Article

Quantitative matrix-assisted laser desorption/ionization mass spectrometry

Division of Endocrinology, Metabolism and Diabetes, School of Medicine, University of Colorado Denver, Mail Stop 8106, 12801 East 17th Avenue, Aurora, CO 80045, USA.
Briefings in Functional Genomics and Proteomics (Impact Factor: 3.67). 10/2008; 7(5):355-370. DOI: 10.1093/bfgp/eln041
Source: PubMed

ABSTRACT

This review summarizes the essential characteristics of matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF MS), especially as they relate to its applications in quantitative analysis. Approaches to quantification by MALDI-TOF MS are presented and published applications are critically reviewed.

Full-text

Available from: Heinrich Roder, Mar 04, 2015
Quantitative matrix-assisted laser
desorption/ionization mass
spectrometry
Ma rk W. Dunc an, He in ri ch Rode r and Stephen W. Hunsucker
Abstract
This review summarizes the essential characteristics of matrix-assisted laser desorption/ionization (MALDI) time-
of-flight mass spectrometry (TOF MS), especially as they relate to its applications in quantitative analysis.
Approaches to quantification by MALDI-TOF MS are presented and published applications are critically reviewed.
Keywords: quantificati on; quantitativ e anal ysis; MALD I; mass spectrometry ; biomark ers
OVERVIEW
Since its inception in 1987 [1], matrix-assisted laser
desorption/ionization (MALDI) time-of-flight mass
spectrometry (TOF MS) has been applied for the
analysis of a wide range of biomolecules. Initial
applications were almost exclusively for the qualita-
tive analysis of biopolymers because MALDI-TOF
MS provided a fast and accurate approach to mole-
cular mass and purity information: Is my material the
right mass and is it free from contaminants? Because
of the speed of analysis, ease of use, relative low
equipment cost, ease of data interpretation and
limited potential for cross-contamination between
samples/users, MALDI-TOF MS systems were con-
sidered walk-up instruments where investigators run
their own samples and determine, within minutes,
whether they were on the right track with their work.
There were, however, two specific areas of
application where MALDI-TOF MS was initially
viewed as impractical: i.e. the analysis of low mass
analytes and quantitative applications. Low mass
analysis is complicated by the vast molar excess of the
matrix that can swamp any analyte-specific signal in
the low m/z region of the spectrum. Quantitative
analysis was viewed as implausible because crystal-
lization does not yield a uniform distribution of the
analyte and matrix across the target surface and this
gives rise to regions where the analyte signal is
especially intense relative to other locations on the
target surface. MALDI MS was therefore viewed
as inherently irreproducible because, for a given
amount of analyte loaded onto the target, the
measured ion intensity varies.
Subsequently, it has been shown that both these
perceived limitations can be overcome and quanti-
fication can be routine, both for low and high mass
analytes. Now, an extensive list of published appli-
cations includes quantification of amino acids, lipids,
natural products, drugs, polymers, herbicides, metab-
olites, toxins, oligonucleotides, carbohydrates, pep-
tides and proteins. (Selected applications from these
areas are discussed at the end of this review.) Here,
we focus on the quantitative applications of MALDI
and illustrate applications relating to both low and
high-molecular mass analytes. We also discuss the
strategies for applying MALDI to relative and
absolute quantification. While MALDI is most com-
monly combined with TOF analysers, other analyser
options are possible and applications employing these
are also presented. Our aim is not to provide an
exhaustive catalogue of published applications, but
to discuss the general principles and to illustrate the
Mark W. Duncan, Division of Endocrinology, Metabolism and Diabetes, University of Colorado Denver, Mail Stop 8106, 12801 East
17th Avenue, Aurora, CO 80045, USA. Tel: (303) 724-3343; Fax: (303) 393-5271;
E-mail: mark.duncan@ucdenver.edu
MarkW. Duncan is a professor in the School of Medicine, University of Colorado, Denver. He also holds an appointment as professor
within the Obesity Research Center, King Saud University, Riyadh, Saudi Arabia.
Heinrich Roder is chief technology officer, Biodesix Inc., Broomfield, CO, USA.
StephenW. Hunsucker was previously an assistant professor at the University of Colorado, Denver, but he has recently relocated to
Biodesix Inc, CO, USA.
B RIEFINGS IN FUNC TIONAL GENOMICS AND P ROTEOMICS . VOL 7. NO 5. 355 ^370 doi: 10.1 093/bfgp/eln0 4 1
ß The A ut ho r 2008. Pub lished by Oxford Universit y Press. All rights reserved . For perm iss i ons, please e mail: jou r nals .per missi ons@oxford j ou r na ls.o r g
Page 1
central considerations in quantitative MALDI by way
of examples. As with any approach, MALDI offers
some advantages and some limitations relative to the
available alternatives. Performance characteristics of
the system and the assay such as throughput, pre-
cision, accuracy, cost, sensitivity, selectivity, linear
dynamic range, and, of course, practicality, are dis-
cussed for some of these applications.
A REVIEW OF THE ESSENTIAL
FEATURES OF MALDI A S THE Y
RELATE TO QUANTIFIC ATION
For a typical MALDI analysis, solutions of a sample
(pure or a mixture; 10 mM) and matrix (10 mM)
are pre-mixed and a small volume (1 ml) is applied
directly to the sample plate. (The matrix is selected
such that it has a large extinction coefficient at the
emission wavelength of the laser.) The solvent is
generally allowed to evaporate at room temperature
and pressure to yield a heterogeneous crystalline
surface. Inside the mass spectrometer the laser is
pulsed onto the crystalline surface and the matrix
immediately sublimes, concomitantly desorbing and
ionizing the analyte molecules. The m/z values for
the analyte ions are then measured. The MALDI
mass spectrum is a plot of intensities on the ordinate
axis and the corresponding mass/charge values on
the x-axis. The intensity scale is the relative
abundance of ions, but generally the region below
about m/z 500 is not shown because it is saturated
with signals from matrix-derived ions. MALDI mass
spectra of pure compounds are normally dominated
by a single ion corresponding to the protonated
molecule; multiply charged ions are rare, except for
very large molecules and even then they are usually
of low relative abundance. MALDI spectra of
mixtures typically show multiple ions corresponding
to the protonated forms of some if not all of the
molecular components present in the sample. It is
noteworthy that peak heights for equimolar loadings
of different analytes may vary significantly. The
proclivity of MALDI to yield singly charged ions is in
contrast to electrospray and offers significant advan-
tages in quantification, especially for mixtures.
Characteristically, there is substantial variability in
the noise level, baseline and peak intensities in a
collection of MALDI spectra generated from the
same sample. Variations in ion current are observed
with consecutive laser shots fired at the same position
on the target surface (shot-to-shot reproducibility),
across different locations on the target surface
(region-to-region reproducibility) and between
identical loadings of the same sample onto different
targets (sample-to-sample reproducibility). To mini-
mize this variability, multiple spectra are usually
acquired from different locations across the target
surface and these are averaged (or added) to yield a
more representative spectrum.
Fluctuations in laser power and changes in
detector response alter signal intensity, but the
primary contributor to variability is heterogeneous
incorporation of the analyte into the co-crystallized
matrix–analyte complex. This results in ‘hot-spots’
on the target surface where the ratio of analyte to
matrix is optimal and where the analyte signal is high
relative to other locations. This variability can be
circumvented, at least in part, by pre-mixing the
sample and matrix solutions and by facilitating faster
crystallization times. Faster crystallization generates
smaller crystals and a more homogeneous incorpora-
tion of the analytes into the crystalline lattice. For
example, when 2,5-dihydroxybenzoic acid (DHB) is
allowed to crystallize slowly (i.e. from predominantly
aqueous solutions at room temp and pressure) large
needle-like crystals form and sample incorporation is
highly variable. When crystallization is rapid (i.e.
when the matrix is prepared in a volatile solvent and/
or the target is dried at elevated temperature and/or
reduced pressure) the resulting crystals are small and
crystallization of the analyte–matrix is much more
uniform [2].
Competitive ionization/ion suppression is an
additional factor that can obliterate any attempt to
quantify by MALDI, especially in complex samples.
In a mixture, some analytes will have higher affinities
for charge than others (e.g. R-terminated peptides >
K-terminated peptides) and will more successfully
compete for the available protons [3]. Therefore,
when quantifying across a series of samples it is
essential to keep the sample composition (or sample
matrix) constant. Sample preparation methods
should aim to reduce the complexity of the
sample, thereby minimizing the background and
eliminating potentially interfering peaks, but at the
same time, these methods must be highly reprodu-
cible and minimize the potential to introduce
contaminants that result in higher variability and
adversely influence signal intensity (i.e. ion suppres-
sion). Although ion suppression is not unique to
MALDI, in electrospray LC-MS applications online
separation helps minimize the potential for other
356 Duncan et a l.
Page 2
sample components to influence the ion current of
the target analyte. In contrast, few quantitative
MALDI applications employ ‘online’ separation of
the analytes; rather, multiple analytes are present
on the target at the same time and actively compete
for the available charge for each desorption/ioniza-
tion event (i.e. laser shot).
