[3]
tandem time‐of‐flight mass spectrometry
79
[3] Tandem Time‐of‐Flight Mass Spectrometry
By MARVIN L. VESTAL and JENNIFER M. CAMPBELL
Abstract
A new tandem time‐of‐flight (TOF–TOF) instrument has been developed by modifying a standard matrix‐assisted laser desorption ionization
(MALDI)‐TOF instrument to make high‐performance, high‐energy collision‐induced dissociation (CID) MALDI tandem mass spectrometry (MS)
a practical reality. To optimize fragment spectra quality, the selected
precursor ion is decelerated before entering a floating collision cell and
the potential difference between the source and the collision cell defines
the collision energy of the ions. Standard operating conditions for tandem
MS use a 1‐kV collision energy with single‐collision conditions and
increased laser power for ion formation. Hence, both high‐ and low‐energy
fragments are observed in MALDI TOF‐TOF spectra. On standard peptides, sensitivities down to 1 fmol are demonstrated. On a mixture of two
solution tryptic digests at the 25‐fmol level, 23 spectra were sufficient to
result in proper database identification.
Introduction
It is generally accepted that MS is essential for protein identification
and characterization. MALDI‐TOF is typically the first method employed
for protein identification, used for the determination of accurate masses of
peptides formed by enzymatic digestion, and MS‐MS in various forms is
used both as a more definitive method for identification and as the principal means for protein characterization (Eng et al., 1994; Mann and Wilm,
1994). Two‐dimensional (2D) gel electrophoresis is by far the most widely
accepted technique for high‐resolution separation of protein mixtures, and
alternatives such as micro–high‐performance liquid chromatography
(HPLC) and capillary electrophoresis have only recently been seriously
considered (Davis et al., 1995; Olivares et al., 1987). Advances in MALDI‐
TOF MS combined with advances in 2D gel electrophoresis and other
separation techniques promise to revolutionize the speed and sensitivity
of the separation, quantitation, identification, and characterization of
proteins in complex mixtures (Shevchenko et al., 1996).
Tandem MS is now established as the method of choice for characterizing proteins, although no single MS‐MS instrument or technique has
established dominance (Ducret et al., 1998; Shevchenko et al., 1997; Wilm
METHODS IN ENZYMOLOGY, VOL. 402
Copyright 2005, Elsevier Inc. All rights reserved.
0076-6879/05 $35.00
DOI: 10.1016/S0076-6879(05)02003-3
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biological mass spectrometry
[3]
et al., 1996). In these techniques, peptide mixtures are introduced into the
mass spectrometer either as a continuous flow of a liquid solution, such
as in nanospray, or as described later for MALDI‐TOF. A molecular ion of
interest is selected by the first MS, ions are caused to fragment, usually by
collision with a neutral gas, and the fragment ion masses and intensities are
measured using the second MS. Most MS‐MS applications employ triple
quadrupoles (Yost and Enke, 1978), hybrid quadrupole‐TOF systems
(Morris et al., 1996) or ion traps, either quadrupole (Louris et al., 1987)
or magnetic (as in Fourier transform–ion cyclotron resonance [FT‐ICR])
(Cody and Frieser, 1982). These techniques employ low‐energy CID in
which the ions are fragmented by a large number of relatively low‐energy
collisions. An alternative technique is high‐energy CID in which the collision energy is sufficient to cause fragmentation as the result of a single
collision, and the possible number of collisions that the ions undergo is
small (i.e., <10). Before the present work, high‐energy CID was available
only on tandem magnetic sector instruments (Sato et al., 1987) or a hybrid
of a magnetic sector with TOF (Medzihradszky et al., 1996). These instruments are complex and expensive and are not readily interfaced with
sensitive ionization techniques such as MALDI and electrospray.
Description of the Instrument
The new TOF‐TOF instrument is derived from a standard high‐
performance MALDI‐TOF instrument (Vestal et al., 1995) that has been
modified to make high‐performance, high‐energy CID MALDI‐TOF‐TOF
MS‐MS a practical reality. An earlier version of the TOF‐TOF system
was based on a very large MALDI‐TOF instrument (Vestal et al., 2000a).
The instrument described here is based on concepts similar to those
described earlier, but several modifications have been made to improve
sensitivity and to achieve better resolution and mass accuracy in a smaller
format. The ion source optics and electronics are essentially unmodified
from those employed on the commercial instrument, except that the high‐
voltage pulse circuit providing the delayed extraction pulsed voltage has
been modified to operate at higher repetition rates. The construction of
the ion mirror is similar to that used earlier except that the single‐stage
mirror has been replaced with a two‐stage mirror to provide higher resolution over a broad energy range. The additional elements required
for MS‐MS operation are installed in the field‐free region between the
ion source and the ion mirror. These include a set of deflection electrodes
for precursor selection, a deceleration lens, an electrically isolated collision
cell, a second pulsed ion accelerator, a set of deflection electrodes
for metastable suppression, and additional focusing and beam‐steering
[3]
tandem time‐of‐flight mass spectrometry
81
electrodes. An additional 250‐L/s turbo molecular pump was added to the
system to evacuate the housing surrounding the collision cell. Similar
pumps are employed on the source and mirror housing as in the original
MALDI‐TOF instrument.
A schematic diagram of the TOF‐TOF instrument is shown in Fig. 1. For
peptide mass fingerprinting or other applications of MALDI‐TOF, no voltage is applied to the elements added for MS‐MS, and the system performance in both linear and reflector modes is equal to or better than the
conventional system described earlier (Vestal et al., 1995). By applying
appropriate direct current (DC) and pulsed voltages to the added elements,
the system is converted to a tandem combination of a linear delayed extraction MALDI‐TOF system for producing and selecting a precursor ion, a
collision cell for energizing the selected ions to fragment, and a second
reflecting TOF system for focusing and detecting the fragment ions.
A field‐free space is provided between the collision cell and the second
analyzer to allow time for the ions to fragment before they are reaccelerated.
