposition products have measured mass losses of approximately 1878 and 5034 Da, respectively. Interestingly, these fragments may result by successive cleavages of two separate Leu-Ile bonds (residues 16-17, calculated mass loss of 1874 Da; residues 44-45, calculated mass loss of 5025 Da). The latter cleavage produces the major form of porcine pepsin, and we assume that the lower-mass component is actually pepsin. Complete decomposition of pepsinogen to pepsin occurs in TFA in less than 1h (data not shown). In spite of the difficulties experienced with this particular sample, it was included here because it further emphasizes a current limitation with linear time-of(40) Markland, F. S., Jr.; Smith, E. L. In The Enzymes; Boyer, P. D., Ed.; Academic Press: New York, 1971; pp 561-608. (41) Markland, F. S., Jr.; Smith, E. L. J. Biol. Chem. 1967,242,51985211. (42) Jacobs, M.; Eliaason, M.; Uhler, M.; Flock, J. I. NucZ. Acids Res. 1985,13,8913-8926. (43) Jany, K. D.; Lederer, G.; Meyer, B. B i d . Chem. Hoppe-Seyler 1986, 367, 87. (44)Lacey, M. P.; Keough, T. Rapid Commun. Mass Spectrom. 1989, 3, 323-328.
1197
300
/ 200
pepsinogen
-
t1
100
0
dz
300
.-3
I
250
E
I
200
I
I
I
30000
40000
I
m/z
Spectra obtained by (a)dissolving pepsinogen in CHES/KCi and then mixing with an equal volume of sinapinic acM prior to analysis and (b) dissolving pepsinogen in 0.1 % TFA for 5 mln and then mlxing with sinaplnic acM and analyzing. Flgure 2.
flight mass spectrometry-poor mass resolution. In this particular case, the measured molecular mass of pepsin is intermediate between the calculated values for its two known forms. This suggests that the spectrum may consist of unresolved contributions from both forms. The situation is further complicated because, at this mass, the photochemically generated adduct is unresolved and contributes to an upward mass shift of the molecular ion envelope. More accurate mass measurements would be expected using quadrupole electrospray ionization. However, in‘this particular case, positive ion analysis is not feasible because pepsin contains only four basic residues. CZE/LDMS Sensitivity. Sensitivity for the off-lineCZE/ LDMS method was assessed using a-lactalbumin as a test protein. This protein was used in an analogous study assessing the sensitivity of off-line CZE/PDMS.” LD mass spectra produced from isolates following injections of 100,500,1000, and 5000 fmol on column are summarized in Figure 3. In all cases, only 50 % of the isolate was loaded onto the solids probe, and spectra were accumulated for 50 laser shots. Samples were analyzed from the lowest concentration to the highest to minimize any effects of cross contamination. The spectrum of the lowest-concentration sample (Figure 3d) shows a larger contribution of the photochemical adduct than is evident in any of the other spectra, possibly because a higher laser power density was needed to detect the protein at this low concentration. In our previous study we demonstrated a CZE/PDMS detection limit of only 5 pmol for lactalbumin. Five picomoles on column represented the high-level standard in the present study; the lowest-levellactalbumin isolate analyzed here corresponds to no more than 50 fmol loaded into the mass spectrometer. This is a 100-fold sensitivity improvement relative to our earlier PDMS results. The sensitivity enhancement would be even greater for larger proteins which
