RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL.

9 , 1044-1050 (1995)

Delayed Extraction Matrix-assisted Laser

Desorption Time-of-flight Mass Spectrometry
M. L. Vestal,* P. Juhasz and S. A. Martin
PerSeptive Biosystems, Inc., 500 Old Connecticut Path, Framingham, MA 01701, USA

SPONSOR REFEREE: Prof. Kenneth Standing, University of Manitoba, Winnipeg, Manitoba, Canada

A new delayed extraction matrix-assisted laser desorptiodionization (MALDI) time-of-flight (TOF) mass
spectrometer system is described which provides dramatically improved performance over that obtained with
identical TOF analyzers operated with constant electrical fields. In this system the ions formed by MALDI are
produced in a weak electrical field, and subsequently extracted by application of a high voltage pulse after a
predetermined time delay. Three parameters can be independently adjusted to optimize the performance. These
include the field magnitude and direction during and immediately following ion production, the magnitude of the
extraction field, and the time delay between the laser pulse and application of the extraction field. Theoretical
equations are presented which guide the selection of these parameters to optimize the performance.
Results are presented from three TOF analyzers of similar design, but ranging in size from a 1.3 m linear
analyzer to a 6.4 rn reflector. In all cases delayed extraction provides substantially improved resolution over that
obtained from static operation. In the smallest linear analyzer resolution of M/AM (full width at half maximum)
of 4000 is demonstrated for angiotensin I, and with the largest reflecting analyzer, the isotope peaks of bovine
insulin are resolved nearly to baseline. With larger proteins, the isotopic peaks are not resolved, but resolution
approaching the width of the isotopic envelope is obtained. Resolution improvement is demonstrated for proteins
up to 30 kDa. In addition to improved resolution, delayed extraction is shown to improve the quality of MALDI
mass spectra by suppressing matrix background, reducing chemical noise, and minimizing the effect of laser
intensity on performance.

Time-of-flight (TOF) mass spectrometry has been
experiencing a revival since the introduction of soft
ionization techniques compatible with this type of mass
analysis such as plasma desorption,' and matrix-
assisted laser desorption/ionization (MALDI).24 The
appeal of TOF mass analysis is also indicated by its
recent, highly promising combination with continuous
ionization methods such as e l e c t r o ~ p r a y .The
~ , ~ wide-
spread use of MALDI in biological applications has
particularly spurred innovation in the design of TOF
analyzers leading to improved mass resolution, sensiti-
vity, and mass accuracy.
A major limitation of TOF mass analyzers has often
been relatively poor mass resolution. The use of reflect-
ing analyzers, such as that described originally by
Mamyrin7has overcome this limitation for many appli-
cations, but with MALDI in particular, resolution is
often inadequate. Earlier studies by Beavis and Chait,'
and by Zhou, Ens, Standing, and Verentchikov' indi-
cate that the major problem is that the ions produced
by MALDI exhibit a rather broad energy distribution.
Initial velocity of desorbed analyte ions is nearly inde-
pendent of mass; thus the initial kinetic energy is
proportional to mass of the analyte. In addition, when
desorption occurs in a strong electrical field, energy is
lost presumably by collisions with the neutral plume,
and further mass dependent energy dispersion results.
In very early work, Wiley and McLaren'" described a
two-field, pulsed ion source which provided first order
correction for the initial space distribution of ions.
* Author for correspondence.
These authors also described a technique which they
named 'time-lag energy focusing' for correcting for
initial velocity distributions. In this instrument, the ions
are produced in a field-free region, and the accelerating
field is turned on by application of a fast pulse at a
predetermined delay time after initial ion formation.
Recently, Brown, Lennon and Christie,", l2 Colby,
King and Reilly13 and Whittal and Li14 have applied
similar mass analyzers to MALDI, and have shown
significantly improved resolution for selected masses as
a function of delay time and pulsed field intensity in the
ion source.
According to the theory of 'time-lag energy focusing'
as originally developed by Wiley and McLaren,'" the
dependence of ion flight time on initial velocity can be
corrected, to first order, by delaying the extraction of
ions from the source by an appropriate amount. If
higher order terms are insignificant, then the mass
resolution should be determined by the ratio of the
total flight time to the uncertainty in the time measure-
ment. In this case, the observed mass resolution should
increase in proportion to the effective length of the ion
flight path. In the work of Wiley and McLaren, both
the initial space and velocity distribution of the ions
were variables and simultaneous correction for both
terms could not be accomplished. In surface ionization
techniques such as MALDI, space focusing is not a
factor; therefore, corrections for velocity focusing will
greatly improve performance. The present work was
undertaken to determine the extent to which perfor-
mance of MALDI for analysis of peptides and proteins
is improved by delayed extraction, and to explore how
this performance depends on the size of the instrument.