Ions produced in the source are focused in space and time at the timed‐
ion‐selector (TIS in Fig. 1). A time delay generator is programmed to open
the gate of the selector at the time the lightest mass of interest reaches the
gate, and the gate is closed when the highest mass of interest passes
through the gate. Ions outside the selected mass window are deflected away
from the collision cell. Selected ions are retarded by the deceleration lens
and enter the collision cell with the selected laboratory collision energy,
defined by the potential difference between the source and the collision
cell. The laboratory collision energy may be varied by adjusting the ion
FIG. 1. Instrumental apparatus. See text for details of operation.
82
biological mass spectrometry
[3]
source potential relative to that of the collision cell. Fragment ions produced
in the collision cell or in the adjacent field‐free region continue to travel with
essentially the same velocity as the precursor ions until they reach the
second pulsed accelerator. After the ions enter the pulsed accelerator, a
high‐voltage pulse is applied to accelerate the selected precursor and fragment ions, and the amplitude of the pulse and the voltages applied to the ion
mirror are adjusted to produce optimum resolution at the detector. With the
apparatus illustrated in Fig. 1, excellent resolution is obtained over the full
mass range of the fragment spectrum with a single set of parameters.
Fragmentation may also occur in the field‐free region between the
source and the selector or in the region between the pulsed accelerator
and the mirror. Fragments produced in the first region are rejected by the
deceleration lens, but fragments occurring in the second region may produce broad unfocused peaks similar to those observed in conventional
MALDI–post‐source decay (PSD) (Kaufmann et al., 1994). These ‘‘metastable’’ peaks may be removed by applying a deflection pulse to the second
ion deflector (labeled ‘‘metastable suppressor’’ in Fig. 1) at the time that
the remaining precursor ions and fragments produced in this field‐free
region reach the vicinity of the deflector. This deflector can be adjusted
to completely remove the precursor ion, but generally the applied voltage
is set to allow about 3–4% of the precursor ions to be transmitted. This
lower voltage reduces the noise due to metastables to an acceptable level
while still allowing the transmitted precursor ions to be used as reference
peaks for calibration of the fragment spectra.
The TOF‐TOF system may employ any ultraviolet (UV) laser appropriate for conventional MALDI. Our earlier work employed a nitrogen
laser (337 nm) at pulse rates between 2 and 20 Hz (Medzihradszky et al.,
2000). A diode‐pumped Nd:YAG laser operating at 355 nm has been used
at rates between 20 Hz and 2 kHz (Vestal et al., 2000b). Most of the results
presented here were obtained at a laser rate of 200 Hz. Although operating
parameters may need to be slightly altered as the laser rate is changed,
there is no effect of the acquisition speed on the performance of the
instrument. It should be noted, however, that higher laser rates allow data
to be obtained more rapidly, and the ability to store and process the data
may become limiting.
Performance Data
Precursor Selection
The selected ions, along with fragments produced (either by unimolecular dissociation or CID) in this field‐free region, pass through
the collision cell, enter the pulsed ion accelerator (labeled ‘‘2nd Source’’
[3]
tandem time‐of‐flight mass spectrometry
83
in Fig. 1), and are accelerated into the second MS by applying a high‐
voltage pulse (typically 7 kV). The timed ion selector is located in the field‐
free space between the ion source and the collision cell, and the time that
the selector is opened and the later time when the acceleration pulse is
applied are calculated, as has been described previously (Vestal and
Juhasz, 1998). If the timed ion selector is not used, then ions arriving at
the accelerator after the pulse is applied do not enter the second MS. A
spectrum obtained with the selector off and the accelerator pulse timed to
focus the matrix trimer ion, nominal mass 568, is shown in Fig. 2A. The
sharp peaks at lower mass are due to fragments formed both in the collision
cell and in the field‐free region between the collision cell and the accelerator, and the broad peaks are due to lower mass matrix ions, which are
formed in the source and are transmitted, with relatively low efficiency,
through the second source and into the second MS before application of
the acceleration pulse. With the selector off or set to transmit a relatively
wide window, the mass range for precursor ions that can be reaccelerated
and focused spans about 5% of the nominal mass chosen. Precursor ions
and their fragments within this mass range are focused and the masses of
the fragments of all transmitted precursors can be accurately determined.
Thus, it is possible to determine the relative intensities of fragment ions
from nearby precursors. This feature may be useful for quantitation using
isotopic labeling techniques such as isotope‐coded affinity tags (ICATs)
(Gygi et al., 1999).
The spectrum obtained with precursor set to transmit only the matrix
trimer ion is shown in Fig. 2B. Laser intensity and other parameters are
identical to those used in Fig. 2A. As can be seen from comparing Fig. 2A
and B, the m/z 568 is transmitted unattenuated, the isotope peak at m/z 569
is attenuated by about an order of magnitude, and only the focused fragment ions are detected at lower mass. This resolving power for precursor
selection is about 500 using a 10% valley criterion or about 1000 FWHM.
The former value accurately represents the ability to select a particular
precursor with minimal contamination by adjacent precursor masses. This
resolution is sufficient to allow selection of the isotopic envelope of peptides throughout the mass range of interest but is not adequate for selecting
a single isotope at higher mass. A single isotope can be selected up to m/z
1000, but the sensitivity is significantly reduced. The current design of the
timed ion selector provides higher resolving power for fragments than for
the precursor; this ensures that the relative contribution from fragment
ions resulting from precursors with masses similar to the selected ion that
are not completely suppressed is always less than that of the selected ion
itself. As is discussed below, in many cases the ability to transmit and focus
neighboring ions allows more than one peptide to be identified even if they
84
biological mass spectrometry
[3]
FIG. 2. MS‐MS spectrum of the trimer ions of CHCA recorded (A) without and (B) with
the timed ion selector being operated. The insets highlight the precursor region of the spectra.
