Anal. Chem. 1994,66, 1066-1061
Identifying Charge States of Peptides in Liquid Chramatography/Electrospray Ionization Mass Spectrometry Gltte Neubauer Department of Biochemistry, Imperial College, London, UK Robert J. Anderegg' Department of Structural and Biophysical Chemistty, Ghxo Research Laboratories, Research Triangle Park, North Carolina 27709
The addition of submillimolar levels of sodium acetate to the mobile phase during an LC/ESI-MS experiment epcourages the formation of sodium replacement ions in addition to the normally observed protonated species. The spacing of these sodiated species allows unambiguous determination of the charge state of the ions and, hence, their actual mass. Such information is very useful when unknown or modified peptides are being analyzed. At the levels reported here (250 pM), the sodium salt seems to cause no deteriorationin chromatographic performance and does not unduly foul the mass spectrometer. Electrospray ionization (ESI) mass spectrometry (MS) has revolutionized the way mass spectromists analyze peptides and proteins.'-' A relativelygentle technique generally leading to the formation of multiply charged analyte ions, ESI permits the analysis of very large, intact biomolecules. Because the samples are sprayed from a liquid solution, the technique has led to one of the most successful interfaces between liquid chromatography and mass spectrometry.68 Tryptic digests of proteins are particularly popular for mapping by LC/ESIMS,8-11in part because the enzyme very selectively cleaves on the C-terminal side of basic residues, leaving a site for protonation at either end of the peptide: the free N-terminus and the basic residue at the new C-terminus. ESI-MS of the tryptic peptides usually produces an abundant doubly charged ion from which the mass of the peptide is readily determined. When collisionally activated and analyzed by MS/MS, the doubly charged ions decompose easily to produce sequencespecific fragment ions.8-10J1 (1) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M.Science 1989, 246,64-7 1. (2) Covey, T . R.; Bonner, R. F.; Shushan, B. I.; Henion, J. D. Rapid Commun. Mass Spectrom. 1988, 2, 249-256. (3) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Mass Specrrom. Rev. 1990, 9, 37-70. (4) Green, B. N.; Oliver, R. W. A. Biochem. Soc. Trans. 1991, 1 9 , 929-935. (5) Chowdhury, S. K.; Katta, V.;Chait, B. T. Biochem. Biophys. Res. Commun. 1990,167, 686-692. (6) Bruins, A. P.; Covey, T. R.; Henion, J. D. Anal. Chem. 198'1.59.2642-2646. (7) Hemling, M. E.; Roberts, G. D.; Johnson, W.; Carr, S. A,; Covey, T. R. Biomed. Environ. Mass Specrrom. 1990, 19, 677-691. (8) Huang, E. C.; Henion, J. D. J. Am. Soc. Mass Specrrom. 1990,1, 158-165. (9) 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. (10) Covey, T.R.;Huang, E. C.; Henion, J. D. Anal. Chem. 1991,63, 1193-1200. (11) Hail, M.; Lewis, S.; Jardinc, I.; Liu, J.; Novotny, M. J . Microcolumn Sep. 1990, 2, 285-292.