There are, however, several very positive attri-
butes of MALDI, not the least of which is its
sensitivity. There have been several estimates of the
amount of material required to generate a spectrum,
but it is clear that attomoles or less are sufficient to
generate a good quality spectrum [4]. A quick survey
of the literature also illustrates the remarkable
versatality of this ionization method: i.e. thermally
labile, low mass analytes; involatile, high mass
biopolymers; even alkanes and polyethylenes [5]
can all be ionized by MALDI. Additional advantages
of the approach include the ability to analyse com-
plex mixtures, the potential for high-throughput, the
speed of the analysis, ease of automation and the low
cost per analysis.
In summary, MALDI offers some important
attributes, but its application to quantitative analysis
requires careful optimization of the experimental
parameters and a good understanding of how
confounding factors could compromise a study. In
particular, sample preparation (e.g. choice of matrix
compound, concentration, solvents and crystalliza-
tion conditions) is critical and must be optimized in
order to reduce the variability introduced during this
step. It is also important to acquire and average many
single-shot spectra from several positions within a
given sample spot to gain representative sample data.
Ideally, the laser power should be automatically
adjusted to limit the acceptable analyte and internal
standard signal intensity to below saturation, but
above background noise. Consequently, criteria for
spectral acceptance based on minimum and max-
imum peak height and the signal-to-noise ratio
should be established and adhered to before the
spectra are averaged.
Q UANTITATIVE ASSAYS IN
BIOLO GIC AL INVESTIGATIONS:
GENE RAL CON S IDERATION S
Why quantify?
Qualitative analyses of complex mixtures are often
made, but without a quantitative dimension these
determinations are of limited value and can be
misleading. For example, by generating a list of the
components in a sample the analyst is indicating the
presence of specific species and implying the absence
of others. But it is important to appreciate that the
exclusion of an analyte from the list may simply
mean that it was not recovered, was not character-
ized, or that it was present below its limit of
detection. Lists must therefore be interpreted with
caution and this is perhaps no more true than in
proteomics: a field rapidly transitioning from a
qualitative to quantitative science. When analysing
complex biological samples it is important to have
reliable measures of absolute analyte levels, or at least
precise determinations of the differences in analyte
levels between two or more samples.
Quantitative relationships
Quantification requires that the magnitude of the
measured property—in this case the ion current—is
proportional to the concentr atio n or amount of the
analyte in the sample matrix. Ideally, from sample to
sample, the process should be reproducible and the
response identical for equal amounts of analyte.
However, as already discussed, there is significant
variability in the MALDI signal even when the same
sample is analysed repeatedly. The process of
applying the sample to the target is irreproducible,
the target surface is heterogeneous and changes in
laser intensity along with fluctuations in detector
response all contribute to signal variability. There is
therefore a poor correlation between uncorrecte d signal
intensity and analyte amount. But, there are ways to
optimize the experimental conditions and design
experiments that allow for meaningful quantitative
comparisons by MALDI. These approaches are
common to other areas of quantitative analysis and
their essential features are reviewed below.
Relative quanti fication withou t an interna l standard
(or pr o fi le analy sis)
For a given sample run repeatedly, the relative signal
intensities within the mass spectrum are surprisingly
constant. Because of this, comparisons can be made
between similar samples (e.g. a series of plasma
samples) and the changes in relative signal intensities
against an essentially ‘constant’ background can
provide useful information. No meaning is ascribed
to absolute peak intensities and typically hundreds of
unidentified features pe r profile are recorded without
the incorporation of any internal standards. The
approach utilizes classification algorithms to examine
Quantitativ e MALDI-T OF MS 357
Page 3
spectra in a reproducible manner in order to
determine the differences between populations.
The most common iteration of this approach is
MALDI protein profiling [6–13].
MALDI profiling offers exceptionally high-
throughput and has therefore become an attractive
tool for the discovery and validation of biomarkers.
Throughput is critical because for any approach to be
of value in clinical science it must be feasible to
analyse hundreds (or even thousands) of samples so
that sufficient representation of the natural popula-
tion variation in human-derived samples can be
determined. In addition, the method must be
sufficiently precise to observe relative differences
between distinct populations.
Profiling has now been applied in thousands of
studies, on a broad range of sample types, and across
a wide range of disease conditions. (Some specific
examples using this approach are discussed later in
this review.) Early studies based on this approach
showed excellent predictive value, but they were
soon criticized, in large part because of problems in
the study design and implementation [14–16].
However, profiling can be used to make meaningful
comparisons if rigid protocols for both experimental
design and data analysis are followed. Specifically,
pre-analytic variables must be controlled and the
available samples split into a training set (i.e. to
develop a classification algorithm) and a blinded test
set(s) (i.e. to verify the classification is sensitive and
specific).
Albrethsen [17] has reviewed the critical issue of
reproducibility in protein profiling and has discussed
approaches to improve the analytical performance of
MALDI protein profiling including automated
sample processing, pre-fractionation, immunocap-
ture, pre-structured target surfaces, standardized
matrix (co)crystallization, internal standard peptides,
quality-control samples, replicate measurements, and
improved algorithms for normalization and peak
detection. Protein profiling can be combined with
techniques like LC-MALDI and other fractionation
approaches to help accommodate the large dynamic
range of protein abundances in biological fluids such
as serum, plasma and urine. This can enhance
coverage, but the reproducibility and throughput
of these additional steps must be carefully evaluated.
Several strategies have been reported [18–20], but
these are yet to be comprehensively evaluated and
other reproducible, high-throughput sample fractio-
nation methods are certainly needed.
Relative quanti fication with an interna l standard
With this approach a fixed amount of an exogenous
component (i.e. an internal standard) is added to all
samples and peak heights or areas of individual
endogeneous analytes are measured relative to it.
Signals (discrete m/z values) that increase relative to
that of the internal standard when two or more
samples are compared can be considered to reflect
increases in the amounts of the analyte; decreases
correspond to reduced levels. Incorporation of the
standard adjusts for some of the variability inherent
in the process of sample preparation, ionization and
ion detection. A single set of samples run under
identical conditions, at the same time, and all
containing the same amount of internal standard, is
considered a single experiment. In biological studies,
the objective is frequently to establish statistically
significant differences between populations: i.e. to
establish that analyte levels in one group are different
to another. This exercise can be undertaken without
knowing the absolute amounts and by simply
comparing relative amounts between two or more
samples/groups (e.g. different by environment, drug,
age or disease). Because absolute quantities are not
determined by this approach, comparisons between
different experiments must be made with extreme
caution. There is no common point of reference
between the two different studies, the studies may
have been performed at different times, with
different instrument conditions/settings or even on
different instruments by different operators.
Although the absolute amount of the internal
standard need not be known, it must remain
constant, must generate a signal that can be measured
precisely (i.e. above the limit of quantification, but
not saturated) and must not significantly suppress the
signals for the analytes in the sample. In addition, it is
important that the mass of the standard be close to
that of the analyte(s) of interest, but be distinct from
that of any sample components. Multiple analytes
can be compared against a single internal standard by
this approach, but there will be some compromise in
quantitative precision for some.
Abso lu te quanti fication with an interna l standard
Relative changes can be transformed to absolute
amounts through a modification of the above
procedure. In addition to the samples under
evaluation, a calibration curve is prepared that also
contains the same (fixed) amount of the internal
standard and varying amounts of a single specific
358 Duncan et a l.
Page 4
analyte of interest. In this way, the constant of
proportionality for one analyte can be established
and ion abundance (or intensity) ratios can be
converted to absolute amounts. If the objective is
to determine absolute amounts for several different
species, separate calibration curves should be con-
structed for each analyte because the constant of
proportionality will be different in each instance.
The internal standard serves as a mimic of the
analyte and therefore its chemical and physical
properties should match those of the analyte as
closely as possible. In this way, the internal standard
will behave in a manner close to or identical to the
analyte at all stages of the assay process (e.g. extrac-
tion, crystallization, ionization/ion suppression).
Although the conversion from relative to absolute
data requires substantial additional effort, absolute
quantification allows results to be reliably compared
across experiments or between laboratories.
Abso l ute quan ti fication by standard addition
When the matrix is complex or highly variable
between samples, as discussed previously, other
components can influence instrument response. In
these circumstances, standard addition analysis is the
preferred approach. Here, each unknown sample is
divided into two or more portions and known
amount(s) of the analyte are added to these
portions (i.e. a spike). The original sample and the
spiked samples are then analysed. The samples
incorporating the spike(s) will show greater analytical
responses than the unadulterated sample due to
the additional amounts of analyte added to them.
