Anal. Chem. 1996, 68, 542-545
Isotopic Assignment in Large-Molecule Mass
Spectra by Fragmentation of a Selected Isotopic
Peak
Peter B. O’Connor, Daniel P. Little, and Fred W. McLafferty*
Baker Chemistry Laboratory, Cornell University, Ithaca, New York 14853-1301
For large (>5 kDa) ionic species, Fourier transform ion
cyclotron resonance instruments yield by far the highest
mass accuracy. However, this can be compromised by
misassignment of the isotopic content based on predicted
natural abundances of the isotopic peaks. As an alternative method independent of natural abundance variations,
high-resolution isolation and dissociation of a single
isotopic peak yields a distribution of isotopic peak abundances characteristic of the isotopic content of the precursor peak. Accuracy is enhanced if the precursor peak is
abundant and of minimum heavy isotope content, and if
the product species is abundant and of intermediate mass.
In addition, such spectra of the highest mass products
are useful for identifying complementary product pairs,
a key step in sequencing proteins and nucleotides.
For molecules as large as 67 kDa,1 electrospray ionization
(ESI)2 Fourier transform mass spectrometry (FTMS)3 achieves4
a resolving power (RP) of >105, far superior to other methods.
Using an internal mass standard yielded an even better mass
accuracy (<1 ppm) for an apomyoglobin (17 kDa) isotopic peak.4
However, using the isotopic peak mass to calculate the relative
molecular weight (Mr) on the basis of the natural isotopic
abundances requires determination of the heavy isotope content
of the measured isotopic peak. At masses below ∼5 kDa, this is
not a serious problem, as the monoisotopic peak (no 13C atoms
or less abundant isotopes of other elements) is of sufficient relative
abundance to show clearly the absence of an isotopic peak 1 Da
lower. At higher masses, the primary method is to fit the observed
isotopic peak abundances to those expected from an “average”
(1) Speir, J. P.; Senko, M. W.; Little, D. P.; Loo, J. A.; McLafferty, F. W. J. Mass
Spectrom. 1995, 30, 39-42.
(2) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Mass
Spectrom. Rev. 1990, 9, 37-70. Smith, R. D.; Loo, J. A.; Loo, R. R. O.;
Busman, M.; Udseth, H. R. Mass Spectrom. Rev. 1991, 10, 359-451.
(3) Comisarow, M. B.; Marshall, A. G. Chem. Phys. Lett. 1974, 25, 282-283.
Marshall, A. G.; Grosshans, P. B. Anal. Chem. 1991, 63, A215-A229.
Wilkins, C. L.; Chowdhury, A. K.; Nuwaysir, L. M.; Coates, M. L. Mass
Spectrom. Rev. 1989, 8, 67-92.
(4) Beu, S. C.; Senko, M. W.; Quinn, J. P.; McLafferty, F. W. J. Am. Soc. Mass
Spectrom. 1993, 4, 190-192. McLafferty, F. W. Acc. Chem. Res. 1994, 27,
379-386.
(5) Senko, M. W.; Beu, S. C.; McLafferty, F. W. J. Am. Soc. Mass Spectrom.
1995, 6, 229-233. By convention,4 the isotopic assignment of a peak is
designated here as “13C content”, as the main heavy isotope is 13C (13C/12C
) 1.1%); however, these isotopic peaks also include unresolved contributions
from 2H/1H ) 0.015%, 15N/14N ) 0.37%, 18O/16O ) 0.20%, 34S/32S ) 4.4%,
etc.