1056 Analytical Chemistty, Vol. 66, No. 7, April 1, 1994
When multiple ions from the same species are present in an electrospray mass spectrum, the charge state of the ions is determined by solving a simple set of simultaneous equations.12 For small peptides,however, theESI mass spectra areoften dominated by a single charge state. Tryptic peptides are usually assumed to be doubly charged as a result of the two basic sites, but in a significant proportion of cases, a singly or triply charged ion (occasionally even a +4 or higher ion) may be the only ion present. This can lead to confusion of mass assignment, particularly when dealing with peptides of unknown structure. High-resolution mass spectrometry has been suggested as a solutionto this problem,13since the spacing of the isotope peaks associated with an ion will be determined by the ion's charge state. That is, in a singly charged ion,the isotope peaks are 1 Da apart, in a doubly charged ion, they are 0.5 Da apart, etc. Unfortunately, the resolution required to measure this spacing for multiply charged ions is beyond the capabilities of most quadrupole mass analyzers used in ESI-MS. Double-focusing sector or Fourier transform mass spectrometers offer higher resolving power, but LC/ESI-MS on these machines remains problematic. The presence of sodium ions as a ubiquitous contaminant in mass spectrometric samples is generally considered a nuisance. At low levels it produces cationized analyte ions, distributing the ion current over several m/z instead of just one, degrading the sensitivityof the analysis. At higher levels, the sodium ion can compete so effectively with the analyte for charge that complete suppression of the analyte signal can result. The presence of a small amount of sodium does, however, aid in the assignment of charge state. The spacing of the sodiated species,like that of the natural isotope peaks, depends on the charge. Singly charged ions will show a sodium adduct 22 Da higher than the MH+, doubly charged ions at 11 Da higher than (M + 2H)*+, etc. Senko et al.I4 have recently recommended using the spacing of copper ion adducts to facilitate mass assignment in mixtures for MS/MS. We here report that the deliberate doping of a sodium salt into the mobile phase during an LC/MS experiment provides (12) Mann, M.; Meng, C.K.; Fenn, J. B. Anal. Chem. 1989,61, 1702-1708. (1 3) Meng, C. K.; McEwen, C. N.; Lanen, B. S. Rapid Commun. MassSpectrom. 1990, 4, 147-150. (14) Scnko, M.W.; Bcu, S. C.; McLaffcrty, F. W. J. Am. Soc. Mass Spectrom. 1993,4, 828-830. QQQ3-27QQI94lQ36&1Q56~04.50/0
0 1994 American Chemical Society
A.
C.
674.4
60 pM
'"1
600 pM l0Ol
1
+' 685.2
Na +2Na 696.2 +3Na
685.8
707.6 0 650 660 670 680 690 700 710 720 730 740 750
1.2 mM
120 pM
0)
.-c> m $
650 660 670 680 690 700 710 720 730 740 750
m/z
25-
+5 Na +3 Na 707.8 +4 Na 729.6 718.6
0 , I 650 660 670 680 690 700 710 720 730 740 750
m/z
Flguro 1. Electrospraymass spectra of substance P (RPKPQQFFOLMNH2, Y = 1347.7) in the presence of varying levels of added sodlumacetate: (A) 60 pM, (6) 120 pM, (C) 600 pM, and (D) 1.2 mM. The promlnent ion at mlz 674 Is doubly charged.
EXPERIMENTAL SECTION Acetonitrile and TFA for LC mobile phases were of HPLC grade and were used without further purification. Substance P was purchased from Sigma. The synthetic oligonucleotide deoxy(GTG-AAG-ATG-TAC) was prepared at Glaxo. Samples of substance P were prepared at 6 pmol/pL in 50% aqueous methanol containing the level of sodium acetate indicated in the legend of Figure 1. These solutions were infused into the mass spectrometer at a rate of 2 pL/min using a Harvard Model 22 syringe pump (South Natick, MA). The oligonucleotidewas dissolved in 5 mM ammonium acetate, pH 8.5/acetonitrile (1:l) and infused as above. LC/MS was conductedusing an AB1 140A pump to provide gradients at 200 pL/min. The solvent stream was split before the Rheodyne 8 125 injector to deliver a flow of 5 pL/min to an LC Packings Hypercarb capillary column (320 pm i.d. X
25 cm). Gradients were typically 10% B to 70% B in 30 min (2%/min), where solvent A was 0.05% aqueous TFA and solvent B was water/acetonitrile/TFA (10:90:0.05). The column effluent was directed through a Linear UVIS 200 detector set to monitor absorbance at 21 5 nm and, from there, into the mass spectrometer. The mass spectrometer was a Sciex API-111. Operating conditions were as follows: ion spray voltage, 5400 V; orifice potential, 80 V; mass range, m/z 20&1500; step size, 0.2 Da for infusion experiments, 0.5 Da for LC/MS; dwell time, 2 ms for infusion, 1 ms for LC/MS. Peptide spectra were obtained in the positive ion mode, oligonucleotide spectra in the negative ion mode, with corresponding changes in the polarities of the applied voltages. Several scans were usually averaged to improve signal-to-noise ratio. Tissue inhibitor of metalloprotease (TIMP) was recombinant material prepared at Glaxo as reported elsewhere.l6 Reduction and alkylation of cysteines were conducted using standard procedures: 1 nmol of TIMP was dissolved in 100 p L of buffer (6 M guanidine hydrochloride, 1 M TRIS-HC1, 2 mM EDTA,pH 8.5) and incubated under nitrogen for 20 min. A 50-fold molar excess of DTT (over Cys) was added and the solution was maintained under nitrogen at 37 OC for 60 min more. The solution was cooled to 0 OC for 10 min, and a 55-fold molar excess of iodoacetic acid (over Cys) was
(15) Niessen, W. M. A.; van der H w e n , R. A. M.; Tmke, A. P.; van der Grtef, 3. Proceedings of the 40th Annual Conference of the American Society for Mass Spectrometry, Washington,D.C., May 31-June 5,1992; pp 1260-1261.