1588
ANALYTICAL CHEMISTRY, VOL. 64, NO. 14, JULY 15, 19Q2
'U " 300
Y
.$ 200 t
Table I. Calculated and Measured Molecular Masses of Several Proteins Isolated from CZE
I-l
c
3 E
100
loo00
15000
zoo00
d z
mi2
average molecular mass' protein
calculated
ref
measured
error (%)
bovine ribonuclease A bovine a-lactalbumin equine myoglobin bovine lactoglobulin subtilisin BPN' porcine pepsin
13 682.4 14 179.1 16951.5 18 363.1 27 533.7 34 459.9b 34643.9 66 432.3
39 39 46 13 39
13 682.0 14 181.3 16955.6 18 371.7 27 536.2
-0.003 +0.016 +0.023 +0.047 +0.009
39 39
34 592.8 66 795.0
c
bovine serum albumin
x
-
.- 200 B 1
100 100 15000
2ow0
loo00
1 m
mi2
2ow0
mi2
F w e 9. Demonstration of the overall senstthrity of the off-llne CZE/ LDMS methodusing bovine lactalbumin as the analyte. Mass spectrum of isolate from (a)5-pmoi injection on column, (b) l-pmol injection, (c) 500-fmol injection, and (d) 100-fmolInjectlon. Only 50% of the isolate was loaded onto the mass spectrometersolids probe for LDMS analysis. 6600
-
6550
-
6500
-
myoglobin +1, day 0
6450
16000
a The calcdated molecular m w e a or primary structures were taken from the indicated sources listed under ref. Two entries because of the known heterogeneity of this enzyme. The error cannot be determined accurately because both forms may be present. The measurement is +0.38% greater than the lower-mass form and -0.15% lower than the high-mass form of pepsin.
*
E
low0
+0.546
17000
18000
d z
spectra. (Upper) immediately after solutlon preparation; (lower)after 1 month storage in CHESIKCIat - 15
Flgwe 4. Myoglobin LD mass
OC.
show progressively lower response by PDMS.45 Protein Stability in the CZE Buffer. Horse heart myoglobin was used as a model protein to determine the effect of long-term storage in the CHEWKC1 buffer. A 5 pmol/pL solution of myoglobin was prepared and analyzed as a timezero control. The mass spectrum spanning the molecular ion region is shown in Figure 4 (upper). The measured molecular mass of 16 952 f 1.2 Da agrees well with the calculated value (16 952.5 Da), and the measured mass resolution at full width half maximum (fwhm), 225, is close to the expected value for this compound. The myoglobin solution was then aliquoted into three sets of five separate microcentrifugetubes. One set of tubes was stored at ambient temperature while the other two seta were stored at -15 and -70 "C,respectively. One tube from each set was analyzed 1,7,14,22, and 30 days after preparation. All of the resulting mass spectra were very similar to that obtained from the initial analysis. The measured molecular masses and mass resolutions were identical within experimental error. The spectrum from the 30-day sample,stored at -15 "C,is overlaid in Figure 4 (lower) for comparison. Clearly, this protein is stable in the CZE buffer over a wide range of temperatures and for an extended (45) Lafortune, F.; Beavis, R.; Tang, X.; Standing,K. G.; Chait, B. T. Rapid Commun.Mass Spectrom. 1987,1,114-116. (46)Zaia, J.; Annon,R. S.; Biemann, K. Rapid Commun.Mass Spectrom. 1992,6,32-36.