542 Analytical Chemistry, Vol. 68, No. 3, February 1, 1996
Figure 1. SWIFT isolation of the 13C5 isotopic peak from the ESI
molecular ions of ubiquitin. Rectangles, expected abundance values
of the isotopic peaks calculated according to ref 5; arrow, calculated
Mr value for naturally occurring isotopic abundances.
molecule (in the present case, a protein) of that mass.5 For an
unknown, the abundance ratios will vary not only with the actual
elemental composition but also with variation in natural isotopic
abundances; for carbonic anhydrase molecular ions, the mass of
the most abundant isotopic peak increases from 29 024.7 to
29 025.7 Da on increasing the average atomic weight of carbon
from 12.0109 to 12.0111, values well within the natural isotopic
variation. Even for a molecule as small as ubiquitin (8.6 kDa),
fitting abundance measurements of lower accuracy to those
expected (Figure 1, rectangles) could obviously lead to a 1 Da
misassignment, an error far worse (∼1/104) than the resolution
achieved.6 Here, an alternative method for determining the
isotopic content of a peak from the abundances of its dissociation
(MS/MS) products, first suggested by Beynon and independent
of the sample’s isotopic content,7 is investigated.
Illustrating this method, precursor ions containing a single,
randomly distributed 13C atom, when cleaved in half, will yield
equal abundances of products with and without the 13C atom; if
instead this were a 13C2 precursor, three products in a 1:2:1 ratio
would result (Table 1, 50%m, 13Cprc ) 2; only relative, not absolute
abundances are necessary, as isotopic effects should be negligible). Other product ions differing in mass by a few daltons could
(6) A 1 Da sequence correction in apomyoglobin (Zaia, J.; Annan, R. S.; Biemann,
K. Rapid Commun. Mass Spectrom. 1993, 6, 32-36) showed a corresponding error in its original Mr measurement by ESI/FTMS (Henry, K. D.; Quinn,
J. P.; McLafferty, F. W. J. Am. Chem. Soc. 1991, 113, 5447-5449).
(7) Bozorgzadeh, M. H.; Morgan, R. P.; Beynon, J. H. Analyst 1978, 103, 613622. Todd, P. J.; Barbalas, M. P.; McLafferty, F. W. Org. Mass Spectrom.
1982, 17, 78-80.
0003-2700/96/0368-0542$12.00/0
© 1996 American Chemical Society
obviously interfere by producing overlapping isotopic peaks;
fortunately, dual products with and without H loss/rearrangement
appear to be rare for fragmentations of protein1 and nucleotide8
multiply charged ions. Application of this method to large
molecules is made possible experimentally by the recently
reported capability9 for SWIFT10 isolation of a single isotopic peak
in the FTMS using a capacitively coupled11 open trapped ion cell
that eliminates axial components to the radio frequency excitation
field.11,12
EXPERIMENTAL SECTION
The external injection ESI/FTMS instrument with a capacitively coupled cylindrical trapped ion cell11 was described previously.9,13 The ESI source yielded 240 pA of cell current with 4 (
2 eV kinetic energy from 20 µM bovine ubiquitin in 75:20:5
MeOH/H2O/HCOOH. Trapping for 1 s with 4 and 5 V on the
trapping electrodes and a N2 gas pulse peaking at 3 × 10-6 Torr
yielded an average signal/noise of 500:1. Excitation for detection
used a 600-1800 m/z bandwidth (50-150 kHz), a sweep rate of
240 Hz/µs, and an attenuation of 10 dB, corresponding to an
excitation amplitude of 124 Vpp. The cell and trapping electrodes
were coupled with 120 pF SMD capacitors and 10 MΩ inductiveless carbon resistors wrapped with a 1/16 in. copper strip bolted
to the cell as a heat sink. Detection in direct mode used a 300
kHz data acquisition rate and a 175 kHz (3 dB) input filter. An
18 dB attenuator was necessary to prevent clipping of the detected
signal by the ADC.
After trapping, a 30 s delay allowed decay of axial kinetic
energy (and hence frequency modulation) from the ions. Isolation
employed a low-resolution, direct mode broad-band SWIFT10
waveform (43 ms, 1.3 µs dwell time, 35 Vpp) to isolate the isotopic
peaks of a single charge state, followed by a high-resolution
heterodyne waveform (requested resolving power 105, 2.1 s, 8 µs
dwell time, 118 kHz reference frequency, 1-2 Vpp) for isolation
of a single isotope. The heterodyne offset frequency of the highresolution SWIFT allowed easy shifting of the isolation window
to select the desired isotopic peak. Fragmentation was effected
by sustained off-resonance irradiation (SORI),14 with a ∼10 Vpp, 2
s, -1800 Hz off-resonance waveform during a 1 × 10-6 Torr
pressure pulse. Peak heights were used to measure relative ion
abundances.