These response differences provide a calibration
point (or points) to determine the analyte concen-
tration in the original sample. (The process of
calculating the amount in the original sample is
shown in Figure 1.)
FITNESS-FOR-PURPOSE
In biology, the most common objective is to
compare analyte values between distinct populations.
In this setting, the individual data points are
comprised of the analyte values, the associated
errors in the analytical method (i.e. instrument
errors þ sample preparation errors) and the inherent
biological variation in the samples. For any analytical
method, the ‘purpose’ should be precisely defined
and all available resources optimally utilized to
address that purpose—nothing more or less. The
focus should be on the efficiency to deliver what is
required for a specific purpose. Resources should not
be squandered on attempting to produce a higher
quality product than is necessary, nor should they be
under-utilized to yield a method that is inadequate
for the intended purpose. One interpretation of this
principle is that, given the variations inherent in
biological samples, it is unnecessary to measure any
analyte with a precision greater than the variability
seen in the population. The utility of MALDI as a
quantitative tool should be considered in this
context; every task is different and what the
method can offer—its strengths and weaknesses—
must relate to the problem at hand. There are
unquestionably instances where MALDI provides
extremely valuable data that are well suited to the
intended purpose and in subsequent sections we
discuss the ways in which quantitative MALDI has
been applied to the study of biological samples and
comment on their fitness-for-purpose.
THE INT E RNAL STANDARD
Internal standards are incorporated into quantitative
mass spectrometric analyses to compensate for
systematic and random errors during analysis, but
their inclusion in MALDI assays, and certainly those
aimed at absolute quantification, is arguably more
important than in most other applications.
The internal standard is selected based on its
ability to mimic the chemical and physical properties
of the analyte of interest throughout all stages of the
analytical process. For quantification of low mole-
cular mass analytes, either by GC-MS or LC-MS,
Figur e 1 : This figure illustrates the process of convert-
ing analyte signal response to amount by the standard
addition method.
Quantitativ e MALDI-T OF MS 359
Page 5
the optimal internal standard is a stable isotope-
labelled form (isotopomer) of the analyte. The same
principle holds true for MALDI applications: stable
isotope-labelled standards are optimal, at least in the
low mass region. Such a standard guarantees identical
extraction, crystallization and gas-phase behaviour.
But, isotopomers become increasingly impractical as
the molecular mass of the analyte increases. For
example, to generate a stable isotope labelled form of
a protein with a molecular mass of 50 000 and with
an isotopic distribution that is distinct from that of its
native counterpart (i.e. non-overlapping) is complex
and costly. In fact, it is by no means certain that this
additional cost and effort is warranted and that it will
result in a significant improvement in precision rela-
tive to that offered by the careful selection of a more
practical alternative. For example, a structural analo-
gue internal standard that maintains the critical solu-
tion phase properties (e.g. acid–base properties and
solubility) and that is close in mass to the analyte, but
sufficiently distinct to allow for appropriate resolu-
tion, is certainly more practical to design and pre-
pare, and may not offer any significant compromise
in either precision or accuracy. A structural analogue
of a peptide can be prepared where amino acids
with similar chemical and physical properties are
interchanged (e.g. substitution of aspartic acid for
glutamic acid or alanine for glycine) and these have
been shown to compensate for crystallization irregu-
larities and subsequent desorption and gas phase
effects. The importance of a vicinal mass for the
internal standard has also been reported based on
experiments with a series of acylcarnitines having a
fixed charge site and growing alkyl chain length [21].
In the final analysis, however, the acceptable degree
of difference between the internal standard and the
analyte is often only determined by experimentation
and must be viewed in the context of the analytical
objective.
In multi-component assays, a single compound
sometimes serve as the internal standard for several
analytes, but because of their chemical and physical
diversity, this can lead to differing responses to
variations in experimental conditions. For example,
different analytes will give different recoveries during
extraction and different responses during crystal-
lization and ionization. One standard is unlikely to
optimally correct for all these factors, but the hope is
that important biological variations will not be
obscured by the sources of variability introduced in
the experimental design.
DATA ANALYSIS: SPECIAL
CONSIDERATIONS IN PROFILING
Although profiling might not be considered an
orthodox example of quantification, it is, nonetheless,
a quantitative exercise. Profile analysis is one of the
major applications of MALDI-TOF MS and there-
fore we include discussion of the critical role of the
software in the process; to our minds it has rarely
received due attention.
It is important that the necessary spectral pre-
processing methods are performed in such a way that
they do not introduce bias into the resulting data sets.
Different mass spectra can show changes in intensity
and shifts in the m/z axis, often in a non-linear
manner. It is therefore important that mass spectra are
standardized before the peak picking process begins,
but many commercial algorithms begin the analysis
with peak lists generated prior to standardization.
Further, in complex mass spectra, peak widths can be
influenced by the presence of proximate peaks and
the peak picking and the peak intensity measurement
process must take these local effects into account.
With rigorous spectral pre-processing consisting of
regional (in terms of m/z) background determinations
and noise estimation, alignment using common peaks
in training sample sets and normalization methods
using partial ion current techniques, MALDI mass
spectra are highly reproducible across laboratories and
even across slightly different hardware [22].
Coefficients of variation (CVs) for each m/z
value within technical replicates of biological samples
such as crude serum vary markedly and typically
range from a few percent to unusable. The role
of normalization in this process cannot be over-
estimated. The standard approach to normalization
is based on the total ion current (TIC). Here,
each spectrum is divided by the sum (or more
accurately the square root of the sum of the squared
intensities), but this approach is not optimal for
several reasons. First, large peaks are the most
important contributors in the TIC normalization of
spectra, but often they contain little relevant
quantitative information, they are saturated, and/or
their intensities fluctuate dramatically from sample
to sample. When employing TIC normalization,
these fluctuations are telegraphed via the normal-
ization process to other peaks, thereby rendering
the whole analysis less sensitive and artificially
increasing CVs. Even worse, TIC normalization
can lead to an over-training of classification
when large peaks present in one clinical group are
360 Duncan et a l.
Page 6
less prevalent in another. (As an example, lysates
prepared from tumor, rather than normal tissue,
may contain higher levels of haemoglobin and other
blood products.) In these circumstances, TIC
normalization inappropriately suppresses all peaks in
the group. An alternative normalization approach is
based on partial ion current (PIC) and uses only a
suitably selected subset of peaks (i.e. those whose
CVs are below a threshold) for normalization. In its
most trivial implementation, highly fluctuating
regions (sample to sample) are excluded from the
normalization process.
More sophisticated selection tools have been
developed based on an iterative algorithm using
CVs as selection criterion [23]. As an example,
Figure 2 compares PIC to TIC using CVs from
replicate analyses of MALDI-TOF mass spectra of
urine samples, i.e. the same sample was run 19 times
and the peak intensities of these 19 replicate runs
were compared. Based on PIC, the CVs are about
half what they would be if TIC normalization had
been adopted. This improvement in precision
increases the sensitivity of any biomarker analysis
accordingly. This section falls short of addressing
other issues. There are numerous other experimental
and computational considerations that determine the
success (or otherwise) of profiling approaches, but
they are beyond the scope of this review.
MODIFIC ATIONS TO THE
GENE RAL MALDI APPROACH
Variants of the basic MALDI approach have been
reported, most involving modifications relating to
the matrix and its application, and some of these offer
potential benefits for quantification. For example,
desorption/ionization on silicon (DIOS) allows for
the analysis of proteins and related small molecules in
the absence of matrix. In common with MALDI,
DIOS yields little or no fragmentation and is
relatively tolerant of moderate amounts of contami-
nants commonly found in biological samples. The
absence of a matrix offers special benefits in the low
mass region [23].
Gross and colleagues have reported the use
of ionic matrices in quantitative applications of
oligodeoxynucleotides, peptides and small proteins
[24, 25]. They report that good calibrations,
linearity and reproducibility were achieved over a
broad concentration range for all the tested ionic
liquid matrices (ILMs) in spite of their different
physical states. However, the SD is higher for
ILMs than for solids with visible crystals. The experi-
mental results indicate various ILMs yield different
sensitivities owing to changes in their cation
components and the slopes of the calibration curves
correlate with the inverse of the peptide molecular
weights.
Caprioli and colleagues reported on an acoustic
reagent multi-spotter that provides improved repro-
ducibility for depositing matrix onto sample surfaces.
Although primarily developed for imaging of tissue
sections, this approach may also offer benefits in
quantification. Their acoustic droplet ejector provides
better control of conditions affecting protein extrac-
tion and matrix crystallization, and offers the
ability to deposit matrix onto small surface features
accurately. Mass spectral quality and reproducibility
was found to be better than that obtained with manual
pipette spotting [26].