(16) Overton, L. K.; Patel, I.; Bccherer, J. D.; Chandra, G.;Gray, J. G.; Kost, T. A. In Baeulwirus Expression Protocols; Walker, J. D.,Richardson, C., Eds.; Humana Prms: Totowa, NJ, in press.
a simple way to eliminate confusion about charge state and has no obvious effect on chromatographic resolution or mass spectrometer operation. The addition of sodium acetate to the LC mobile phase in anion exchange chromatography/ MS of oligosaccharides has been reported,15but its use was primarily to facilitate the formation of (M + Na)+ ions in thermospray ionization MS.
Ana&tIcaIChemktty, Vol. 66, No. 7, April 1, 1994
1057
added. The solution was incubated another 60 min under nitrogen, desalted, and purified by reversed-phase HPLC.
A.
677.6*
792.5
RESULTS AND DISCUSSION The facile cationization of analytes by sodium in ESI-MS is well-known to any practicing spectrometrist. The observed ions are not simply multiple additions of Na+, which would result in a series of ions described by
mlz = [M + n N a * l / n
+ "a) + nH+l/n
1
(1)
where n = 1, 2, 3, .., but rather represent either successive replacement of protons with Na+ and then multiple proton addition m/z = [(M - mH
lI lI
L"
0
0
500 600 700 800 900 1000 1100 1200 1300 1400
8.
-R77. . .A
'"1
(2)
m/z
~
617.2 m
or addition of some combination of protons and sodium ions: m / z = [(M
+ "a+)
+ (n- m)H+l/n
(3)
where m = 0, 1, 2, 3, .., and n = 1 , 2, 3, .... The distinction between the latter two cases may seem trivial, since they are computationally indistinguishable, but for cases where the number of sodium ions exceeds the charge state (m > n; for example, in Figure IC,D), only some form of sodium replacement (eq 2) is possible. (The notation above is intended to represent a formalization of the process and does not imply any real-time sequence of events). The presence of high levels of sodium can totally suppress the analyte signal, so proteins and, particularly, oligonucleotides must often be desalted before analy~is.1~ In reversephase LC, most salts are eluted at the void volume of the column, and sodium contamination poses less of an annoyance in LC/MS than in infusion ESI or FAB-MS. Unfortunately, the information conveyed by the sodium-replaced analogues of the analyte is also lost in LC/MS. With our current understanding of ESI-MS, the most important information to be lost relates to the charge state of the ion of interest. Figure 1 shows a part of the ESI mass spectra of substance P, a readily available and well-behaved model peptide, infused into the mass spectrometer as a solution containing varying amounts of added sodium acetate. The spectra are dominated by a doubly charged ion at m / z 674. Even with no added Na, there is a small ion at m / z 685, representing a sodium-replaced form of the peptide. The spacing of 11 Da confirms that the ion is doubly charged. As the added sodium concentration is increased to 60 pM, 120pM, 600 pM, and 1.2mM, the sodium replacements increase in abundance and additional replacements of two, three, four, and five protons with sodium ions appear. Above -2 mM added Na+, the peptide signal completely disappears, as the peptide can no longer compete for charge with the electrolyte. Although the charge-state information is useful, one must be concerned that too much sodium might distribute the ion current over too many ions, resulting in a loss of sensitivity. (17) Stults, J. T.;Marstcrs, J. C . Rapid Commun. MassSpecrrom. 1991.3, 359-
363.
fQ50 Analytical Chemistry, Vol. 88, No. 7, April 1, 1994
I
I
=-I 1
9
50
0 500
600
700
800
900 1000 1100 1200 1000 1100 1200
m/z Figure 2. Eiectrospray mass spectra of a portion of the LC/MS analysls of a tryptlc dlgest of TIMP (A) without sodium and (6)with 250 pM sodium acetate added to the LC mbUe phase. Peaks marked with asterisksare discussed inthetext. The spaclng of the sodlumreplaced species in (6)identifiesthe charge state of the ions. Peptldesequences are QFQALGDAADIR (y= 1233.4) and FVGWEVNQlTLYQR (y = 1753.0).