A binomial expansion was used to compute the theoretical
abundance distributions for the fragment ion isotopic peaks
resulting from dissociation of the ions of a single isotopic peak
containing n 13C atoms (13Cn) that are randomly distributed in the
collection of ions dissociated; examples are given in Table 1. For
the selected peaks, ions in which one or more 13C atoms are
replaced by unit-mass-higher isotopes of other elements (randomly
(8) Little, D. L.; Chorush, R. A.; Speir, J. P.; Senko, M. W.; Kelleher, N. L. J.
Am. Chem. Soc. 1994, 116, 4893-4897.
(9) O’Connor, P. B.; McLafferty, F. W. J. Am. Soc. Mass Spectrom. 1995, 6,
533-535.
(10) Stored Waveform Inverse Fourier Transform: Marshall, A. G.; Wang, T.-C.
L.; Ricca, T. L. J. Am. Chem. Soc. 1985, 107, 7893-7897. Chen, L.; Wang,
T. C. L.; Ricca, T. L.; Marshall, A. G. Anal. Chem. 1987, 59, 449-454.
(11) Beu, S. C.; Laude, D. A., Jr. Anal. Chem. 1992, 64, 177-180.
(12) Caravatti, P.; Allemann, M. Org. Mass Spectrom. 1991, 26, 514-518.
(13) Beu, S. C.; Senko, M. W.; Quinn, J. P.; Wampler, F. M., III; McLafferty, F.
W. J. Am. Soc. Mass Spectrom. 1993, 4, 557-565.
(14) Gauthier, J. W.; Trautman, T. R.; Jacobson, D. B. Anal. Chim. Acta 1991,
246, 211-225. Senko, M. W.; Speir, J. P.; McLafferty, F. W. Anal. Chem.
1994, 66, 2801-2808. Huang, Y.; Pasa-Tolic, L.; Guan, S.; Marshall, A. G.
Anal. Chem. 1994, 66, 4385-4389.
Table 1. Product Isotopic Composition (13Cpdt) versus
Fractional Mass (%m) and Precursor 13C Content
(13Cprc)a
13C
13C
%m
10
20
30
1
2
3
4
5
2
3
4
5
6
11
12
17
18
39
40
100
100
100
100
100
82
75
53
50
8
7
22
33
44
56
67
100
100
100
100
35
32
1
4
7
12
19
56
61
89
94
73
69
1
1
3
19
22
49
56
100
97
4
6
19
23
100
100
1
1
6
7
78
80
2
3
4
5
6
11
12
17
18
+13C1b
+13C1
100
100
100
80
67
29
24
40
35
50
75
100
100
100
80
73
80
75
6
19
38
50
63
100
100
100
100
2
6
12
21
75
83
88
94
2
4
38
47
57
66
13
19
28
36
+13C1
+13C1
+13C3
+13C3
100
78
58
47
36
36
30
60
52
86
100
100
100
93
78
70
89
83
18
43
64
86
100
100
100
100
100
6
18
37
57
86
96
86
93
2
8
18
51
66
58
68
1
3
22
33
31
40
+13C2
+13C2
+13C4
+13C4
75
50
38
23
15
38
28
41
32
100
100
100
75
60
75
63
72
61
33
67
100
100
100
100
94
95
88
15
44
67
89
93
100
100
100
7
22
44
62
78
83
92
3
12
30
44
56
68
+13C3
+13C3
+13C6
+13C6
+13C17
+13C18
50
33
17
10
5
36
24
51
38
74
64
100
100
67
50
30
71
54
80
65
91
82
50
100
100
100
75
100
86
100
90
100
95
33
67
100
100
100
100
100
100
100
100
17
50
75
71
86
80
90
90
95
10
30
36
54
51
65
74
82
2
3
4
5
6
11
12
17
18
40
2
3
4
5
6
11
12
17
18
50
2
3
4
5
6
11
12
17
18
39
40
60-90
pdt
0
prc
inverse of 40-10%m
distributed), such as 2H, 15N, and 17O, will give the same product
abundances. The probability, p, of a given 13C residing in a given
fragment is assumed to be the ratio of the fragment’s mass to the
mass of the precursor.7 The ratio of the number of carbons in
the fragment to those in the precursor, although more accurate,
would not be suitable for unknowns. For the average variation
of protein composition, this approximation gives an error of <2%.