period of time. The resulta suggest that the integrity of at least some protein isolates will not be compromised upon storage prior to LDMS analysis. Mass Measurement Accuracy. Table I summarizesthe CZE/LDMS mass measurementa obtained in the present study. The mass accuracies for several of the compounds such as ribonuclease A, bovine a-lactalbumin, equine myoglobin, and subtilisin BPN' average about 0.015%, which is close to the accuracy previously reported for proteins having masses less than 30 OOO Da.30 However, a larger error was observed for bovine lactoglobulin. This spectrum was quite weak, and its signal to noise would clearly have benefited had more of the isolate been loaded into the mass spectrometer, The analysis of the lactoglobulin isolate was further complicated because it was a mixture. Reanalysis of 2 pmol of lactoglobulin (not electrophoresed), using bovine trypsinogen as the internal mass standard, gave a much more intense mass spectrum and a measured molecular mass of 18 366.3 Da. The mass error in this analysis (+0.012 7% 1 is more in line with expectation^.^^ In order to obtain mass measurementa accurate to within 0.01-0.02%, an internal standard must be added to CZE isolates prior to LDMS analysis. The standard must be added directly to the analyte since the LD mass spectrometer we use does not have an xy-moveable sample stage that allows the standard and analyte to be prepared separately on the same substrate. The amount of internal standard added to each isolate must be experimentally optimized by trial and error as illustrated in Figure 5 for bovine lactoglobulin A. In Figure 5a, 250 fmol of myoglobin were added as a standard to an isolate containing no more than 400 fmol of the analyte. The singly charged myoglobin molecular ion is clearly evident while the doubly charged ion, which is also needed for mass calibration, is not resolved from a low-mass impurity. Reanalysis of another portion of the isolate, after addition of 500 fmol of myoglobin, gave the spectrum in Figure 5b. In this case, both internal standard ions are readily detected, enabling proper calibration of the mass scale. Finally, it should be pointed out that mass accuracy can deteriorate if the ratio of internal standard/analyte is either too large or too small. We prefer to work with standard and analyte signal intensities that are within about a factor of 5 of one another. The mass measurement accuracies obtained for molecules weighing more than about 30 OOO deteriorate with current LDMS time-of-flight instruments, in part because of the unresolved contributions of matrix adducts. The effect is clearly noticeable for large proteins such as bovine serum albumin. Smaller proteins such as porcine pepsin can also
ANALYTICAL CHEMISTRY, VOL. 64, NO. 14, JULY 15, 1902 {a)
6650
1199
lactoglobulin t1 myoglobin t1
6600 h
.a
'I
6550
6500 I
I
10000
myoglobin +2
6650
-
.-
-8
15000 dz
I
20000
5
myoglobin +1
6600
'
6550
6500
L
I
lo000
I
15000
20000
25000
3oooo
mlz
+ +
lactalbumin
4 Time (min)
h'
20000
Flgwo 5. LD mass spectrum of (a) 5 4 0 0 fmol of lactoglobulin Isolate 250 fmol of myoglobin and (b) 1 4 0 0 fmol of lactoglobulln isolate 500 fmol of myoglobin.
\1
loo carbonic anhydrase +l
L
I
I
d z
2
10 Time (min.)
Lactoglobulin
I
6
Flguro 6. Electropherogram of a test mixture of myoglobin, lactalbumin, and lactoglobulin.
be problematic if they consist of two or more closely related forms having unresolved masses. SimpleMixture Analysis. The electropherogramshown in Figure 6 demonstrates the quality of protein separation that can be achieved with untreated columns at high pH. This particular separation, which was optimized, was obtained with a buffer pH of 9.0. Proteins were loaded at +20 kV for 10 8, the separation voltage was +25.0 kV,and detection was carried out at a wavelength of 214 nm. Approximately 450 fmol of myoglobin, 500 fmol of lactalbumin, and 400 fmol of ladoglobulin were injected. Baseline resolution is evident between the three major components, and the analysis time is less than 5 min. The measured molecular masses and separation efficiencies (in parentheses) for the three proteins are myoglobin 16951.2 Da (14280 plates), lactalbumin 14 176.4Da (11870plat.m) andlactoglobulin18 367.0Da(9770 plates). The root-mean-square of the mass measurement errors obtained from analysis of these isolates is 0.015%.
Flgwo 7. Electropherogram (a) of a test mixture of myoglobin, lactalbumln, and carbonic anhydrase. (b) LD mass spectrum of the unresolved components.