The sensitivity of the signal in our experiment remains relatively constant up to - 1 mM Na+ and then began to decrease. At 1.2 mM Na+, there is a loss of -30% of the signal, in part due to the larger number of sodiated species over which the charge was distributed. We selected 250 pM as an acceptable compromise: the sodium-replacedforms were readily discernible, yet not so large as to interfere. We then prepared our standard LC solvents, but added 250 pM NaOAc to both solvents A and B. The separation of a tryptic digest of the tissue inhibitor of metalloprotease (TIMP) was used to evaluate the LC/MS performance of the sodium-containing mobile phase. Our original concern in adding the salt to the mobile phase was less with the sodium than with the acetate counterion, since the peptides normally ion pair with trifluoroacetate, and the presence of a second counterion might alter the ion-pairing behavior. However, the TFA was present at 0.05% (-4.4 mM) and the NaOAc at 250 pM, so no adverse effect in the separation of the digest was observed (data not shown). Figure 2A shows a mass spectrum obtained during the on-line LC/MS analysis of a tryptic digest of TIMP. Signals fromseveralpeptidesareobservedat m/z618,792,877,1081, and 1188, indicating the coelution of multiple tryptic peptides. The signals at m/z 792 and 1 188 can be related as the doubly and triply charged ions of the same peptide, but the charge state of the remaining ions is unclear. Upon addition of sodium
734.6 594.2
552
+3
1101.6
+2
1112.6
I
741.8 0 600
w 500
660
7b0
8bO
960
Id00
1lbO
1200
mlz
593.8
c
m
500
II
600
1889.6 760
8bO
960
800
900
1000
1100
1200
1300
mlz Figure 4. Electrospray mass spectrum from the LC/MS analysls of a tryptic digest of TIMP. Pep= 2201 (LQSGTHCLWlDQlLa(l SEK). The protonated trlply charged lon (mlz 735) la more abundant than the doubly charged ion (mlz 1102), but the triply chargedsodium replaced ion (mlz 742) is less abundant than its doubly charged counterpart (mlr 1113).
'.2
I1
700
Id00 11'00 1200
mlz Flgure 3. Eiectrospray mass spectra of another portion of the LC/MS analysis of a tryptic digest of TIMP (A) without sodium and (B) with 250 pM sodium acetate added to the LC mobile phase. Ions at mlz 552 and 594 are singly charged, as demonstrated by the spacing of 22 De betweenthepratonatedandsodiatedionsin(B).Peptidegwereldentifled as YEIK (y = 551.6) and QFQSR (Ad, = 593.6).