RESULTS AND DISCUSSION
Selection of Individual Isotopic Peaks. As previously
described,9 the capacitively coupled cell allows selective SWIFT
excitation to remove all ions except a single isotopic peak for large
molecules such as ubiquitin (8565 Da, Figure 1) and carbonic
Analytical Chemistry, Vol. 68, No. 3, February 1, 1996
543
Figure 2. (a) Partial ESI mass spectrum of the 50-mer oligonucleotide T50. (b,c) Ions remaining after SWIFT excitations applied in the
dashed box regions of the frequency domain spectrum.
anhydrase (29 kDa). An obvious application of this capability is
for MS/MS of individual molecular ions in mixtures, or of
individual fragment ions from the ionic mixtures formed by
dissociation of large, multiply charged molecular ions. For
example (Figure 2, top spectrum), the ESI/FTMS mass spectrum
from nozzle-skimmer dissociation15,16 of the synthetic oligonucleotide T50 (15.1 kDa) shows (M - 23H + 3Na)20- molecular ions,
m/z ∼759, overlapping the main isotopic peaks of the fragment
ion (T5 - H2O)2- at m/z 759.05 and 759.55. High-resolution
SWIFT excitation (dashed lines, Figure 2, middle spectrum)
isolates the latter two ions out of the mixture, retaining roughly
20% of their abundance. However, the removal of high-abundance
peaks near those of low abundance can be more difficult. In the
reverse attempt to isolate the 20- ion distribution, the two 2isotopic peaks were only attenuated to ∼25% of their original
abundance relative to that of the 20- ions, and the adjacent 20isotopic peak at m/z 758.99 was ejected. SORI collisional activation14 was found to be more efficient than infrared multiphoton
dissociation (IRMPD)17 following high-resolution SWIFT.
MS/MS Assignment of Isotopic Composition. The conventional method5 finds the best match of the isotopic peak
abundances predicted (Figure 1, rectangles) from natural isotopic
abundances versus the experimental values. For ions as large as
ubiquitin, the isotopic peak compositions will be clearly assignable
only if sufficiently high abundance accuracy has been achieved.
For Beynon’s alternative method described here,7 the third and
fifth isotopic peaks (designated 13C3 and 13C5)5 were isolated by
SWIFT;10 with our present methodology,9 this reduces the signal
by a factor of 4. Fragmentation of each of these peaks yielded
the spectra of Figure 3 for the products y18, b52, y58, and y24, %m
(Table 1) ) 24.5, 68.2, 76.3, and 31.8, respectively. The experimental isotopic distributions for b52, y58, and y24 agree well with
those calculated (O; Table 1 gives predicted abundances versus
the 13C content of the precursor and the fraction of its mass (%m)
in the product ion). These values are clearly distinguishable from
those calculated for formation from precursor ions with one 13C
atom less or more (4 or 0, shown for best fit). The y18 data,
which are of much lower signal/noise, agree within their experi(15) Loo, J. A.; Edmonds, C. G.; Smith, R. D. Anal. Chem. 1991, 63, 24882499.
(16) O’Connor, P. B.; Speir, J. P.; Senko, M. W.; Little, D. P.; McLafferty, F. W.
J. Mass Spectrom. 1995, 30, 88-93. Aaseruud, D. J.; Little, D. P.; O’Connor,
P. B.; McLafferty, F. W. Rapid Commun. Mass Spectrom. 1995, 9, 871876.
(17) Little, D. P.; Speir, J. P.; Senko, M. W.; O’Connor, P. B.; McLafferty, F. W.
Anal. Chem. 1994, 66, 2809-2815.