Gross and colleagues also developed an alternative
small droplet deposition method using an induction-
based fluidics (IBF) technique. This allowed the
investigators to dispense nanolitre drops and they
report that this improves the signal intensity and
sensitivity achieved in MALDI-MS [27].
Solvent-free or dry prepared sample methods
have also been developed and show some advantages
over traditional wet sample preparation methods
[28]. For example, solvent-free sample preparation
methods enable analysis of insoluble materials and
reportedly provide higher quality mass spectra. The
surfaces of these samples have been examined and
the authors report remarkably smooth and homo-
geneous sample morphologies [26].
Figur e 2: A comparison of CVs obtained using TIC
(open circles) and PIC (solid circles).
Quantitativ e MALDI-T OF MS 361
Page 7
There are numerous other refinements to the
general MALDI process that have the potential to
improve the precision and accuracy of quantification,
but they are yet to be assessed rigorously in a
quantitative setting.
EXAMPLES OF QUANTITATIVE
MALDI APP LICATION S FROM THE
LIT ERATURE
Quantitative MALDI has been employed to analyse a
wide range of low and high mass molecules from
many different sample types. The published applica-
tions cover the spectrum of quantitative analyses
from relative to absolute, but few of the assays have
been applied in a practical setting and even fewer are
used on a routine basis. In the text that follows, we
present some examples of where MALDI has been
employed as the ionization method and the applica-
tion is quantitative in nature. In almost all instances,
these reports amount to a proof-of-principle, rather
than an approach that is likely to be applied in a
routine setting.
Low molecular mass analytes
These studies mirror more conventional quantitative
applications of mass spectrometry (i.e. GC-MS and
LC-MS) and typically incorporate stable isotope-
labelled internal standards. In some of the first reports
in this area, we set out to demonstrate that MALDI
can be applied to the analysis of low molecular mass
analytes and yield reliable quantitative data [29].
We reported three examples: a standard curve for
3,4-dihydroxyphenylalanine (DOPA) was prepared
by using an isotopomer (i.e. [
13
C
6
]DOPA) as
the internal standard; [
2
H
16
]-acetylcholine was emp-
loyed as an internal standard for the quantification of
acetylcholine; and in the final example, the peptide
H-Ser-Ala-Leu-Arg-His-Tyr-NH
2
was quantified
by employing a structural analog as the internal
standard (i.e. Ac-Ser-Ile-Arg-His-Tyr-NH
2
). In each
instance Pearson correlations for calibration curves
(r
2
> 0.95) demonstrated that MALDI can be a viable
approach for the quantitative analysis of low
molecular mass analytes. This early work helped to
dispel the notion that MALDI could not deliver
quantitative results, but we did not undertake any
practical sample analysis at this stage.
MALDI-TOF MS has also been applied to the
quantification of lysine, alanine and glucose [30]. The
method is based on using stable isotopes as internal
standards and the authors report fast, sensitive and
reproducible quantification of these compounds. The
quantitative method was demonstrated for aqueous
standard solutions with concentrations of the analytes
between 10 mM and 100 mM. The mean SDs from
five replicate measures of each analyte were 4.3%
(lysine), 3.7% (alanine) and 3.2% (glucose). This level
of precision is impressive and comparable to that
achievable with the best of quantitative methods.
A noteworthy additional feature of this study is that
the authors also demonstrated excellent agreement
between these data and those obtained by more
conventional quantitative techniques.
In 2002, we reported a more comprehensive and
practical study in which automated MALDI-TOF
MS was used to quantify a range of components in
biological extracts and fluids [31]. Growth hormone
was measured in rat pituitary tissue; insulin in human
pancreatic tissue; homovanillic acid in human urine;
and LVV-hemorphin-7, epinephrine and norepi-
nephrine in human adrenal and pheochromocytoma
tissues. Internal standards included compounds of
similar molecular mass, structural analogues and
isotopomers. These were incorporated into each
assay and the practical limitations of quantitative
MALDI-TOF MS are discussed. Although we had
no need to routinely apply any of these assays, they
were cost-effective, fast and offered excellent
performance characteristics. Based on this study,
these or similar methods could be applied in a
routine setting.
Sleno and Volmer [32] have also addressed some
of the practical and theoretical aspects of quantifica-
tion of low mass analytes by MALDI-TOF MS.
They quantified quinidine, danofloxacin, ramipril
and nadolol by MALDI and carefully selected the
internal standards to match the structure and
fragmentation pathways of the analytes. The impact
of different matrices was also examined as well as the
role of laser energy and pulse rate. Results for the
quantification of small molecules with molecular
masses <500 Da by MALDI were generally good. In
a companion study, the same authors focused their
attention on the selection of internal standards for
low mass quantification by MALDI and discussed the
importance of a vicinal mass for the internal standard
[21]. Both these studies were performed on a
prototype MALDI-triple quadrupole instrument
equipped with a high repetition rate laser.
Several more recent reports also describe key
issues for the successful application of MALDI-TOF
362 Duncan et a l.
Page 8
MS to quantifying drugs. van Kampen and collea-
gues [33] examined such issues as the matrix (i.e.
nature and preparation), the inclusion of Li
þ
to
facilitate formation of cationized drug species,
automation and data analysis procedures. Of special
note, they utilized a high molecular mass matrix,
meso -tetrakis (pentafluorophenyl) porphyrin because
it eliminated chemical noise in the low m/z range.
Their method was applied to the quantification of
clinically relevant concentrations of lopinavir, an
HIV protease inhibitor, in extracts of peripheral
blood mononuclear cells.
Notari and colleagues [34] recently applied
MALDI-TOF/TOF MS to the quantification of
abacavir, amprenavir, didanosine, efavirenz, nevir-
apine and stavudine in the plasma of HIV-infected
patients. They used standard additional analysis and
therefore it was not necessary to incorporate stable
isotope-labelled analogues to achieve precise and
accurate quantification. Standard addition is a
powerful approach, but it requires that each sample
be analysed multiple times, and consequently is slow
and labour-intensive relative to approaches based on
the use of an internal standard and calibration curve.
The authors point out, however, that the high-
throughput and full automation achievable with
MALDI makes it feasible to adopt this approach in
a routine setting, especially when the matrix is
complex, or highly variable between samples.
Kovaric and colleagues [35] also investigated
aspects of the quantitative analysis of pharmaceutical
compounds in human plasma based on MALD-TOF
MS operated in the multiple reaction monitoring
mode. Talinolol was selected as a model analyte in
these studies and liquid–liquid extraction and protein
precipitation were evaluated, both with and without
chromatographic separation. They report acceptable
precision and accuracy when liquid–liquid extraction
was employed without any LC separation and note
that a full calibration curve and its quality control
samples (20 samples) can be analysed within a
minute. Combining LC with MALDI analysis
improves the linearity down to 50 pg/ml, but
reduces the throughput by (at least) 2-fold. These
authors argue that matrix effects are still a significant
issue with MALDI but can be monitored in a similar
way to that used for LC-MS analysis based on
electrospray.
Wagner and colleagues [36] have also highlighted
the potential for high-throughput in their studies.
These authors quantified saquinavir in human plasma
samples without prior chromatographic separation.
They report that the method was precise and
accurate, but that the spotting and crystallization
processes need to be automated and are one of the
main sources of analytical variation. They too point
out that compared to approaches requiring online
LC separation or infusion, quantitative analysis by
MALDI combined with selected reaction monitor-
ing (SRM) can be undertaken within a few seconds,
and consequently, potentially thousands of measure-
ments can be undertaken per day.
Peptid es and pro t eins
Analy sis of one or a few proteins
Peptides and proteins can be measured directly as
intact molecules, or for proteins, it is also possible to
base quantification on one or more chemical or
enzymatic cleavage products. Examples of both
approaches have been reported. Kiernan and collea-
gues [37] developed and applied a sensitive mass
spectrometric immunoassay to the quantitative
analysis of C-reactive protein (CRP) in human
plasma. In this study, multiplexed antibody-based
retrieval and MALDI-TOF MS were used to target
retinol-binding protein, C-reactive protein, serum
amyloid P component and an added exogenous
internal reference standard (i.e. staphylococcal enter-
otoxin B). The three analytes of interest and the
exogenous internal standard were isolated from 35
plasma samples using a high-throughput robotic
system and eluted directly onto the MALDI target.
Their approach allowed semi-quantitative analysis
of both retinol-binding protein and serum amyloid
P component, and absolute quantification of
C-reactive protein. The CRP values measured by
this approach were compared to CRP values deter-
mined by a high-sensitivity latex immunoturbido-
metric assay. While the values between assays
showed the same general trend, the mass spectro-
metric assay consistently returned lower numbers.