[3]
tandem time‐of‐flight mass spectrometry
85
have the same nominal mass or differ by an amount that is insufficient to
allow complete separation by the precursor selector.
Sensitivity
Under ideal conditions, the ionization efficiency of MALDI is very
high. In peptide mass fingerprinting, absolute ionization efficiency is not a
primary concern because chemical noise from matrix components and
impurities is usually the major factor limiting sensitivity. In MS‐MS, the
quality of the fragment spectrum is determined by the total number of ions
accumulated in that spectrum; only at very low sample concentrations is
the chemical noise the limiting factor. Using ‐cyano‐4‐hydroxycinnamic
acid (CHCA) matrix, we have measured overall efficiencies of about 1%
for peptides loaded at low concentrations by desorbing all of the sample
loaded and integrating the total number of ions detected. These efficiency
measurements include the transfer efficiency of the analyzer (estimated to
be 40% when operated in MS‐MS mode). Efficiencies for other popular
matrices (e.g., 2,5‐dihydroxybenzoic acid [DHB]) may be as much as an
order of magnitude lower. Examples of some results showing the sensitivity
in MS‐MS mode that is routinely achieved on simple mixtures of peptides
are shown in Figs. 3–5. These results were obtained from a mixture of three
peptides (des‐Arg1 bradykinin, angiotensin I, and Glu1 fibrinopeptide B) at
equimolar concentrations. These samples were loaded at total amounts
ranging from 1 pmol to 1 fmol each in 3‐mm diameter sample spots using
a standard dried droplet method. The resulting average surface concentrations range from approximately 1 fmol/100 micron diameter laser spot
down to 1 attomole/laser spot. At the laser intensities used for these
measurements, about 1000 laser shots can be accumulated from each laser
spot before that portion of the sample spot is depleted, and the relative
response from each laser spot within the sample spot varies by about a
factor of five.
The result shown in Fig. 3B represents the accumulation of 1000 shots
on a ‘‘good’’ laser spot from the 10 fmol sample. Figure 3C shows the result
of 10,000 shots accumulated from several laser spots from the 1 fmol
sample, and Fig. 3A is the accumulated spectrum from 5000 shots from
the 1 pmol sample. In each case, a spectra from all laser shots fired
were averaged into the final result; selective averaging was not employed.
Approximately 10 attomoles of sample was consumed in obtaining the
spectra shown in Fig. 3B and C, and 5 fmol was consumed to obtain
spectrum A. In all spectra, internal fragment ions are indicated by asterisks,
and neutral losses from the labeled ions are indicated by plus signs. These
spectra were obtained with the metastable suppressor in operation, thereby
86
biological mass spectrometry
[3]
FIG. 3. MS‐MS spectra of Glu1 fibrinopeptide B (EGVNDNEEGFFSAR) recorded with
the laser operated 20% above threshold, N2 as a collision gas, and single‐collision conditions
for (A) 5000 shots from a 1 pmol sample spot, (B) 1000 shots from a 10 fmol sample spot, and
(C) 10,000 shots from a 1 fmol sample spot.
reducing the precursor ion intensity by about a factor of 30. The total
integrated ion intensity of the precursor ion (before attenuation) is approximately equal to the total integrated fragment ion intensity in these spectra.
The total number of fragment ions integrated in Fig. 3C is approximately
50,000; about 100,000 in Fig. 3B; and 5,000,000 in Fig. 3A. At the highest
concentration, the ionization efficiency is lower by about a factor of 10
[3]
tandem time‐of‐flight mass spectrometry
87
FIG. 4. Expansion of the m/z 70–200 region of Fig. 3. In all spectra, internal fragment ions
are indicated by asterisks, and neutral losses from the labeled ions are indicated by plus signs.
because of depletion of the matrix ions. Reliable detection and mass
measurement of a fragment ion requires collection of at least 10 ions in
the two or three digitizer channels that define the peak. The relative
intensities of several of the structurally significant y and w ions (e.g., 8,
10, 11, and 12) in this particular case are in the range of 1–3% of the most
88
biological mass spectrometry
[3]
FIG. 5. Expansion of the m/z 650–1100 region of Fig. 3.
intense fragment peak. Nevertheless, these ions are detectable and the
dominant fragment masses can be determined with an uncertainty of
less than 50 ppm. Portions of the spectra are expanded in Figs. 4 and 5 to
better illustrate the quality of the spectra. A representative set of masses,
intensities, and mass errors is summarized in Table I.
[3]
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tandem time‐of‐flight mass spectrometry
TABLE I
MASS ACCURACIES FOR THE GLU1 FIBRINOPEPTIDE B SPECTRUM SHOWN IN FIGS. 3–5,
V
L
F
R
y1‐NH3
y1
y2‐NH3
y2
y3‐NH3
y3
y4
y6
y7
w8
w9
y9‐NH3
y9
y13
a
PANEL
Aa
m/z
Observed
Height
(Da)
(ppm)
72.0813
87.0558
120.0813
129.1140
158.0930
175.1195
229.1301
246.1566
316.1621
333.1886
480.2571
684.3469
813.3895
867.4001
996.4427
1039.4485
1056.4750
1441.6348
72.0973
87.0708
120.0946
129.1190
158.1003
175.1194
229.1310
246.1540
316.1512
333.1801
480.2445
684.3455
813.3967
867.4621
996.5135
1039.4786
1056.4893
1441.7113
2517
908
3735
1078
1067
8039
833
1639
1411
1171
1168
4810
2122
856
1195
830
7817
1411
0.0160
0.0150
0.0133
0.0050
0.0073
0.0001
0.0009
0.0026
0.0109
0.0085
0.0126
0.0014
0.0072
0.0620
0.0708
0.0301
0.0143
0.0765
221.7
172.3
110.9
38.8
46.4
0.9
4.0
10.6
34.5
25.4
26.3
2.0
8.9
71.4
71.0
29.0
13.5
53.1
Columns denote the identity of the fragment, the theoretical mass of the peptide, the
observed mass using default instrumental calibration, the height of the peak, and mass
errors in m/z and in ppm.