544
Analytical Chemistry, Vol. 68, No. 3, February 1, 1996
mental uncertainty with both 13C4 and 13C5 for the precursor. Also,
note that the y24 and b52 fragments are complementary products
of the 76-residue protein (24 + 52 ) 76) and thus have inverted
isotopic patterns (same abundances but in reverse mass order)
from those expected; also, these data are nearly inverted for the
y18/y58 pair, whose mass sum is <1% greater than the (M +
10H)10+ mass. The data in Table 1 are given only for m ) 10%50%, as these represent the inverse of the 90%-50% values.
For the optimum application of this method, a first criterion is
a high relative abundance for both the precursor isotopic peak
(Figure 1: 13C1, 19%; 13C2, 46%; 13C3, 76%; 13C5, 100%) and the
fragment ion chosen; the summed product abundances are b52,
81%; y58, 100%; y24, 61%; y18, 33%. A second criterion is minimization
of the precursor 13Cn content. The relative differences in abundance for the correct and incorrect 13Cn(1 assignments are
obviously larger for the spectra of the 13C3 precursor than those
of the 13C5 (Figure 3 and Table 1), more than offsetting the lower
13C (76%) precursor abundance. Third, Table 1 shows that the
3
relative abundance differences are also less favorable if the
fractional mass of the product ion is quite low (∼10%) or quite
high (∼90%, complementary data of 10%). For example, 13C2 (and
13C ) precursors yielding 10%, 20%, and 50% fractional mass
3
products should give 13C0-4 product ratios of 100:22:1.2 (100:33:
4:0), 100:50:6.3 (100:75:19:1.6), and 50:100:50 (33:100:100:33), with
a signal/noise of 5:1, the 10% product data for 13C2 and 13C3 would
be indistinguishable, but the 50% product data would provide a
clear assignment (this is an additional reason for the uncertain
assignment from the y18 data of Figure 3). Note that even the
abundance differences for the 20% products are larger than those
caused by a 1 Da shift in the calculated isotopic abundances of
the precursor (Figure 1, rectangles).
Nonrandom Isotopic Distributions. This method is based
on the assumption that the higher mass isotopes (13C, 2H, 15N,
18O, etc.) are randomly distributed. The sulfur isotopes (32-34S,
100:0.8:4.4) are an exception for proteins, as their location in
product ions is determined by the location of the Cys and Met
amino acids in the protein. As an example, if ubiquitin was
modified to contain these residues at positions 1, 2, and 3 (and
thus only in the b52 product), the dissociation of the 13C3 and 13C5
precursors including 33S and 34S would cause a maximum change
of 2.1% in the b52 abundances of Figure 3; this would not affect
the precursor isotopic assignment from these data.
Complementary Fragment Ions. As an alternative, the
isotopic content of the precursor can be assigned by summing
the values of a complementary pair.4,16 Considering such a pair,
the measured masses of y24 (13C1), 2726.50, and b52 (13C4), 5838.13,
yield the sum 8564.63; thus, the precursor peak measured as
8564.61 Da should represent 13C5 (the value expected from the
ubiquitin structure is 8564.64). For this, the current5 isotopic
composition assignment of the larger fragment (e.g., b52 or y58)
formed from the 13Cn all-isotope precursor (Figure 3, top spectrum)
can be made more certain with the fragment ion data from the
13C and 13C peaks. Again, this can be enhanced by the choice
5
3
of the precursor isotopic peak. For y58, the 13C3 and 13C2
precursors should yield 13C0-4 product ratios of 3.0:29:93:100 and
9.7:62:100:0; although the abundance of the 13C2 precursor is only
60% of that of the 13C3 precursor, the abundance of the 13C2
monoisotopic product would be double that from 13C3.
(18) Chen, R.; Grosshans, P. B.; Limbach, P. A.; Marshall, A. G. Int. J. Mass
Spectrom. Ion Processes, in press.