The electropherogramshown in Figure 7a was obtained by injection of about 2.1 pmol of myoglobin, 3.6 pmol of bovine a-lactalbumin, and 2.9 pmol of bovine carbonic anhydrase. The efficiency of this separation was intentionally degraded (pH = 9.0, separation voltage +18.0 kV) so that myoglobin and carbonic anhydrase coeluted. The mass spectrum obtained from the isolate of coeluting components (the earlier eluting peak) is given in Figure 7b. This experiment demonstrates the obvious advantage of obtaining "full scan" data on each isolate as opposed to selected ion monitoring, the approach that has been previously demonstrated for on-line CZE/MS using uncoated columns and high buffer pH'e.4~5 Clearly, the LDMS approach is capable of detecting coeluting components,whether they are relatively close in mass as shown in Figure l a or differ widely in mass as shown here. Protein Identification by Use of CZE/LDMS and Edman Sequencing. To demonstrate the possibility of identification of proteins isolated from CZE, we collected a 6pmol isolate of bovine a-lactalbumin onto a prewetted PVDF membrane. The membrane was directly inserted into an automated sequencer and 18 cycles were carried out. The results are summarized in Table I1 (upper). Of the 18cycles, 13residues were correctly identified. The glutamicacids (E's) could not be observed in the present experiment because of an HPLC interference from the CHESiKCl buffer. The cysteines were not modified, so nothing was detected in cycle 6. The sequence found in the present experiment was wed, exactly as shown, to search against previously published sequences in three protein information resources data banke (PSQ,PSQ NEW, and PSQ N R L 3 D 3 . Undefined residues were entered at cycles 1,6,7,10, and 11. The search strategy allowed for as many as three mismatches. Of the 21 930 proteina searched (more than 6 million residues), only three matching proteins were identified: bovine, goat, and water buffalo (fragment)lactalbumin. The molecular ma88 of water buffalo lactalbumin is not reported in the data base; however, the molecular mass of bovine a-lactalbumin is 14 179.1 Da
1800
ANALYTICAL CHEMISTRY, VOL. 64, NO. 14, JULY 15, 1992
Table 11. N-Terminal Sequence Analysis of Bovine a-Lactalbumin and Subtilisin BPN’ Isolated from CZE Bovine a-Lactalbumin correct sequence experimentalresult
EQLTK CEVFR ELKDL KGY -QLTK --VF- -LKDL KGY
molecularmass (Da) searchresults bovine a-lactalbumin EQLTK CEVFR ELKDL KGY EQLTK CEVFQ KLKDL KDY goat lactalbumin water buffalo EQLTK CEVFR ELKDL KNY lactalbumin (fragment)
calculated measured 14,179.1 14,187.1
14,181.3
a
These enzymes do not have a methionine at residue 50 and do not yield this CNBr fragment.
Subtiliiin BPN’ correct sequence experimentalresult
Table 111. Calculated and Measured Average Masses (Da) of Some CNBr Digestion Products from the Proteins Identified by Edman Seauencinrt calculated measured molecular mass molecular mass protein (1-50) (223-end) (1-50) (223-end) A23624 BPN’ a 5347.1 lSOl BPN’ 4 5599.3 subtilisin BPN’ 4948.5 5598.3 4949.9 5598.7 SUBSS BPN’ a 5646.3 550517 Natto kinase a 5644.4
AQSVP YGVSQ IKAPA LHSQG AQSVP YGVSQ IKAPA LHSQG
molecularmess (Da) A23824 BPN’ lSOl BPN‘
subtilisinBPN’ SUBSS BPN’ JS0517 Natto kinase
search results
calculated
measured
AQSVP YGISQ IKAPA LHSQG AQSVP YGVSQ IKAPA LHSQG AQSVP YGVSQ IKAPA LHSQG AQSVP YGISQ IKAPA LHSQG AQSVP YGVSQ IKAPA LHSQG
27 385.4 27 477.7 27 533.7 27 671.7 27 724.8
27 536.2
Not reported.