The minimum measurable peak in these examples ranges from 0.02%
of total fragment intensity in Fig. 3C, 0.01% in Fig. 3B, and 0.0002% in
Fig. 3A. The more intense peaks in the fragment spectra are typically 1–2%
of the total fragment intensity; thus, the dynamic range in the spectra
obtained at low sample levels is about 100. By accumulating a large number
of laser shots, the dynamic range may exceed 10,000 at sample concentrations above approximately 1 fmol/laser spot. Unfortunately, as the dynamic
range exceeds about 1000, a peak is observed at virtually every possible
fragment mass, and these additional peaks may be of little utility in elucidating structure. At very low concentrations, increasing the number of ions
integrated beyond about 100,000 may not improve the quality of the
spectra significantly, because ions due to chemical noise from matrix components and impurities within the selected precursor mass window may be
the limiting factor.
The total number of laser shots that must be accumulated to achieve a
particular result depends on several factors. If the goal is to measure
the masses of only the most intense peaks for peptide identification by
90
biological mass spectrometry
[3]
database searching, then accumulation of a few thousand ions may be
sufficient. For samples containing fewer than 10 peptides at surface concentrations higher than about 10 attomoles/laser spot, this requires only
about 100 laser shots. On the other hand, acquiring the same number of
ions from a particular component present at this concentration in a mixture
of 100 peptides requires 10 times as many laser shots. If the goal is de novo
sequencing or determination of posttranslational modifications, then a
larger number of ions and laser shots is required. Accumulation of 10,000
shots is usually sufficient for even the most demanding case. As several
hundred thousand shots are generally required to significantly deplete the
sample with standard dried droplet sample preparation, there is no problem recording several high‐quality MS‐MS spectra from each sample spot.
Fragmentation
The fragmentation processes occurring in the TOF‐TOF instrument are
closely related to those occurring in the PSD process (Kaufmann et al.,
1994) employed in most MALDI‐TOF mass spectrometers equipped with
reflecting analyzers. Ions may be excited sufficiently in the MALDI source
to spontaneously decay after extraction, or they may fragment as the result
of an energetic collision with a neutral molecule. This technique has been
useful, but its utility has been limited by a number of instrumental factors.
As discussed earlier in this chapter, these limitations on speed, sensitivity,
resolution, and mass accuracy, as well as ease of use, have been overcome
in the TOF‐TOF system.
Fragmentation of peptide ions in various MS‐MS instruments has been
extensively reviewed (Shulka and Futrell, 2000). The energy imparted to
the ions during the formation process, which leads to unimolecular fragmentation, is strongly dependent on the ionization method. In MALDI,
both the laser intensity and the properties of the matrix affect the distribution of internal energy in the peptide ions. Near threshold for ionization,
little fragmentation is observed, but as the laser intensity is increased above
threshold, significant unimolecular fragmentation may occur. More fragmentation is generally observed for ‘‘hot’’ matrices such as CHCA than for
‘‘cooler’’ matrices such as DHB. At modest laser intensities (10–20%
above threshold), the unimolecular fragmentation is dominated by simple
peptide bond cleavages to yield the y and b ions observed in low‐energy
CID and in PSD. At high laser intensities, the fragments due to small
neutral losses (e.g., ammonia and water) from these y and b ions predominate, and a ions may also increase with laser intensity. In general, immonium ions and internal fragments are weak in the unimolecular spectra
even at high laser intensity. Increasing the laser intensity beyond 20–30%
[3]
tandem time‐of‐flight mass spectrometry
91
above threshold rapidly increases the rate of sample consumption, but the
observed ion intensities tend to approach a saturation value, indicating
reduced ionization efficiency under these conditions. Spectra recorded
in the TOF‐TOF when no gas is added to the collision cell are very similar
to those obtained in low‐energy CID experiments on singly charged ions in
other MS‐MS instruments such as hybrid quadrupole TOF (Anderson
et al., 1998) or quadrupole ion traps (Doroshenko and Cotter, 1996).
Fragmentation in the TOF‐TOF instrument may also be produced by
high‐energy collisions with a neutral gas. This approach is similar to that
employed in the tandem magnetic sector instruments, and the results
obtained are comparable even though fast atom bombardment ionization
was employed in the earlier work (Johnson et al., 1988). The major differences appear to be that the TOF‐TOF provides much higher detection
efficiency and resolution over the entire mass range of the fragments, while
the sector instruments detected the low mass ions with rather low efficiency. In high‐energy collisions, the possible internal energy gain after the
collision is determined by the laboratory kinetic energy of the ions, the
mass of the neutral partner, and the number of collisions that occur. This
energy is added to that imparted by the ionization process. The effect from
all of these variables has been studied over limited ranges, but most of the
routine work has employed a laboratory collision energy of 1 kV, argon,
neon, or nitrogen as collision gas, and collision gas pressures corresponding
to average collision numbers between zero and three. These conditions
appear to be satisfactory for a wide range of peptides observed in protein
digests, but there are some indications that either higher laboratory energy
or higher mass collision partners may be desirable for producing more
extensive fragmentation of peptides with masses above m/z 3000. A few
selected examples of results of these studies on fragmentation are given
below; the complete results are published elsewhere (Campbell, 2003).
The fragment spectra obtained at different collision gas pressures for
Glu1 fibrinopeptide B (EGVNDNEEGFFSAR), a typical pseudo‐tryptic
peptide with a C‐terminal arginine and no other basic residues, are shown
in Figs. 6–10. The spectrum in Fig. 6A was obtained with no collision
gas added, Fig. 6B at a pressure corresponding to about one collision,
on average, and Fig. 6C to about two collisions. Because Poisson statistics
apply, for the condition used for recording Fig. 6B, about 37% of the
ions undergo one collision, and 26% two or more. In Fig. 6C, 27% of
the ions undergo one collision, and 59% two or more. With no collision
gas, the observed intensities of immonium ions and internal fragments are
very weak, and no v and w ions due to side‐chain cleavages are observed.