CCC 0951-4198/95/ 1 11044-07 Received 24 July 1995

@ 1995 by John Wiley & Sons, Ltd. Accepted 24 July 1995
 DELAYED EXTRACTION MALDI TOF MASS SPECTROMETRY 1045

Grids first-order time focus occurs near the entrance to the

drift space. In this case
w = x(do/d,) - 1 (3)
and both first- and second-order velocity focusing can
be achieved.
Adjustment of a delayed extraction ion source to
optimize resolution can be accomplished by the aid of
I I-%* Eqn (1). At a given extraction field (determined by
V, ), increasing extraction delays are required to focus
higher masses, and at low-to-intermediate mass the
delay is approximately proportional to the square root
HV switch Delay of m/z. Alternatively, higher masses can be brought
into focus by increasing the extraction field, while
keeping the extraction delay constant.
U

Figure 1. Block diagram of the electronics used to provide extraction EXPERIMENTAL

of ions. This system allows independent control of the initial field in
the ion production region, the extraction field, and the delay between
ion production and extraction.
THEORY
Following the approach outlined by Wiley and
Mclaren," the optimum time lag (in microseconds) for
first order velocity focusing at the detector plane is
given by
z=0.144d,(m/~V,)"~[l/w + (To/Vx)'"] (1)
where d, is the length of the first field in mm, m is the
mass in daltons, z is the number of elemental charges
on the ion, V, is the amplitude of the extraction pulse in
volts, To is the most probable initial kinetic energy of
the ions in electronvolts, and w is given by
w = (xii +x)3/2[(~/2d,)- (do/d0)(i +4+x(doid,)- 1
(2)
where x = V,/Vl , with V, being the potential applied to
the intermediate grid. L is the field-free drift distance
and dois the length of the second accelerating field (Fig.
1). For space focusing, the potentials are adjusted so
that w vanishes; and for velocity focusing, w must be
greater than zero.
This theory also applies to reflecting analyzers, but
with adjustment of the operating conditions so that the
Mass Spectrometry
The coaxial geometry TOF analyzer employed in this
work is shown schematically in Fig. 2. Ths system
employs a single-stage mirror and two-stage ion accel-
eration. The annular detector for the reflected ions is
located immediately adjacent to the mirror, and the
grid which defines the field at the front of the multiplier
also provides the boundary of the mirror. An ion guide
wire occupies almost the entire drift space between the
source and the back of the detector for reflected ions.
The guide-wire voltage is controlled from the data
system and is programmed in proportion to the acceler-
ating voltage. Ion flight distances for the three
analyzed5 used in these studies are summarized in
Table 1.
A schematic diagram of the electronics used to
provide delayed extraction of ions from the two-stage
source is shown in Fig. 1. In this arrangement, three
important parameters are independently variable.
These are (i) the intensity and direction of the electric
field in the ion source during and immediately after ion
production, (ii) the time delay between ion production
and application of the extraction field, and (iii) the
intensity of the extraction field. The high voltage sup-
plies are independently variable up to 30 kV and are
computer controlled via 16 bit digital-to-analog con-
verters (DACs). The time delay is set by a computer-
controlled delay generator and the rise-time for

Sample Linear
plate Extraction
Timed ion detector
grids
\ A
I
selector
\ Reflector i
? 1"111111"
I
I

-.= i
Pum;ing
Camera
Beam guide
I11111111111
Reflector
detector
Pumping
Figure 2. Schematic diagram of time-of-flight mass analyzer employed in this work.
1046 DELAYED EXTRACTION MALDI TOF MASS SPECTROMETRY