acetate to the LC mobile phase, a corresponding portion of thechromatogram yielded thespectrumin Figure 2B. Because of differences in preparing the mobile phases, the peptides in Figure 2B were chromatographicallymore completelyresolved, so the spectrum is somewhat simpler; but the signals at m / z 617 and 877 remain, now with sodium-replaced forms at m / z 628 and 888. The spacing of 1 1 Da in each case confirms that the ions are doubly charged and allows assignment of the molecular masses of the two peptides as 1234 and 1753. Because the protein sequence of TIMP is known, the masses of the tryptic peptides can be predicted, and the two masses do correspond to expected tryptic peptides. Figure 3A shows another spectrum from the same digest. In this case, two prominent ion signals are observed at m / z 552 and 594. The lack of signals from differently charged members of either ion series leads to some uncertainty about charge state. When Na+ is added to the mobile phase, the ambiguity is resolved by the appearance of sodium-replaced forms at m / z 574 and 616. The spacing of 22 Da between the original ions and their sodium-replaced analogues demonstrates that the ions are singly charged. Again, tryptic peptides of these masses are predicted from TIMP. The observed level of sodium replacement in the LC/MS analysis of the TIMP peptides was somewhat greater than expected based on the data from substance P. Upon examination of the sequence of substance P, we realized that
the peptide has no acidic functionality. The C-terminus is amidated, and there are no acidic residues in the sequence. The unfortunate choice of substance P as a model probably caused us to overestimate the amount of sodium necessary, but also demonstrates that sodium replacement will occur even for protons that are not particularly acidic. Examination of mass spectra where multiple charge states of the same peptide occur revealed another interesting observation. Figure 4 shows the mass spectrum of another TIMP tryptic peptide; in this case both the doubly charged ( m / z 1102) and triply charged ( m / z 735) ions are observed, along with their corresponding sodium-replaced counterparts at m / z 1 1 13 and 742. The spacing of 1 1 and 7 Da confirms the charge states, should any confirmation be necessary. However, the relative abundance of the sodium-replaced ion for the triply charged species is significantly lower than the relative abundance of the corresponding doubly charged ion. If charge-state distributions can be modeled as Gaussian curves,l* this implies that the Gaussian distribution of the fully protonated species maximizes at a slightlydifferent charge state than that of the sodium-replaced form. This trend is observed in virtually all peptides we have examined, with one important exception, discussed below. We have also noted this trend in larger molecular mass proteins and in oligonucleotides. Figure 5 is the negative ion ESI mass spectrum of an oligonucleotide, deoxy(GTG-AAG-ATG-TAC). The sample as received was contaminated with sodium, so the sodium concentration is unknown, but the spectrum shows a large number of sodium-replaced ions. As with the peptides, the spacing of the sodium-replaced ions reveals their charge state, although that information is readily calculatedby other means. What is interesting about this spectrum is the charge-state distributions of the protonated and variously sodium-replaced species. Figure 6 shows the spectral data replotted as abundancevs chargeand curve fit to a Gaussian distribution.18 It is clear that there is a trend, as more protons are replaced by sodium ions, toward a distributionfavoringthe lower charge states. (18) Anderegg, R. J.; King, G. Rocdings of the 40th Annual Conference of the American Society for Mass Spectrometry, Washington,D.C., May 31-June 5, 1992; pp 623624.
Ana&tIcalChem~try, Vol. 66, No. 7, Aprll 1, 1994
IOSQ
100
1
1001
-51
I.5 W
W
.-> i;j $
I
25
0
500 600 700 800 900 1000 1100 1200 1300 1400
m/z Flgurr 5. Negative ion electrospray mass spectrum of a synthetic oligonucleotide, deoxy(QTQ-AAG-ATGTAC), showlng multlpiesodiumreplacedspecles. The calculated& = 3709.5 basedonthe sequence. The measured value, using only the first ion Ineach charge-state cluster, is 3708.4 1.2.
-
/7
0
I
900
1000
1100
1200
L
1300
. 1400
1500
m/z Figure 7. Electrospray mass spectrum from the LC/MS analysis of a tryptic digestof TIMP. The glycopeptiderepresentedby thls spectrum has 4 = 2791 and is -35% carbohydrate. The carbohydrate sequence has not been reported, but our data suggest It is a simple high-mannose structure with no sialic acid.*I