The authors speculate that the different values
between assays result from the different reagents
(antibodies and standards) used in the two
approaches.
There are several notable features to this study.
First, the automated approach—both sample pre-
paration and mass spectrometry were automated—
allows for high-throughput analysis and provides the
highest level of reproducibility (coefficient of varia-
tion for CRP <15%); this is a critical consideration
if these types of assays are to enter the clinical
Quantitativ e MALDI-T OF MS 363
Page 9
diagnostic arena. Second, the authors collected and
averaged 500 spectra from different regions within
each sample spot, thereby minimizing the shot-to-
shot and region-to-region variability. Finally, the
mass spectrometric immunoassay has the capacity to
independently quantify variant forms of the proteins
of interest. This is generally not feasible with
immunoaffinity-based assays that use either spectros-
copic or turbidometric detection.
Our group has also quantified protein isoforms, in
both relative and absolute terms, based on MALDI-
TOF MS [38]. The utility of the approach was
demonstrated by quantifying the and b protein
isoforms of myosin heavy chain (MyHC) in human
atrial tissue. Isoform-specific tryptic peptides of
-MyHC (726–741) and b-MyHC (724–739) were
identified and calibration curves were constructed by
plotting ion current ratios against molar ratios for the
two synthetic peptides. For atrial samples, MyHC
was digested by trypsin, the ion current ratio was
determined for the two tryptic peptides, and this was
converted to the peptide ratio, then the isoform
ratio. The molar ratio was converted to absolute
values by employing a third peptide as an internal
standard (i.e. an analogue of the two peptides being
measured). A known quantity of the IS was added to
the MyHC digests and the measured ion current
ratios were converted to the actual quantities of the
isoform-specific peptides and then the quantity of
each protein isoform. The accuracy of the method
was confirmed by comparing these results to those
determined by an established method for the MyHC
isoform ratio determination. This approach is gen-
erally applicable to the analysis of protein isoforms
that have differences in primary sequence or
chemical modification. This is often difficult to
achieve with traditional antibody-based assays.
Recently, Bizzarri and colleagues [39] undertook
absolute quantification of troponin T in mouse
cardiac tissue by MALDI-TOF MS. After protein
extracted from heart tissue, cardiac troponin T was
isolated by high-performance liquid chromatography
and digested with trypsin. Quantification was based
on measurement of a cardiac troponin T-specific
tryptic peptide (YEINVLR). Calibration curves were
generated in the sample matrix by adding varying
amounts of the cardiac troponin T-specific synthetic
peptide and a fixed amount of an internal standard,
which was a structural analogue of the native peptide
(YEIQVLR). The authors investigated critical
aspects of assay performance including enzymatic
digestion efficiency, matrix effects, recovery, linear-
ity, precision, limit of detection and limit of
quantification. Although one limitation of the assay
is the requirement for extensive sample clean-up,
this study represents an excellent example of assay
development and validation. Nevertheless, this work
too is more of a proof-of-principle, rather than a
practical routine application.
Co m prehen si v e peptide and/or pr otein app l ication s
(pep tidomics and pr oteom ics)
A strategy based on isotope labelling of peptides
and liquid chromatography combined with MALDI
has been employed to accurately quantify and
identify differentially expressed proteins between an
E-cadherin-deficient human carcinoma cell line
(SCC9) and its transfectants expressing E-cadherin
(SCC9-E) [40]. Proteins extracted from each cell line
were digested with trypsin and the resulting peptides
were labelled individually with either [
2
H
0
]- or
[
2
H
2
]-formaldehyde. The labelled peptides were
combined and the peptide mixture was separated
and fractionated by a strong cation exchange (SCX)
column. Peptides from each SCX fraction were
further separated on a reversed phase column. The
effluents were spotted onto a MALDI target with a
heated droplet LC-MALDI interface and after
mixing with a MALDI matrix, individual sample
spots were analysed by MALDI on a quadrupole
TOF MS. An initial MS scan was used to quantify
the dimethyl-labelled peptide pairs and MS/MS
analysis was performed on the peptide pairs demon-
strating a relative peak intensity change of greater
than 2-fold. The MS/MS spectra were subjected to
database searching for protein identification. The
strategy was employed to detect and compare
relative peak intensity changes for 5480 peptide
pairs and amongst these, 320 peptide pairs showed
changes of >2-fold. MS/MS analysis led to the
identification of 49 differentially expressed proteins
between the parental SCC9 cells and SCC9-E
transfectants. The accuracy of the MS quantification
and subcellular localization for six of the differentially
expressed proteins were validated by immunoblot-
ting and immunofluorescence assays.
Gutierrez et al. [41] have also quantified peptides
by MALDI-TOF MS. They examined the influence
of factors such as crystal heterogeneity, peptide
ionization efficiency and data handling. In their
studies, they report an overall coefficient of variance
364 Duncan et a l.
Page 10
of 4.4% and a quantitative working range of
0.58–37.5 ng for bovine insulin.
Off-line capillary liquid chromatography was
combined with MALDI-TOF MS and TOF/TOF-
MS for the identification and quantification of
neuropeptides in microwave-fixed rat brain tissue
[42]. The effluent was mixed with matrix solution
and transferred to a MALDI target plate by pulsed
electric field deposition. Consistent with other
reports, their detection limits were in the low amol
range and they detected over 400 distinct peaks.
TOF/TOF-MS allowed identification of 10 pep-
tides, including three novel peptides. Quantification
was evaluated using substance P as the analyte and
[
15
N
3
]-labelled substance P as an internal standard.
Measured levels for substance P were about 7-fold
higher than previously reported in the rat striatum
and the authors attributed this to the unique
properties of microwave fixation. They conclude
that their approach could serve as a versatile tool for
neuropeptide analysis in brain tissue.
A variant of stable isotope labelling combined
with mass spectrometry, termed isotope-coded
protein label (ICPL), has been reported to yield
high accuracy, reproducibility and good sequence
coverage. The utility of the approach was demon-
strated by comparative analysis of two differentially
spiked proteomes [43].
Pan et al . [44] employed a targeted quantitative
proteomics approach based on MALDI TOF/TOF
MS to search for, identify and quantify selected
peptides in human cerebrospinal spinal fluid (CSF).
The approach involved isotopically labelled synthetic
peptides as references for targeted identification,
quantification and mass spectrometric analysis, and
LC MALDI-TOF/TOF was used to selectively
identify and quantify the targeted peptides in the
proteome of CSF without prior depletion of
abundant proteins. Candidate markers were quanti-
fied in CSF samples of patients with Parkinson’s
disease (PD) and Alzheimer’s disease (AD) along with
normal controls.
Griffin et al. [45] described an approach to the
quantitative analysis of complex protein mixtures by
MALDI on a quadrupole TOF (QqTOF) mass
spectrometer [46]. Proteins were labelled on cystei-
nyl residues with an isotope-coded affinity tag
reagent, enzymatically digested, the labelled peptides
purified by a multi-dimensional procedure, and then
eluted directly onto a MALDI sample target. After
the addition of matrix, the sample spots were
analysed by MALDI-QqTOF MS. The effectiveness
of the approach was demonstrated by the quantifica-
tion and identification of protein expression in
Saccharo myce s ce r ev i siae grown on two different
carbon sources.
Liao and Turko [47] recently reported another
approach to proteome analysis based on ammonium
sulphate depletion of serum, labelling of unfolded
proteins with native acrylamide and deuterated
[
2
H
3
]-acrylamide, separation of the unfolded
acrylamide-modified proteins, trypsinolysis and rela-
tive quantification by MALDI MS. There is minimal
sample handling before isotopic labelling and
extensive separation of unfolded proteins after
isotopic labelling. The authors report the quantifica-
tion of a large number of serum proteins, including
those with an abundance of 10
–5
less than albumin,
and suggest that this is a robust and inexpensive
workflow suitable for the quantitative profiling of
protein changes in the serum proteome.
Pr o tei n pr ofi l ing studies
There are hundreds of examples in the literature of
where protein profiling has been used to uncover
different protein signatures in two or more popula-
tions. Studies have utilized many sample types
including serum, plasma and urine. As discussed in
previous sections, many of the early studies were not
appropriately validated or replicated. We recently
reported a study that establishes the reproducibility of
the profiling approach both within and between
laboratories. We developed and tested the ability of a
predictive algorithm based on MALDI-TOF MS
analysis of pre-treatment serum to identify patients
who are likely to benefit from treatment with
epidermal growth factor receptor (EGFR) tyrosine
kinase inhibitors (TKIs) [22]. Some but not all
patients with non-small cell lung cancer (NSCLC)
respond to treatment with EGFR TKIs. Sera
collected from NSCLC patients before treatment
with gefitinib or erlotinib were analysed by MALDI
independently at two institutions. An algorithm to
predict outcomes after treatment with EGFR TKIs
was developed from a training set of 139 patients
derived from three cohorts. The algorithm was then
tested in two independent validation cohorts of 67
and 96 patients who were treated with gefitinib and
erlotinib, respectively, and in three control cohorts
of patients who were not treated with EGFR TKIs.