As collision gas is added, the pattern of the y ions is only slightly affected,
with the low mass ions increasing in intensity relative to the higher masses.
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biological mass spectrometry
[3]
FIG. 6. MS‐MS spectrum of Glu1 fibrinopeptide B (EGVNDNEEGFFSAR) recorded
with the laser operated 20% above threshold and with (A) no gas added to the collision cell,
and N2 added to the collision cell such that the average number of collisions was (B) one and
(C) two.
[3]
tandem time‐of‐flight mass spectrometry
93
FIG. 7. Expansion of the m/z 10–200 region of Fig. 6.
In contrast, the immonium ions, internal fragment ions, and side‐chain
cleavage ions all increase approximately in proportion to the average
collision number. This behavior appears to be independent of the peptide
sequence and can be used to determine whether a proposed assignment of
a fragment peak is plausible. In these measurements, the dynamic range is
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biological mass spectrometry
[3]
FIG. 8. Expansion of the m/z 225–275 region of Fig. 6.
greater than 1000, and in the range where dipeptide and tripeptide internal
fragments are observed (Figs. 8 and 9), a detectable peak is observed at
each nominal mass. These peaks of low relative intensity are generally not
useful for determining structure. On the other hand, the intensities of
several of the y, v, and w ions in the high mass region (Fig. 10) are 1% or
[3]
tandem time‐of‐flight mass spectrometry
95
FIG. 9. Expansion of the m/z 350–400 region of Fig. 6.
less of the most intense fragment peaks, and these peaks, which are essential for determining the sequence, may not be reliably measured if the
dynamic range is less than 100.
Substance P (RPKPQQFLM‐NH2) has been studied using a wide variety of ion‐activation methods, including surface‐induced (Nair et al., 1996),
photo‐induced (Barbacci and Russell, 1999), and electron capture–induced
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biological mass spectrometry
[3]
FIG. 10. Expansion of the m/z 1075–1500 region of Fig. 6.
(Axelsson et al., 1999) dissociations. Additionally, Substance P has been
widely adopted as a ‘‘test molecule’’ for CID, and several studies describing the fragmentation of substance P in sector instruments are available
(Hayes and Gross, 1990). With arginine at the N‐terminus, lysine at residue
3, and no basic residue near the C‐terminus, the positive charge tends to
reside on the N‐terminal fragments, in contrast to the previous example,
[3]
tandem time‐of‐flight mass spectrometry
97
and the fragment spectrum is dominated by a, b, and d ions. An extensive
study of the fragmentation of substance P has been completed to quantitatively determine the dependence of fragmentation in TOF‐TOF
on operating parameters (Campbell, 2003). Results from that study are
selected to demonstrate some specific attributes.
Total fragmentation efficiency as a function of gas pressure in the
housing surrounding the collision cell is shown for laboratory collision
energies of 1 and 2 kV in Fig. 11. The pressure in the collision cell is
estimated to be about three orders of magnitude higher but is not measured
directly, and the ionization gauge readings are uncorrected for its response
to a particular gas relative to air. In this example, neon was the collision
gas, but qualitatively similar results were obtained for all of the gases
studied including He, Ar, and Kr. These results were obtained by integrating the total fragment ion intensity, and separately, the total precursor ion
intensity over the isotopic envelope. For all the experiments, the laser
was operated near threshold; thus, the contribution of unimolecular reactions to the total fragment ion intensity is minimal. Although there is
considerable scatter in these data, it is clear that fragmentation efficiency
FIG. 11. Fragmentation efficiency as a function of collision cell housing pressure for MS‐
MS of substance P using near‐threshold laser intensity, Ne as a collision gas, and 1 kV (s) and
2 ktV () laboratory frame collision energies.
98
biological mass spectrometry
[3]
in the 1–2 collision pressure regimen is enhanced at the higher collision
energy. Inspection of the spectra shows that most added fragment ion
intensity is observed as immonium ions and internal fragments. This trend
is further illustrated in Fig. 12, where the total immonium and the total
precursor ion intensities relative to total ion intensity are plotted as a
function of housing pressure for neon at 2 kV. In this case, the total ion
intensity is confined to those peaks that are assigned to expected fragments
of the peptides. As a result, the low‐intensity peaks that are not assigned
are neglected in this example.
A portion of the substance P fragment spectrum obtained under three
operating conditions is shown in Fig. 13. In Fig. 13A, a sample spectrum
from the data of Fig. 11, the laser was operated near threshold, and the gas
pressure was adjusted so that the precursor was attenuated by about 70%.
The data in Fig. 13B were obtained under identical conditions except that
the laser intensity was increased by about 20%, and the data in Fig. 13C
used the same high laser intensity, but the collision gas was removed.
Except for the higher relative intensity of the a‐17 ions, the spectrum in
Fig. 13A is virtually identical to that obtained by high‐energy CID in a
four‐sector instrument (Hayes and Gross, 1990). Clearly, the b ions are
FIG. 12. For the data of Fig. 11, 2‐kV laboratory frame collisions, the fraction of total
assigned ion intensity that is observed as immonium () and precursor (s) ions.
[3]
tandem time‐of‐flight mass spectrometry
99
FIG. 13. The expansion of the m/z 550–900 portion of the MS‐MS spectra of substance P
recorded with (A) a threshold laser and gas pressures operating such that 70% of the
precursor ion was suppressed, (B) laser intensity 20% above threshold with the same gas
pressure as (A), and (C) laser intensity as in (B), with no gas in the collision cell.
primarily due to excitation in the ion source, whereas the d ions are only
produced by high‐energy collisions. The a ions are produced by both
processes, as are some of the internal fragments, whereas the immonium
ions are primarily produced by high‐energy collisions.