RESULTS AND DISCUSSION

Table 1. Effective flight paths for the mass analyzers
employed in this work
Analyzer Linear (m)
RP 1.3
EL 2.0
XL 4.2
switching on the extraction field is 1:ominally 20 ns.
This system is applicable to both linear and reflecting
analyzers.
Materials
Matrix materials were purchased from Aldrich (Mil-
waukee, WI, USA) and used without further purifica-
tion. Solvents and concentrations for the matrices
were: a-cyano-4-hydroxycinnamic acid (CHCA),
10 g/L in 1:1acetonitrile +water, sinapinic acid, 10 g/L
in 1:2 acetonitrile+water, and a mixture of 2,5
dihydroxybenzoic acid and 5-methoxysalicylic acid as
described by Karas et a1.16 Peptide and protein
standards, angiotensin I ,bovine insulin, cytochrome c,
ribonuclease B, and carbonic anhydrase were pur-
chased from Sigma and used without further purifica-
tion. Samples were diluted to 1-5 pmol/pL final con-
centration with the matrix preparations and 0.5-1 pL of
the solution was placed on the sample plate accommo-
dating 100 samples in a 10 X 10 format.
The results of a series of experiments using angiotensin
Reflector (m) I as a test compound are summarized in Fig. 3 for all
2.0 three analyzers run both in static mode and using
3.0 delayed extraction. The enhancement in resolution of
6.6
the molecular ion region of angiotensin I is plotted
from top to bottom as a function of the analyzer length
(RP, EL, XL) and from left to right as a function of
static versus delayed extraction. The matrix was a-
cyano-4-hydroxycinnamic acid (CHCA). The experi-
mental conditions were optimized independently for
each instrument. (i) Measurements done using the
static linear mode (data not shown) indicated that the
resolution is more dependent on laser intensity than on
the geometry of the analyzer. In all cases the isotope
peaks could be resolved near the laser intensity thres-
hold for production of sample ions, but at higher
intensities the isotopes were not resolved in any of the
analyzers. (ii) With the static reflector (column 1,
Figure 3), mass resolution was less dependent on laser
intensity, the resolution increasing as a function of the
length of the analyzer. (iii) The addition of delayed
extraction further enhanced the resolution obtained
from each analyzer. As shown in the middle column of
Fig. 3, isotope resolution could be obtained-in the
linear mode and improved with analyzer length. (iv)
The combination of delayed extraction and a reflector
(right-hand column, Fig. 3) illustrates the enhanced
resolution as compared with column 1. In this latter
mode, the resolution is remarkably tolerant of higher

I I
(RPI- 1 lRPl
Res.:4000 - Res.:4600

1 %
I I I I I I I I I I I t I I

I
El I m- 11 E l
I
Res.: 5800 Res. :6600 - Res.23600
1

Iv J
I I 1 I I I 4 I I I I I I I I I I I

1290 1300 1290 1300 1290

m/Z m/Z
Figure3. Comparison of the resolution and signal-to-noise (S/N) ratio obtained for the molecular ion region of angiotensin I
(MH', monoisotopic m / z = 1296.68) in three different operating modes (static linear, delayed extraction linear and delayed
extraction reflector) as a function of the analyzer geometry (RP, EL, XL) as given in Table 1. Matrix: a-cyano-4-hydroxycinnamic
acid.
 DELAYED EXTRACTION MALDI TOF MASS SPECTROMETRY 1047

laser irradiance. As a consequence, signal-to-noise

ratio with delayed extraction is substantially improved im
compared to static MALDI. For example, in the spec- ./I. Res.: 5000
trum obtained from delayed extraction with the XI,
system in reflector mode, the major peaks were
approaching saturation at 1V full scale and S/N ratio
was about 1000. As can be seen from Fig. 3, the
resolution in all three operating modes increases with
increasing drift distance.
Examples of results on a somewhat larger peptide,
ACTH clip (18-39), in CHCA matrix are shown in Fig. Res.: 6000
4. Frames (a) and (b) compare the resolution and S/N
ratios for the molecular ion region of ACTH clip (18-
39) obtained using static ion extraction in the reflector
mode on the XL with that obtained with delayed
extraction in the linear mode on the RP. The effective
flight path of the RP in the linear mode is five times less
than that of the XL in the reflector mode, yet the
observed resolution is similar. In addition, the delayed
extraction (DE)-MALDI mass spectrum exhibits a bet-
nlhl Res.: 12500

ter S/N ratio. Similar results have been obtained on all

of the peptides tested. Isotopic resolution can be routi-
nely obtained up to about m l z 3000 in the linear mode
using the smallest instrument, and up to about m / z 5710 5720 5730 5740 5750 5760
5000 with the largest. With the reflecting mode, resolu- d Z
tion for peptides vanes from about M/AM (FWHM) =
Figure 5. Mass spectra of the molecular ion region of bovine insulin
4500 with the short flight tube to more than 10 000 for (average) M,= 5733.5) obtained with delayed extraction in the reflec-
the largest analyzer (Fig. 4(c)). tor mode of the RP, EL and XL instruments, respectively, with
Performance of these reflecting analyzers with sinapinic acid matrix, illustrating the increasing resolution as a
delayed extraction for bovine insulin in sinapinic acid is function of analyzer length.