while that of goat lactalbumin is 14 187.1 Da. The experimentally measured mass of 14 181.3 Dais in closer agreement with the value for bovine lactalbumin than that for goat lactalbumin. In the present experiment, data obtained from 18 cycles of Edman degradation were used, along with the molecular mass measurement,to tentatively identify the isolated protein as bovine a-lactalbumin. In this case, one could arrive at the same conclusion with much less N-terminal sequence information. For example, if one uses only residues 2-5 to search against the data banks, one finds 97 proteins containing the sequence QLTK (assuming no mismatches). However, only three of those proteins have the QLTK sequence localized at the N-terminus beginning at residue 2; they are the same three proteins identified above. A 5-pmol isolate of subtilisin BPN’ was similarly collected onto a PVDF membrane for subsequent N-terminal sequence analysis. In this experiment, the membrane was thoroughly washed with deionized water prior to insertion into the sequencer. The results of 20 cycles of Edman degradation are summarized in Table I1 (lower). The experimentalresults, which matched exactly with the known sequence, were again searched against the three data banks assuming three mismatches. A number of proteins were identified by this procedure including two complexes of subtilisin BPN’ with inhibitors, subtilisin BPN’ inactivated with peroxide, a subtilisin NOVO fragment (only 55 residues),and the five proteins listed in Table I1 (lower). In the cases listed, 19 of the fiist 20 amino acid residues are identical for the candidateproteins. The proteins fall into twoclasses: subtilisin BPN’, lSOl BPN’, and 550517 Natto kinase have identical N-terminalsequences (V at residue 8) while A23624 BPN’ and SUBSS BPN’ have identical N-terminal sequences (I at residue 8). Fortunately, the five proteins have significantly different molecular masses. The experimentallymeasured mass of 27 536.2 Da is in close agreement with the value calculated for subtilisin BPN’ (27 533.7 Da) and quite different from the calculated values for the other species. Additional structural confirmation can also be obtained by selective chemical or enzymaticdegradation. To illustrate, CNBr digestion waa carried out on a portion (90%,or ca. 3.2 pmol) of the BPN’ isolate remainingafter the LDMS analysis. The molecular masses of the C-terminal fragments, Table 111, differentiate A23624 BPN’, SUBSS BPN’, and 550517 Natto kinase from the remainingtwo BPN’s. Differentiation
so00
10000 m/z
Flgure 8. LD mass spectrum of the CNBr digest of subtilisin BPN’ that had been Isolated by CZE.
of lSOl BPN’ and subtilisin BPN’ is more difficult on this basis because their C-terminal fragments differ in mass by only 1 Da. However, subtilisin BPN’ is the only one of the five enzymes that contains a methionine at residue 50. Therefore, it is the only one of the enzymes expected to yield a relatively small N-terminal fragment (1-50) upon CNBr digestion. The average molecular mass of BPN’ (1-50) is calculated to be 4948.5 Da. The mass spectrum of the unseparated CNBr digest of the BPN’ isolate, given in Figure 8, demonstrates strong signals for both the (1-50) N-terminal fragment and the (223-275) C-terminal fragment. The measured average masses of 4949.9 Da for (1-50) and 6698.7 Da for (223-275) agree well with the values expected for subtilisin BPN’. These latter results illustrate the complementary nature of N-terminal sequence analysis and mass spectrometry. The results obtained from Edman sequencing are specific enough that the identity of an isolate can be narrowed down to only a few possible proteins. However, in some cases like subtilisin BPN’, lSOl BPN’, and JS0517 Natto kinase, direct identification of the isolate is not possible based on Edman sequencing alone because the N-terminal amino acid sequences of the candidates are identical. Final identification can be made by mass spectrometry if the molecular masses or peptide maps are sufficiently distinct. In the present example, both the molecular masses and the masses of the CNBr digestion products are sufficiently unique to allow identification of the isolate as subtilisin BPN’.
ACKNOWLEDGMENT We gratefully acknowledge R. A. Grant and J. 5.Whitten who provided the N-terminal sequence analyses.
RECEIVED for review January 28, 1992. Accepted April 20, 1992. Registry No. CHES, 103-47-9;TFA, 76-05-1;KCl, 7447-407; ribonuclease, 9001-99-4; subtilisin, 9014-01-1; pepsin, 900175-6; pepsinogen, 9001-10-9; carbonic anhydrase, 9001-03-0.