100
biological mass spectrometry
[3]
Applications to Protein Identification and Characterization
Although the TOF‐TOF system is relatively new, applications to identification and characterization of proteins have been reported (Juhasz et al.,
2002). One example is presented here to demonstrate the current performance of the technique. For this experiment, 5 pm each of ‐galactosidase
(Escherichia coli) and glycogen phosphorylase (rabbit muscle) were digested with bovine trypsin and lyophilized. One‐hundred microliters of
CHCA was added to the separate dry samples, and 0.5 l of each resulting
solution were mixed on a 3‐mm diameter sample spot. Thus, the nominal
sample loading on the MALDI plate, assuming no losses and complete
digestion, is 50 fmol total, or about 50 attomoles/laser spot. The peptide
mass fingerprint obtained from this sample is shown in Fig. 14. At least 100
peptides are observed that can be assigned to these two proteins. As noted
earlier, the ionization efficiency for any component in such a complex
mixture is substantially reduced by competition for the available protons;
FIG. 14. Peptide mass fingerprint of a mixture of tryptic digests of 25 fmol of
‐galactosidase (Escherichia coli) and 25 fmol of glycogen phosphorylase (rabbit muscle)
on a 3‐mm sample spot.
[3]
tandem time‐of‐flight mass spectrometry
101
nevertheless, 50 fmol of sample loaded onto a single 3‐mm spot is sufficient
to allow excellent fragment spectra to be obtained on a large number of
these peptides. For this example, 10,000 laser shots were accumulated for
each of 22 selected mass peptides, as indicated by the mass numbers in
Fig. 14. The region between m/z 1050 and 1090 is expanded in the inset
showing the presence of at least six peptides in this mass range; the four
labeled peaks were selected for MS‐MS analysis. Spectra obtained for these
peaks are shown in Fig. 15. These spectra were adequate for unambiguous
identification of the peptide via database searching. Only the most intense
peaks are labeled in Fig. 15, and the number of fragment peaks matching
expected fragments of the peptide found is indicated in Table II.
No fragment peaks from adjacent precursors were observed, and the
unassigned masses were plausible low mass fragments not included in the
set used in the database searching program.
All of the more intense peaks in the region below m/z 2500 provided
MS‐MS of sufficient quality to allow de novo sequencing, although in this
FIG. 15. MS‐MS spectra for each of the four peptides labeled on the inset of Fig. 14.
Results of the database search for these spectra are shown in Table II.
102
[3]
biological mass spectrometry
TABLE II
DATABASE SEARCHING RESULTS FROM THE PEPTIDES NOTED ON FIG. 14a
m/z
Unmatched/
submitted
Immonium
ions?
Rank
Sequence of
peptide
869.45
1053.57
1067.49
1072.54
1083.52
1299.62
1355.67
1394.73
1428.68
1440.73
1442.70
1550.77
1566.79
1689.88
1742.90
1776.11
1787.73
1886.90
2265.20
2465.19
2522.23
2847.41
3292.61
1/11
0/15
3/9
1/9
1/10
9/35
0/16
2/36
10/32
9/21
1/9*
0/13
5/13
2/20
1/33
8/30
2/10
6/24
2/26
0/13
2/20
0/26
2/40
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
No
No
No
Yes
No
Yes
Yes
No
No
No
Yes
Yes
Yes
Yes
Yes
1
1
1
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
FAAYLER
VIFLENYR
WVGYGQDSR
EIWGVEPSR
GDFQFNISR
ELNYGPHQWR
DYYFALAHTVR
LPSEFDLSAFLR
DWENPGVTQLNR
LLSYVDDEAFIR
VLYPNDNFFEGK
IGEEYISDLDQLR
DFNVGGYIQAVLDR
ARPEFTLPVHFYGR
LSGQTIEVTSEYLFR
IENGLLLLNGKPLLIR
HSDYAFSGNGLMFADR
GYNAQEYYDRIPELR
DVSLLHKPTTQISDFHVATR
IDGSGQMAITVDVEVASDTPHPAR
VVQPNATAWSEAGHISAWQQWR
VTVSLWQGETQVASGTAPFGGEIIDER
GVNRHEHHPLHGQVMDEQTMVQDILLMK
a
The MS‐Tag (version 3.6) portion of the Protein Prospector package was used to search
the Swiss Prot. 5.10.2001 protein database. The columns denote the calibrated mass of the
peptide in MS mode, the portion of unmatched to submitted fragment ions (with m/z >
140), whether or not the detected immonium ions were used in the search, and what rank
MS‐Tag gave to the known sequence of the ions (shown in the last column).
case, the fragment masses alone were sufficient to tentatively assign the
sequence. One example is shown in Figs. 16 and 17. The masses submitted
the database search are shown in Table III. Of the 36 fragment peaks with
m\z values more than 150 that were submitted, 34 matched known fragments of the tryptic peptide LPSEFDLSAFLR from ‐galactosidase within
the specified mass error of 100 millimass units. It should be noted that both
unmatched peaks were of relatively low intensity and that the peak at m/z
1184.68 can be assigned to y11 , although its mass error is greater than 100
millimass units. The fragment ions provide complete coverage of the amino
acid sequence of the peptide, so it would be possible to use the spectrum
for de novo sequencing. At higher mass (e.g., m/z 3292 shown in Fig. 18),
sequence coverage is incomplete, but the spectrum is sufficient to unambiguously identify the peptide from the database. The arginine at position 4 due to the missed cleavage causes the spectrum to be dominated
by a and b ions, with particularly intense ions due to cleavage at the
[3]
tandem time‐of‐flight mass spectrometry
FIG. 16. Exemplary MS‐MS spectrum of the peptide of mass 1394 from Fig. 14.
FIG. 17. Expansion of the m/z 150–300 region of Fig. 16.