(4 I
#

Res.: 5100

(M+H)+
d r

(M+2H)Z+

I -25Vbias I

(M+H)+

2455 2465
d Z
2475
I (M+2H)2+

Figure 4. Examples of the mas spectra of ACTH clip (18-39) (MH' ,

1000 3000 5000 7000
monoisotopic mlz = 2465.2) obtained with (a) static reflector XL, (b)
delayed linear RP, and (c) delayed reflector XL systems. The delayed d Z
extraction-linear R P with a 5X shorter effective flight path exhibits
similar resolution and improved SIN ratio as compared with the static Figure6. Mass spectra of bovine insulin from a linear analyzer RP
reflector XL. Enhanced resolution (>lOOOO) may be obtained by showing matrix ion suppression by delayed extraction as a function of
combining the extended flight length of the XL with delayed extrac- the initial retarding field; field free (0 V), -25 V and -50 V, respecti-
tion (c). Matrix: a-cyano-4-hydroxycinnamic acid. vely. Matrix: a-cyano-4-hydroxycinnamic acid.
1048 DELAYED EXTRACTION MALDI TOF MASS SPECTROMETRY

[M+H~+ 1 (M+207)'

2000 6000 10000 14000

d Z
Figure7. Delayed extraction linear mass spectrum of cytochrome c (Mr= 12360.1) in linear R P
analyzer. The improvement in resolution is highlighted by the separation of the two sinapinic acid
+
adducts differing by 18 mass units (M 207, M + 225, inset). The sample was biased 70 V below the
extraction grid prior to application of the extraction field.

shown in Fig. 5. With the shortest analyzer (RP) the
isotopes are not resolved, but with the intermediate
system (EL) the isotopes are clearly resolved, corres-
ponding to a resolution (M/AM) (FWHM) of slightly
more than 6000, while in the largest system (XL) the
isotopic peaks are resolved nearly to baseline. In this
latter case the measured resolution, M/AM (FWHM) =
12 500.
For larger proteins the isotopes are not resolved, but
nearly isotopically limited resolution can be obtained
with delayed extraction in both linear and reflecting
analyzers, and the results are almost independent of the
drift distance. This is due to the fact that all the
analyzers in the delayed extraction mode have an
inherent resolution specification of greater than M/AM
(FWHM) = 3000, while the isotopically limited resolu-
tion of larger proteins is approximately M/AM
(FWHM) = 2000.
One of the problems that is frequently encountered
in application of MALDI to larger proteins is that the
intense matrix peaks, and associated fragments and
neutrals may interfere with the detection of larger
proteins at low levels. Ion gating systems have been
developed to overcome this problem, and the analyzers
used in the present work are equipped with a deflection
gate for removing unwanted low-mass ions, and vari-
able voltage detector gates for preventing detector
saturation by low-mass neutrals. Nevertheless, chemi-
cal noise may still limit the sensitivity for higher-mass
species. We have found with delayed extraction that
the matrix background can be very efficiently sup-
pressed by applying a retarding field (reverse bias) to
the sample prior to extraction. An example is presented
in Fig. 6, where mass spectra of bovine insulin were
recorded in CHCA matrix at three different initial
sample potentials using delayed extraction with the
extraction field applied about 700ns after the laser
pulse. The same spot was irradiated in all the three
examples. Figure 6(a) corresponds to approximately
zero field prior to ion extraction. Equally strong signals
from the peptide and matrix are observed. In Fig. 6(b)
the sample was biased approximately 25 V below the