Figure 3. SWIFT isolation and dissociation of the 13C3 and 13C5 (M + 10H)10+ peaks of ubiquitin (13C5, 8564.6 Da, Figure 1) to yield the
fragment ions (all charge states summed) y18 (13C1, 2097.3 Da), b52 (13C4, 5838.1 Da), y58 (13C4 , 6531.5 Da), and y24 (13C1, 2726.5 Da). In each,
the spectra shown are from dissociation (top) of the full isotopic distribution, (second), of 13C5, and (third, if present) of 13C3, with best fit of
calculated abundances for dissociation of ∆13Cn-1; O, 13Cn; and 0,13Cn+1.
Unequal Complementary Pairs. Although a proportionately
larger fragment ion from the dissociation of a 13Cn peak provides
decreasing abundance differences for its 13Cn(1 products, this does
increase greatly the abundance of the product 13Cn peak. Even
the dissociation of a 13C18 peak should produce (Table 1) a 50%
relative abundance for the 13C18 peak of the 90% product (as well
as for the 13C0 peak of the 10% product); this 13Cmax assignment is
aided by the fact that the next higher peak must be absent. Note
that the data for the b52 ion (Figure 3) show clearly the 13C5 peak
from the 13C5 precursor. With the 13C0 peak of the complementary
y24 also designated clearly, the sum of these peak masses gives
the mass of the 13C5 precursor isotopic peak. This should simplify
the identification of complementary pairs, a valuable first step in
MS/MS characterization of proteins,16 with increasingly large
precursors requiring increasingly unequal masses. However, this
does not provide the 13C content of either the precursor or the
larger fragment, although this conceivably could be sought from
the product abundances of an isotopic peak of the larger fragment
(MS/MS/MS).
Larger Molecules. Extending this method to much larger
proteins requires increasing accuracy in abundance measurements
(Table 1). Although isolation and fragmentation of the most
abundant (13C17) isotopic peak of carbonic anhydrase (29 kDa)
was successful, isotopic assignment was not possible because the
adjacent isotopic peaks of the precursor could only be reduced
to ∼30% of their original value and the MS/MS spectrum was
weak.9 Calculated isotopic distributions of the y25, y76, and b135
fragment ions16 from 13C17 and 13C18 are similar to those for the
10%, 30%, and 50% values in Table 1. Again, the differences in
fragment isotopic abundances for 13C17 and 13C18 precursors are
more favorable in the order 50% > 30% > 10%. The 13C17/13C18
differences for the 50% product are more distinctive than the
differences caused by a 1 Da shift in the (M + nH)n+ natural
isotopic abundances (13C14-13C20) of 72, 85, 100, 99.9, 95, 86, 75.
Extending this method to proteins as large as albumin (67 kDa)1
could be possible with 13C40 dissociation (Table 1). The required
improvements in experimental intensity values will be sought with
the implementation of a radially linearized cell18 and remeasurement19 to increase signal/noise.
CONCLUSIONS
Product abundances from isolation and fragmentation of a
single isotopic peak can provide an accurate assay of its isotopic
content. This avoids the 1 Da error that can result from
conventional 13C content assignment5 for >5 kDa ions that
assumes natural isotopic abundances for the sample. This
assumption is unnecessary for isotopic peak fragmentation, which
makes possible mass accuracies greater than the instrument
resolution. This is a special advantage for ESI/FTMS,4 with its
uniquely high resolution of 1/105-1/106. Increases in measurement sensitivity, SWIFT10 selectivity, and MS/MS efficiency will
be necessary for the routine application of this methodology to
>20 kDa ions.
ACKNOWLEDGMENT
The authors thank Troy D. Wood, Ziqiang Guan, and Russell
A. Chorush for experimental assistance and valuable discussions
and the National Institutes of Health (Grant GM16609) for
generous financial support.
Received for review June 15, 1995. Accepted November
7, 1995.X
AC950599Z
(19) Williams, E. R.; Henry, K. D.; McLafferty, F. W. J. Am. Chem. Soc. 1990,
112, 6157-6162. Speir, J. P.; Gorman, G. S.; Pitsenberger, C. C.; Turner, C.
A.; Wang, P. P.; Amster, I. J. Anal. Chem. 1993, 65, 1746-1752.
X
Abstract published in Advance ACS Abstracts, December 15, 1995.
Analytical Chemistry, Vol. 68, No. 3, February 1, 1996
545