103
104
[3]
biological mass spectrometry
TABLE III
DETAILS OF THE MS‐TAG DATABASE SEARCH FOR THE SPECTRUM OF THE PEPTIDE
LPSEFDLSAFLR (m/z 1394) SHOWN IN FIG. 16 AND 17a
Assignment
m/z
PS‐28
PS‐18
y1
a2
LS
b2
wa2
FL‐28
FD
y2‐17
LSA
y2
b3
SAF
PSE
v3
a4
y3‐17
b4
y3‐17
PSEF‐28
PSEF
y4
a5
y5‐17
y5
wa6
b6
y6
a7
y7‐17
y7
y8
y10
157.1
167.08
175.12
183.14
201.11
211.15
229.13
233.16
263.11
271.18
272.16
288.19
298.16
306.14
314.15
343.22
399.24
418.24
427.23
433.25
435.27
461.22
506.33
546.34
576.26
593.33
647.39
689.36
706.46
774.43
804.46
821.49
968.59
1184.68
1227.75
1281.76
a
(millimass units)
2.4
2.5
2.6
9.9
11.3
6.8
0.4
8.8
10.6
2.9
0.9
11.4
14.7
0.8
13
7.1
16.1
7.5
9.2
10.2
37.4
15.2
19.4
51.3
52.8
8
40.1
38.8
33.7
29.2
35.8
33.7
71.9
80.8
Column denotes the identity of the fragment ion, the submitted mass, and the
calculated mass error in millimass units.
[3]
tandem time‐of‐flight mass spectrometry
105
FIG. 18. Sample high‐mass MS‐MS spectrum of peak with m/z 3292 from Fig. 14.
C‐terminal side of aspartic acid (b16 and b23) and at the N‐terminal side of
proline (b8).
One of the more difficult examples is illustrated in Fig. 19, corresponding to a region of the peptide mass fingerprint containing a number
of relatively weak peaks close together as indicated in Fig. 14. It was
not possible to separate the two components at nominal masses 1440
and 1442 in the precursor selection without sacrificing too much intensity;
nevertheless, it was possible to identify both peptides by database searching as indicated in Table II. The major fragments corresponding to each
are indicated in Fig. 19. A total of 21 fragments were submitted for
the search, and all except 9 matched the component at m/z 1440. Eight
of the nine remaining fragments matched the peptide with molecular
weight 1442.
The results of the database search on the 23 peptides are summarized in
Table II. At least one MS‐MS spectrum was obtained on each of these
masses, and 10,000 laser shots were summed for each. For the more intense
peaks, a few hundred laser shots were sufficient for identifying the peptide
106
biological mass spectrometry
[3]
FIG. 19. MS‐MS spectrum recorded for m/z 1440 from Fig. 14. Inset of precursor region
demonstrates that fragments are formed from two equally abundant precursor ions.
Fragments from LLSYVDDEAFIR are shown in bold type.
by searching the database, but the signal/noise ratio and dynamic range is
improved by summing more shots. In most cases, all of the more intense
peaks in the spectra were assigned to expected fragments from the peptide
found. In cases in which a significant number of unassigned fragments are
observed, the peptide mass fingerprint is rather congested and it appears
that several relatively low‐intensity peptide peaks may also be present
within the selected precursor window.
Conclusion
Work has established the MALDI‐TOF‐TOF system as a useful tool
for protein identification using database searching, as well as for protein
characterization and de novo sequencing. This system is now available
commercially. Further work on software for automatic interpretation of
the spectra is required to make the high‐throughput promise a reality.
[3]
tandem time‐of‐flight mass spectrometry
107
References
Anderson, U. N., Colburn, A. W., Makarov, A. A., Papakis, E. N., Davis, S. C., Hoffman,
A. D., and Thomson, S. (1998). In‐series combination of a magnetic‐sector mass
spectrometer with a time‐of‐flight quadratic‐field ion mirror. Rev. Sci. Instrum. 69,
1650–1660.
Axelsson, J., Palmblad, M., Hakansson, K., and Hakansson, P. (1999). Electron capture
dissociation of substance P using a commercially available Fourier transform ion cyclotron
resonance mass spectrometer. Rap. Comm. Mass. Spectrom. 13, 474–477.
Barbacci, D. C., and Russell, D. H. (1999). Sequence and side‐chain specific photofragment
(193 nm) ions from protonated substance P by matrix assisted laser desorption ionization
time‐of‐flight mass spectrometry. J. Am. Soc. Mass Spectrom. 10, 1038–1040.
Campbell, J. M. (2003). Mapping the properties of center of mass collision energy on a
MALDI TOF/TOF mass spectrometer—fundamentals and applications. In ‘‘Proceedings
of the 51st ASMS Conference.’’ Montreal, P.Q., June 8–12.
Cody, R. B., and Frieser, B. S. (1982). High‐resolution detection of collision‐induced dissociation
fragments by Fourier transform mass spectrometry. Anal. Chem. 54, 1431–1433.
Davis, M. T., Stahl, D. C., Hefta, S. A., and Lee, T. D. (1995). A microscale electrospray
interface for on‐line capillary liquid chromatography/tandem mass spectrometry of
complex peptide mixtures. Anal. Chem. 67, 4549–4556.
Doroshenko, V. M., and Cotter, R. J. (1996). Pulsed gas introduction for increasing CID
efficiency in a MALDI/quadrupole ion trap mass spectrometer. Anal. Chem. 68, 463–472.
Ducret, A., Van Oostveen, I., Eng, J. K., Yates, J. R., III, and Aebersold, R. (1998). High
throughput protein characterization by automated reverse‐phase chromatography/
electrospray tandem mass spectrometry. Protein Sci. 7, 706–719.
Eng, J. K., McCormack, A. L., and Yates, J. R., III (1994). An approach to correlate tandem
mass spectral data of peptides with amino acid sequences in a protein database. J. Am.
Soc. Mass Spectrom. 5, 976–989.
Gygi, S. P., Rist, B., Gerber, S. A., Turacek, F., Gelb, M. H., and Aebersold, R. (1999).
Quantitative analysis of complex protein mixtures using isotope‐coded affinity tags. Nat.