of the sodium-replaced species now seems to maximize at the +3 state, while that of the protonated form maximizes at the +2 state. This is exactly the opposite of the trend observed for other peptides. It remains to be determined whether this behavior is observed for other glycosylated peptides. One possible explanation for this seemingly anomalous behavior might be that sodium is playing more than one role in the glycopeptide. The polyhydroxy1 functionality of the sugars is known to cationize readily, and perhaps we are seeing a combination of sodium replacement and sodium addition. There is no difference in mass between a triply (H+) charged ion with two sodium replacements and an ion with one sodium replacement, charged with two protons and one sodium ion. Oe+O I 0 2 4 6 8 10 A final interesting feature of the spectrum in Figure 7 is Charge the relatively high abundance of the sodium-replaced material as compared to the protonated forms. This spectrum resulted Flgurr 8. Charge-state distrlbutlons of the ions from Figure 5 and from the same LC/MS conditions that produced the spectra corresponding Gaussian curves1* for the protonated (0)and d~ ( 0 ) and tetra (A)sodlum-replaced species. The center of the distribution in Figures 2-4, yet the amount of sodium replacement appears moves to lower charge state (higher mlz) as the level of sodlation to be substantially greater. This is probably a result of the increases. numerous hydroxyl functionalities present in the sugar portion of the glycopeptide, allowing many more potential sites for This effect has been noted before in negative ion ESI mass sodium replacement or addition. As above, at this time, spectralgand was attributed to the presence of a common set generalizations must be made with caution. of sites for both deprotonation and H/Na substitution. It is In summary, the addition of low levels of a sodium salt to difficult to makea similar argument for our positive ion results. the mobile phase during an LC/ESI-MS experiment encourThere is no reason to suspect that the sites that would readily ages the formation of sodium-replaced ions in addition to the protonate should also be the sites of likely H/Na substitution. normally observed protonated species. The spacing of these Our results suggest that the effect may result from an sodiated species allows unambiguous determination of the ionization-based or an instrumental-based bias toward lower charge state of the ions and, hence, their actual mass. Such charging of sodium-replaced ions. information is very useful when unknown or modified peptides The important exception to this trend occurred in the mass are analyzed. At the levelsreported here (250 pM), the sodium spectrum of a glycopeptide. TIMP is a glycoprotein, with salt seems to cause no deterioration in chromatographic two N-linked glycosylation sites.20 The mass spectrum of a performance and does not unduly foul the mass spectrometer. tryptic peptide encompassing one of the glycosylation sites is We have used this technique with infusion samples as well. shown in Figure 7. Once again, the doubly and triply charged Occasionally a sample is analyzed for which a signals are ions are apparent, along with their sodium-replaced analogues. observed at the expected molecular mass and at approximately However, unlike all the cases discussed above, the distribution twice the expected molecular mass. In these cases, there can be ambiguity about whether the signals represent a real (19)Loo,J.A.;OgorzalekLoo,R.R.;Light,K. J.;Edmonds,C.G.;Smith,R.D. covalent dimer or just a nondissociated cluster of the molecule. Anal. Chem. 1992,64, 81-88. (20) Docherty, A. J. P.; Lyons, A.; Smith, B. J.; Wright, E. M.; Stephens, P. E.; The predicted m / z values in both cases would be identical, Harris, T. J. R.; Murphy, G.; Reynolds, J. J. Nature 1985, 318, 66-69. (21) Ncubauer, G.; Anderegg, R. J., unpublished results. but the charge state would be different. In one case, the signals p5.7
4e+51
I
1080
I
I
I
Analyrical Chemistry, Vol. 66,No. 7, April 1, 1994
+
+
would represent (M H)+ and (2M H)+, and in the other case, they would represent (Mz 2H)2+ and (M2 H)+, where M is the expected molecular mass. Addition of sodium to the solution produces sodium-replaced species, and the charge state of both ions is readily determined. For example, if a molecule had a molecular mass of 500, the protonated and sodium-replaced monomer would be observed at m / z 501 and 523, respectively, while the protonated and sodium-replaced (noncovalent) dimer would beobservedat m / z 1001 and 1023, respectively. If there was in fact a covalent dimer (mass lOOO), the singly charged species would be observed at m / z 1001 and 1023, as before, but the doubly charged ions would occur at m / z 501 and 512 (not m / z 523). Although not specifically studied here, knowledge of the charge state of ions would also be useful for conducting LC/ MS-MS experiments, because one could know in advance the
+
+
likely mass range over which to scan for product ions. For example, with a singly charged precursor ion, one need only scan to the m / z of the precursor. With a doubly charged precursor ion, singly charged product ions could occur up to twice the m / z of the precursor. ACKNOWLEDGMENT TIMP was provided by Anne Hassel of Glaxo. Helpful discussions with Dan Kassel, Larry Shampine, and Jianmei Ding are gratefully acknowledged. Received for review December 8, 1993. Accepted January 17, 1994.' ~~
a
~
Abstract published in Advance ACS Abstracts. March 1, 1994.
Analytical Chemism, Vol. 66,No. 7, April 1, 1994
1001