The clinical outcomes of survival and time to
progression were analysed. An algorithm based on
Quantitativ e MALDI-T OF MS 365
Page 11
eight distinct m/z features was developed based on
outcomes after EGFR TKI therapy in the training set
patients. Classifications based on spectra acquired at
the two institutions had a concordance of 97.1%,
showing that high reproducibility can be achieved
using MALDI-TOF MS. For both validation
cohorts, the classifier identified patients who
showed improved outcomes after EGFR TKI
treatment. In one cohort, median survival of patients
in the predicted ‘good’ and ‘poor’ groups was 207
and 92 days, respectively [hazard ratio (HR) of death
in the good versus poor groups ¼ 0.50, 95%
confidence interval (CI) ¼ 0.24–0.78]; in the other
cohort, median survivals were 306 versus 107 days
(HR ¼ 0.41, 95% CI ¼ 0.17–0.63). The classifier did
not predict outcomes in patients who did not receive
EGFR TKI treatment. This MALDI algorithm
was not merely prognostic but could classify
NSCLC patients for good or poor outcomes after
treatment with EGFR TKIs and it may assist in
the pre-treatment selection of appropriate sub-
groups of NSCLC patients for treatment with
EGFR TKIs. This is only one example of a rapidly
expanding literature on profile analysis. Early studies
were marred by design problems and lacked
thorough validation but increasingly the potential
of the approach is being realized in newly published
work.
Phosphorylation analy sis
There are few mass spectrometric approaches to
quantify the extent of phosphorylation of a substrate,
in large part because this is a difficult problem
confounded by the physiochemical differences
between unphosphorylated and phosphorylated pep-
tides. In one of the first practical applications,
Matsumoto and colleagues [48] used MALDI-TOF
MS to quantify the phosphopeptide produced by
calcium/calmodulin-dependent protein kinase II
(CaMK II). They measured both the substrate
peptide and the resulting product directly by
MALDI to improve accuracy, and their approach
eliminated the need for radiolabelled materials. They
found that the phosphorylation ratio obtained from
MALDI-TOF was consistently smaller than that
obtained from HPLC and they attributed this to
non-linearity of the detector under the analysis
conditions.
Huang and colleagues [49] coupled stable isotope
dimethyl labelling with immobilized metal affinity
chromatography (IMAC) enrichment to quantify
protein phosphorylation at MS-determined phos-
phorylation sites. They studied two model phospho-
proteins and also applied their analysis to pregnant
rat uteri, both with and without treatment with
8-bromo-cGMP. They acknowledge some limita-
tions with their approach, notably low-throughput,
but suggest that they have a working approach to this
challenging task.
Parker and colleagues have generated calibration
curves from a set of synthetic peptides of known
input ratios. Towards developing a universally appli-
cable approach to phosphorylation analysis they used
these to determine subtleties in sequence-dependent
differences for relative desorption/ionization effi-
ciencies and to predict relative signal strengths for
other peptide sequences [50].
Each of these applications provides useful data in a
given setting, but no universally applicable, high-
throughput and accurate approach to quantitative
phosphorylation analysis has yet been developed.
Oligosaccharide analysis
MALDI has been applied to both the qualitative and
quantitative analysis of oligosaccharides of lichenase-
hydrolysed water-soluble b-glucan from barley [51].
For this application, MALDI proved to be a rapid,
accurate and sensitive approach that could also offer
primary structural features on water-soluble b-glucan
from different barley varieties.
Inulin, a class of fructo-oligosaccharide derived
from plants, is an additive in baked goods, dairy
products, infant formula and dietary supplements. In
order to gain a better understanding of the role of
inulin, MALDI combined with Fourier transform ion
cyclotron resonance MS was used for qualitative and
quantitative analysis of bacterial growth. The method
employed an internal standard and reference to a cal-
ibration curve to quantify the consumption of fructo-
oligosaccharide by Bifidob acterium longum bv. infantis.
The method described was designed to be more rapid,
precise and robust than other existing methods [52].
In a similar application, MALDI-MS has also been
applied to the analysis of oligosaccharides in aqueous
food extracts. Fructo-oligosaccharides were measured
by standard addition and the assay was applied to
samples including red onions, shallots and elephant
garlic [53].
Bile acids
Six cholic acid derivatives: taurocholic acid
(TCA), taurochenodeoxycholic acid (TCDCA),
366 Duncan et a l.
Page 12
taurolithocholic acid (TLCA), glycocholic acid
(GCA), glycochenodeoxycholic acid (GCDCA)
and glycolithocholic acid (GCDCA) have been
quantified by MALDI-TOF MS. Urine samples
were pre-concentrated and purified by solid phase
extraction (SPE). The method was optimized to
eliminate ion suppression and proved to be repro-
ducible from day to day [54]. The same group has
applied essentially the same procedure to the
quantification of bile acids in plasma [55].
Lipids
Fujiwaki et al. [56] employed MALDI-TOF MS to
quantify sphingolipids in cardiac valve tissue from a
patient with Fabry disease. Crude lipids were
extracted from the tissue with chloroform/methanol
and then saponified with base. Six different species of
ceramide trihexose were detected in the tissue
extracts. The peak heights for these species were
compared to a fixed amount of the exogenous inter-
nal standard sphingosylphosphorylcholine. A linear
response was reported for one of the species from 0
to 50 ng with a good Pearson correlation (r > 0.95).
The authors showed that ceramide trihexose species
were markedly increased in tissue obtained from a
patient with Fabry disease, relative to a healthy
control, and they plan to apply the method to body
fluids and other tissues in the hopes of developing a
diagnostic test.
The phospholipid composition of serum lipopro-
teins has been quantified by MALDI-TOF MS [57].
Lipoproteins were isolated from serum and the
bound lipids were extracted and analysed by
MALDI. A variety of lipids were detected including
phospholipids (PLs), lysophospholipids (lysoPLs),
sphingolipids (SLs), triglycerides (TGs), cholesteryl
esters (CEs) and free cholesterol. MALDI analysis
also allowed for the characterization of individual
fatty acid chains; however, as expected, the correla-
tion between ion peak intensity and lipid concentra-
tion was poor due to the diverse nature of the lipid
mixture (i.e. the wide variation in polarity). The
authors quantified lipids with similar chemical
characteristics but with different fatty acid chains by
using 4-cholesten-3-one as an internal standard.
They also investigated the influence of sample
solvent, laser shot frequency and laser intensity on
assay performance.
MALDI-TOF MS was recently used to determine
the SLs in liver and spleen specimens from patients
with Niemann-Pick disease types A and C, and
Gaucher disease. Crude lipids were extracted from
tissue with chloroform and methanol, mild alkaline
treatment of the crude lipids was performed and a
SL fraction was prepared and analysed. Sphingosyl-
phosphorylcholine was used as the internal standard
for quantification of sphingomyelin and ceramide
monohexoside (CMH). The authors report that
accumulated sphingomyelin and CMH in small
amounts of tissues from sphingolipidosis patients
can be quantified for diagnostic purposes and also for
biochemical pathophysiology evaluation [58].
Quantification of other compounds
by MALDI
There are numerous other examples of quantifica-
tion by MALDI. For example, quantification of
DNA oligonucleotides by MALDI-TOF MS has
been demonstrated [59]. The authors used the
sequence of interest and a synthetic oligonucleotide
internal standard with a single base mutation. The
natural and mutant sequences were co-amplified by
PCR and a third primer was designed to anneal to
the region immediately upstream of the mutation
site. Depending on the specific mutation introduced
and the ddNTP/dNTP mixtures used, either one or
two bases were added to the third primer to produce
two extension products from the natural and mutant
templates. Finally, the two extension products were
detected and quantified by MALDI-TOF MS. This
method is generally applicable to the quantification
of DNA oligonucleotides.
MALDI-TOF MS has been used to identify and
quantify coccidiostats in poultry feeds. DHB was
found to be the best matrix and the coccidiostats
were shown to form predominantly [M þ Na]
þ
ions,
with some [M þ K]
þ
and [M H þ 2Na]
þ
ions.
A Sep-pak C18 cartridge purification procedure was
developed for sample work-up and limits of
detection for lasalocid, monensin, salinomycin and
narasin standards were reportedly 251, 22, 24 and
24 fmol, respectively [60].