Biotechnol. 17, 994–999.
Hayes, R. N., and Gross, M. L. (1990). Collision‐induced dissociation. Methods Enzymol. 193,
237–263.
Johnson, R. S., Martin, S. A., and Biemann, K. (1988). Collision‐induced fragmentation of
(M þ H)þ ions of peptides. Side chain specific sequence ions. Int. J. Ion Proc. Mass
Spectrom. 86, 137–154.
Juhasz, P. J., Campbell, J. M., and Vestal, M. L. (2002). MALDI TOF‐TOF technology for
peptide sequencing and protein identification. In ‘‘Mass Spectrometry and Hyphenated
Techniques in Neuropeptide Research’’ (J. Sibberring, R. Ekman, D. M. Desiderio, and
N. M. Nibbering, eds.). Wiley, Hoboken, NJ.
Kaufmann, R., Kirch, D., and Spengler, B. (1994). Sequencing of peptides in a time‐of‐flight
mass spectrometer: Evaluation of postsource decay following matrix‐assisted laser
desorption ionisation (MALDI). Int. J. Mass Spectrom. Ion Proc. 131, 355–385.
Louris, J. N., Cooks, R. G., Syka, J. R. P., Kelley, P. E., Stafford, G. C., Jr., and Todd, J. F. J.
(1987). Instrumentation, applications, and energy deposition in quadrupole ion‐trap
tandem mass spectrometry. Anal. Chem. 59, 1677–1685.
Mann, M., and Wilm, M. (1994). Error‐tolerant identification of peptides in sequence
databases by peptide sequence tags. Anal. Chem. 66, 4390–4399.
Medzihradszky, K. F., Adams, G. W., Bateman, R. H., Green, M. R., and Burlingame, A. L.
(1996). Peptide sequence determination by matrix‐assisted laser desorption ionization
108
biological mass spectrometry
[3]
employing a tandem double focusing magnetic‐orthogonal acceleration time‐of‐flight mass
spectrometer. J. Am. Soc. Mass Spectrom. 7, 1–10.
Medzihradszky, K. F., Campbell, J. M., Baldwin, M. A., Falick, A. M., Juhasz, P., Vestal, M. L., and
Burlingame, A. (2000). The characteristics of peptide collision induced dissociation using a
high‐performance MALDI TOF/TOF tandem mass spectrometer. Anal. Chem. 72, 552–558.
Morris, H. R., Paxton, T., Dell, A., Langhorne, J., Berg, M., Bordoli, R. S., Hoyes, J., and
Bateman, R. H. (1996). High sensitivity collisionally‐activated decomposition tandem
mass spectrometry on a novel quadrupole/orthogonal‐acceleration time‐of‐flight mass
spectrometer. Rap. Comm. Mass Spectrom. 10, 889–896.
Nair, H., Somogyi, A., and Wysocki, V. H. (1996). Effect of alkyl substitution at the amide
nitrogen on amide bond cleavage: electrospray ionization/surface–induced dissociation
fragmentation of substance P and two alkylated analogs. J. Mass Spectrom. 31, 1141–1148.
Olivares, J. A., Nguyen, N. T., Yonker, C. R., and Smith, R. D. (1987). On‐line mass
spectrometric detection for capillary zone electrophoresis. Anal. Chem. 59, 1230–1232.
Sato, K., Asada, T., Ishihara, M., Kunihiro, F., Kammei, Y., Kubota, E., Costello, C. E.,
Martin, S. A., Scoble, H. A., and Biemann, K. (1987). High‐performance tandem mass
spectrometry: Calibration and performance of linked scans of a four sector instrument.
Anal. Chem. 59, 1652–1659.
Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996). Mass spectrometric sequencing of
proteins from silver stained polyacrylamide gels. Anal. Chem. 68, 850–858.
Shulka, A. K., and Futrell, J. H. (2000). Tandem mass spectrometry: Dissociation of ions by
collisional activation. J. Mass Spectrom. 35, 1069–1090.
Vestal, M. L., Juhasz, P., and Martin, S. A. (1995). Delayed extraction matrix assisted laser
desorption time‐of‐flight mass spectrometry. Rapid Comm. Mass Spectrom. 9, 1044–1050.
Vestal, M., and Juhasz, P. (1998). Resolution and mass accuracy in matrix assisted laser
desorption ionization time‐of‐flight. J. Am. Soc. Mass Spectrom. 9, 892–911.
Vestal, M. L., Juhasz, P., Hines, W., and Martin, S. A. (2000a). A new delayed extraction
MALDI TOF MS‐MS for characterization of protein digests. In ‘‘Mass Spectrometry in
Biology and Medicine’’ (A. L. Burlingame, S. A. Carr, and M. A. Baldwin, eds.), pp. 1–16.
Humana Press, Totowa, NJ.
Vestal, M. L., Campbell, J. M., Hayden, K. M., and Juhasz, P. J. (2000b). Performance
Evaluation of Improved MALDI‐TOF MS‐MS System. In ‘‘Proceedings of the 48th ASMS
Conference.’’ Palm Beach, CA. June 11–15.
Wilm, M., Shevchenko, A., Houthaeve, T., Briet, S., Schweigrer, L., Fotsis, T., and Mann, M.
(1996). Femtomole sequencing of proteins from polyacrylamide gels by nano‐electrospray
mass spectrometry. Nature 379, 466–469.
Yost, R. A., and Enke, C. E. (1978). Selected ion fragmentation with a tandem quadrupole
mass spectrometer. J. Am. Chem. Soc. 100, 2274–2275.
Further Reading
Shevchenko, A., Chernushevich, I., Ens, W., Standing, K. F., Thomson, B., Wilm, M., and
Mann, M. (1998). Rapid ‘‘de novo’’ peptide sequencing by a combination of
nanoelectrospray, isotopic labeling and a quadrupole/time‐of‐flight mass spectrometer.
Rapid Comm. Mass Spectrom. 11, 1015–1024.