27000 28000 29000 30000 31000 32000

d Z
Figure 8. Delayed extraction linear MALDI mass spectrum of carbonic anhydrase (M, = 29 024) on RP
analyzer illustrating the enhanced resolution which may be obtained for higher mass proteins. Matrix:
9 : 1 2,5 dihydroxybenzoic acid + 5-methyoxysalicylic acid.
 DELAYED EXTRACTION MALDI TOF MASS SPECTROMETRY
Res.: 1100 -
(RNAse A ) '
13000 14000
d z
Figure 9. Delayed extraction linear MALDI mass spectrum of RNAse B acquired in the linear mode on
the RP analyzer. Peak at mlz 13 683 corresponds to the protonated unglycosylated form (RNAse A).
The other major peaks are assigned to the various glycoforms as indicated. O-mannose, D-N-
acetylglucosamine.
grid potential. The matrix ions are slightly less abun-
dant, and, in particular, monomer ions of the matrix
are suppressed. Figure 6(c) illustrates the effect of
applying a 50 V reverse bias on the sample; the matrix
ions are completely absent from the mass spectrum.
Mass resolution in all cases was in the range M/AM
(FWHM) = 950-1000, corresponding approximately to
the width of the isotopic distribution.
This method for suppression of matrix background is
also effective for larger analytes. As an illustration, a
DE-MALDI mass spectrum of cytochrome c in a sina-
pinic acid matrix is shown in Fig. 7. Sample voltage was
about 70 V below the grid potential prior to application
of the extraction field. The normal low-mass gating
system was not used in these measurements. The back-
ground is very clean and matrix ions are of low abun-
dance in the mass spectrum, even using sinapinic acid,
which normally gives a very strong matrix background.
Mass resolution in this example is M/AM (FWHM) =
1150. As the peak labels indicate, it was possible
to resolve two matrix adducts to the molecular ion
+
differing by only 18 mass units (M 207 and M 225, + by resolution of the time measurement rather than by
respectively).
The largest protein to date for which data have been
obtained, that clearly show the advantages of DE, is
carbonic anhydrase; an example is shown in Fig. 8. In
this case the measured resolution is M/AM (FWHM) =
840, and a fragment or impurity peak 44 mass units less
than the molecular mass is partially resolved (-CO,).
Since the loss of small neutral fragments has frequently
been observed in MALDI of small proteins,' the mea-
sured resolution may be limited by unresolved frag-
ments such as that produced by loss of ammonia, or
carbon dioxide.
The advantages of DE are more readily apparent
with more complex samples. Figure 9 presents a
DE-MALDI mass spectrum of ribonuclease B, a small
glycoprotein with an inhomogeneous, high-mannose
type glycan. The matrix was sinapinic acid. The mass of
the unglycosylated protein, ribonuclease A, is
13 682 Da, and it is present as a minor component. The
glycoforms are easily identified in this mass spectrum as
(GlcNAc),Man, , (GlcNAc),Man, , (GlcNAc),Man, ,
1049
15000 16000
(GlcNAc),Man,, and (GlcNAc),Man, , with calculated
masses of 14899.1, 15 061.3, 15 223.4, 15 385.5, and
15 547.7, respectively. The last component is often
undetected under continuous extraction MALDI
~0nditions.l~Measured mass resolution for all five gly-
coforms is in excess of MIAM (FWHM) = 1100. The
unglycosylated protein is slightly out of the focus and
the resolution is 'only' M/AM (FWHM) = 900.
CONCLUSIONS
As predicted by theory, delayed extraction
MALDI-TOF provides substantial improvement in
resolution over that obtained under static conditions
for both linear and reflecting analyzers. For the three
analyzers tested, the resolution in reflector mode was
very nearly proportional to total effective ion flight
path, but the resolution of the linear analyzers
increased rather more slowly with increasing flight
path. Using delayed extraction, the mass resolution for
peptides was apparently limited in the shortest analyzer
the initial velocity distribution, but with the larger
analyzers higher order terms which are independent of
flight distance become more important.
In addition to improved mass resolution, delayed
extraction provides other improvements in the quality
of MALDI spectra, which are more difficult to quan-
tify. These include a lower level of chemical noise due
to fragmentation in the ion accelerating fields, reduc-
tion or elimination of matrix background, and signifi-
cant reduction in the dependence of ion flight times on
laser intensity. With delayed extraction, peak widths
and centroids are much less dependent on the absolute
intensity of the ion signal; thus, it is not necessary to
operate near the threshold for ion production to obtain
both high resolution and improved mass accuracy.
In this work, we have demonstrated advantages of
delayed extraction MALDI-TOF for applications to
peptides and proteins. We anticipate that this new
technique may prove to be of even greater value for
applications to more fragile biopolymers such as oligo-
nucleotides, where the performance of conventional
1050 DELAYED EXTRACTION MALDI TOF MASS SPECTROMETRY
MALDI has not been nearly as good as it is for
polypeptides. Work on the application of DE-MALDI
to DNA sequencing is in progress and results will be
presented elsewhere.
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