Wang and Sporns [61] used a MALDI-TOF MS
method to quantify four flavonol glycosides in
almond seedcoats (i.e. isorhamnetin rutinoside, iso-
rhamnetin glucoside, kaempferol rutinoside and
kaempferol glucoside) by incorporating rutin
(quercetin-3-rutinoside) as the internal standard; and
a method has been reported to detect and quantify
curcumin and two curcuminoid metabolites in mouse
serum and mouse lung cell cultures. Standard curves
were prepared in serum and showed correlation
Quantitativ e MALDI-T OF MS 367
Page 13
coefficients of 0.94–0.99. Alcoholic extraction, con-
centration and addition of dilute hydrochloric acid to
stabilize the curcumin were found to be essential to
the reproducibility of the protocol. Untreated and
curcumin-treated mouse lung fibrotic and non-
fibrotic cell cultures were analysed by MALDI and
curcumin uptake was calculated. Curcumin was not
detected in untreated cells [62].
A combinatorial extraction method and an auto-
mated MALDI-TOF MS procedure were used to
improve the clinical analysis of the immunosuppres-
sant drug cyclosporin A. Cyclosporin extracts from
whole blood were analysed by MALDI and electro-
spray mass spectrometry, allowing for their iden-
tification and quantification. The combinatorial
approach was devised to optimize the extraction by
generating an array of solvent systems to be used for
extraction from blood, and an automated analysis
was performed by MALDI-TOF MS to identify
successful extractions [63].
FUNDING
National Institutes of Health (to M.W.D. and
S.W.H.); Cystic Fibrosis Foundation.
References
1. Karas M, Bachmann D, Bahr U, et al. Matrix-assisted
ultraviolet laser desorption of non-volatile compounds. IntJ
Mass Spectrom Ion Process 1987;78:53.
2. Nicola AJ, Gusev AI, Proctor A, et al. Application of the
fast-evaporation sample preparation method for improving
quantification of angiotensin II by matrix-assisted laser
desorption/ionization. Rapid Commun Mass Spectrom 1995;9:
1164–71.
3. Krause E, Wenschuh H, Jungblut PR. The dominance of
arginine-containing peptides in MALDI-derived tryptic
mass fingerprints of proteins. Anal Chem 1999;71:4160–5.
4. Cerpa-Poljak A, Jenkins A, Duncan MW. Recovery of
peptides and proteins following matrix-assisted laser deso-
rption ionization mass spectrometry. Rapid Commun Mass
Spectrom 1995;233–9.
5. Wallace WE. Reactive MALDI mass spectrometry: applica-
tion to high mass alkanes and polyethylene. Chem Commun
2007;4525–7.
6. Rubin RB, Merchant M. A rapid protein profiling system
that speeds study of cancer and other diseases. Am Clin Lab
2000;19:28–9.
7. Zhou H, Roy S, Schulman H, etal. Solution and chip arrays
in protein profiling. Trends Biotechnol 2001;19:S34–9.
8. Petricoin EF, Ardekani AM, Hitt BA, et al. Use of
proteomic patterns in serum to identify ovarian cancer.
Lancet 2002;359:572–7.
9. Petricoin EF, Liotta LA. Proteomic analysis at the bedside:
early detection of cancer. Trends Biotechnol 2002;20:S30–4.
10. Petricoin EF III, Ornstein DK, Paweletz CP, et al. Serum
proteomic patterns for detection of prostate cancer. J Natl
Cancer Inst 2002;94:1576–8.
11. Conrads TP, Zhou M, Petricoin EF III, et al. Cancer
diagnosis using proteomic patterns. Expert Rev Mol Diagn
2003;3:411–20.
12. Grizzle WE, Adam BL, Bigbee WL, et al. Serum protein
expression profiling for cancer detection: validation of a
SELDI-based approach for prostate cancer. Dis Markers
2003;19:185–95.
13. Sidransky D, Irizarry R, Califano JA, et al. Serum protein
MALDI profiling to distinguish upper aerodigestive tract
cancer patients from control subjects. J NatlCancer Inst 2003;
95:1711–7.
14. Baggerly KA, Morris JS, Coombes KR. Reproducibility
of SELDI-TOF protein patterns in serum: comparing
datasets from different experiments. Bioinformatics 2004;20:
777–85.
15. Baggerly KA, Morris JS, Edmonson SR, etal. Signal in noise:
evaluating reported reproducibility of serum proteomic tests
for ovarian cancer. J Natl Cancer Inst 2005;97:307–9.
16. Diamandis EP. Serum proteomic profiling by matrix-
assisted laser desorption-ionization time-of-flight mass
spectrometry for cancer diagnosis: next steps. Cancer Res
2006;66:5540–1.
17. Albrethsen J. Reproducibility in protein profiling by
MALDI-TOF mass spectrometry. ClinChem 2007;53:852–8.
18. Villanueva J, Philip J, Entenberg D, et al. Serum peptide
profiling by magnetic particle-assisted, automated sample
processing and MALDI-TOF mass spectrometry. Anal
Chem 2004;76:1560–70.
Key Point
Quantitative applications of MALDI present some additional
challenges over and above those a ssociated with alternative ioni-
zation methods. Despite this, following quickly after the devel-
opment of technique, there were reports of quantitative
applications that spanned a wide range of compounds. Most
app licat ions , at l east in the earl y phase, we r e de monst rati ons of
the potential of MALDI to provide precise quantitative data in
the low atto- to femtomole range; few of these reports detail
what might eventually become a practical assay. Subsequently, a
second generation of studies demonstrated the practical bene-
fits of MALDI ionization, not the least of which are its high-
throughput, versatility, low cost, reliability and ease of use.
These attributes make MALDI attractive as a tool, especially
for profile analysis and targeted quantification by standard addi-
tion. It is fair to say that each of these manuscripts covers some
of the impor tant issues in the application of MALDI as a quanti-
tative tool, but most fall short of providing rugged assays that
have been thoroughly tested in a routine setting. There is little
doubt that practical applications are feasible, even optimal for
some tasks, but we are yet to see wide adoption of MALDI-TOF
MS in a routine analytical setting.This is in part because commer-
cial l y avail ab le hardware and software have been de v e loped wit h
qualitative applications in mind, and when these are applied to
quantification, they deliver suboptimal performance. In future,
we look forward to seeing applications of MALDI to quantifica-
tion that are fit for their intended purpose and that take advan-
tage of the unique feature of MALDI ionization, especially its
except iona l th rou g h p ut when it is comb i ned w it h TOF analys is .
These developments will best be realized through symbiotic col-
laboration between biological scientists and instrument
developers.
368 Duncan et a l.
Page 14
19. Harkins JBt, Katz BB, Pastor SJ, etal. Parallel electrophoretic
depletion, fractionation, concentration, and desalting of 96
complex biological samples for mass spectrometry. Anal
Chem 2008;80:2734–43.
20. Righetti PG, Boschetti E, Lomas L, et al. Protein equalizer
technology: the quest for a ‘democratic proteome’.
Proteomics 2006;6:3980–92.
21. Sleno L, Volmer DA. Assessing the properties of internal
standards for quantitative matrix-assisted laser desorption/
ionization mass spectrometry of small molecules. Rapid
Commun Mass Spectrom 2006;20:1517–24.
22. Taguchi F, Solomon B, Gregorc V, etal. Mass spectrometry
to classify non-small-cell lung cancer patients for clinical
outcome after treatment with epidermal growth factor
receptor tyrosine kinase inhibitors: a multicohort cross-
institutional study. J Natl Cancer Inst 2007;99:838–46.
23. Thomas JJ, Shen Z, Crowell JE, et al. Desorption/ionization
on silicon (DIOS): a diverse mass spectrometry platform for
protein characterization. Proc Natl Acad Sci USA 2001;98:
4932–7.
24. Armstrong DW, Zhang LK, He L, et al. Ionic liquids as
matrixes for matrix-assisted laser desorption/ionization mass
spectrometry. Anal Chem 2001;73:3679–86.
25. Li YL, Gross ML. Ionic-liquid matrices for quantitative
analysis by MALDI-TOF mass spectrometry. JAmSocMass
Spectrom 2004;15:1833–7.
26. Hanton SD, McEvoy TM, Stets JR. Imaging the
morphology of solvent-free prepared MALDI samples.
J Am Soc Mass Spect r om 2008;19:874–81.
27. Tu T, Sauter AD, Jr, Sauter AD, III, et al. Improving the
signal intensity and sensitivity of MALDI mass spectrometry
by using nanoliter spots deposited by induction-based
fluidics. J Am Soc M ass Spectr om 2008;00.
28. Hanton SD, Parees DM. Extending the solvent-free
MALDI sample preparation method. JAmSocMass
Spectrom 2005;16:90–3.
29. Duncan MW, Matanovic G, Cerpa-Poljak A. Quantitative
analysis of low molecular weight compounds of biological
interest by matrix-assisted laser desorption ionization. Rapid
Commun Mass Spectrom 1993;7:1090–4.
30. Wittmann C, Heinzle E. MALDI-TOF MS for quantifica-
tion of substrates and products in cultivations of Coryne-
bacterium glutamicum. Biotechnol Bioeng 2001;72:642–7.
31. Bucknall M, Fung KY, Duncan MW. Practical quantitative
biomedical applications of MALDI-TOF mass spectro-
metry. J Am Soc Mass Spectr om 2002;
13:1015–27.
32. Sleno L, Volmer DA. Some fundamental and technical
aspects of the quantitative analysis of pharmaceutical
drugs by matrix-assisted laser desorption/ionization mass
spectrometry. Rapid Commun Mass Spectrom 2005;19:
1928–36.
33. van Kampen JJ, Burgers PC, de Groot R, et al. Qualitative
and quantitative analysis of pharmaceutical compounds
by MALDI-TOF mass spectrometry. Anal Chem 2006;78:
5403–11.
34. Notari S, Mancone C, Alonzi T, et al. Determination of
abacavir, amprenavir, didanosine, efavirenz, nevirapine, and
stavudine concentration in human plasma by MALDI-
TOF/TOF. J Chromatogr B Analyt Technol Biomed Life Sci
2008;863:249–57.
35. Kovarik P, Grivet C, Bourgogne E, et al. Method
development aspects for the quantitation of pharmaceutical
compounds in human plasma with a matrix-assisted laser
desorption/ionization source in the multiple reaction
monitoring mode. Rapid Commun Mass Spectrom 2007;21:
911–9.
36. Wagner M, Varesio E, Hopfgartner G. Ultra-fast quantita-
tion of saquinavir in human plasma by matrix-assisted laser
desorption/ionization and selected reaction monitoring
mode detection. J Chromatogr B AnalytTechnol Biomed Life Sci
2008.
37. Kiernan UA, Addobbati R, Nedelkov D, et al. Quantitative
multiplexed C-reactive protein mass spectrometric immu-
noassay. JProteomeRes 2006;5:1682–7.
38. Helmke SM, Yen CY, Cios KJ, et al. Simultaneous
quantification of human cardiac alpha- and beta-myosin
heavy chain proteins by MALDI-TOF mass spectrometry.
Anal Chem 2004;76:1683–9.
39. Bizzarri M, Cavaliere C, Foglia P, et al. A label-free method
based on MALDI-TOF mass spectrometry for the absolute
quantitation of troponin T in mouse cardiac tissue. Anal
Bioanal Chem 2008.
40. Ji C, Li L, Gebre M, et al. Identification and quantification
of differentially expressed proteins in E-cadherin deficient
SCC9 cells and SCC9 transfectants expressing E-cadherin
by dimethyl isotope labeling, LC-MALDI MS and MS/MS.
JProteomeRes 2005;4:1419–26.
41. Gutierrez JA, Dorocke JA, Knierman MD, et al.
Quantitative determination of peptides using matrix-assisted
laser desorption/ionization time-of-flight mass spectrome-
try. Biotechniques 2005;Suppl:13–17.
42. Wei H, Nolkrantz K, Parkin MC, et al. Identification and
quantification of neuropeptides in brain tissue by capillary
liquid chromatography coupled off-line to MALDI-TOF
and MALDI-TOF/TOF-MS. Anal Chem 2006;78
:4342–51.
43. Schmidt A, Kellermann J, Lottspeich F. A novel strategy for
quantitative proteomics using isotope-coded protein labels.
Proteomics 2005;5:4–15.
44. Pan S, Rush J, Peskind ER, et al. Application of targeted
quantitative proteomics analysis in human cerebrospinal
fluid using a liquid chromatography matrix-assisted laser
desorption/ionization time-of-flight tandem mass spec-
trometer (LC MALDI TOF/TOF) platform. JProteomeRes
2008;7:720–30.
45. Griffin TJ, Gygi SP, Rist B, et al. Quantitative proteomic
analysis using a MALDI quadrupole time-of-flight mass
spectrometer. Anal Chem 2001;73:978–86.
46. Gygi SP, Rist B, Gerber SA, et al. Quantitative analysis of
complex protein mixtures using isotope-coded affinity tags.
Nat Biotechnol 1999;17:994–9.
47. Liao WL, Turko IV. Strategy combining separation of
isotope-labeled unfolded proteins and matrix-assisted laser
desorption/ionization mass spectrometry analysis enables
quantification of a wide range of serum proteins. Anal
Biochem 2008;377:55–61.
48. Matsumoto H, Kahn ES, Komori N. Nonradioactive
phosphopeptide assay by matrix-assisted laser desorption
ionization time-of-flight mass spectrometry: application to
calcium/calmodulin-dependent protein kinase II. Anal
Biochem 1998;260:188–94.
49. Huang SY, Tsai ML, Wu CJ, et al. Quantitation of protein
phosphorylation in pregnant rat uteri using stable isotope
dimethyl labeling coupled with IMAC. Proteomics 2006;6:
1722–34.
Quantitativ e MALDI-T OF MS 369
Page 15
50. Parker L, Engel-Hall A, Drew K, et al. Investigating
quantitation of phosphorylation using MALDI-TOF mass
spectrometry. J Mass Spectrom 2008;43:518–27.
51. Jiang G, Vasanthan T. MALDI-MS and HPLC quantifica-
tion of oligosaccharides of lichenase-hydrolyzed water-
soluble beta-glucan from ten barley varieties. JAgricFood
Chem 2000;48:3305–10.
52. Seipert RR, Barboza M, Ninonuevo MR, et al. Analysis
and quantitation of fructooligosaccharides using matrix-
assisted laser desorption/ionization Fourier transform
ion cyclotron resonance mass spectrometry. Anal Chem
2008;80:159–65.
53. Wang J, Sporns P, Low NH. Analysis of food oligosacchar-
ides using MALDI-MS: quantification of fructooligosac-
charides. J Agric Food Chem 1999;47:1549–57.
54. Mims D, Hercules D. Quantification of bile acids directly
from urine by MALDI-TOF-MS. Anal Bioanal Chem 2003;
375:609–16.
55. Mims D, Hercules D. Quantification of bile acids directly
from plasma by MALDI-TOF-MS. Anal Bioanal Chem
2004;378:1322–6.
56. Fujiwaki T, Tasaka M, Takahashi N, et al. Quantitative
evaluation of sphingolipids using delayed extraction matrix-
assisted laser desorption ionization time-of-flight mass
spectrometry with sphingosylphosphorylcholine as an
internal standard. Practical application to cardiac valves
from a patient with Fabry disease. J Chromatogr B Analyt
Te c h n o l B i o m e d L i f e S c i 2006;832:97–102.
57. Hidaka H, Hanyu N, Sugano M, et al. Analysis of human
serum lipoprotein lipid composition using MALDI-TOF
mass spectrometry. Ann Clin Lab Sci 2007;37:213–21.
58. Fujiwaki T, Tasaka M, Yamaguchi S. Quantitative evalua-
tion of sphingomyelin and glucosylceramide using matrix-
assisted laser desorption ionization time-of-flight mass
spectrometry with sphingosylphosphorylcholine as an
internal standard Practical application to tissues from patients
with Niemann-Pick disease types A and C, and Gaucher
disease. J Chromatogr BAnalytTechnol Biomed Life Sci 2008;870:
170–6.
59. Ding C. Qualitative and quantitative DNA and RNA
analysis by matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry. Methods Mol Biol 2006;
336:59–71.
60. Wang J, Sporns P. MALDI-TOF MS quantification of
coccidiostats in poultry feeds. J Agric Food Chem 2000;48:
2807–11.
61. Frison-Norrie S, Sporns P. Identification and quantification
of flavonol glycosides in almond seedcoats using MALDI-
TOF MS. J Agric Food Chem 2002;50:2782–7.
62. May LA, Tourkina E, Hoffman SR, et al. Detection and
quantitation of curcumin in mouse lung cell cultures by
matrix-assisted laser desorption ionization time of flight mass
spectrometry. Anal Biochem
2005;33 7:62–9.
63. Wu J, Chatman K, Harris K, et al. An automated MALDI
mass spectrometry approach for optimizing cyclosporin
extraction and quantitation. Anal Chem 1997;69:3767–71.
370 Duncan et a l.
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    • "In conclusion, the PGC methodology affords reliable detection, validation and quantification of all oligomer species that may emerge from treating cellulose with LPMOs. In cases where ultrahigh sensitivity is required, internal standards either as heavy isotope labeled oligosaccharides or by standard addition [31] could be used for quantification by MS. "
    [Show abstract] [Hide abstract] ABSTRACT: Lytic polysaccharide monooxygenases play a pivotal role in enzymatic deconstruction of plant cell wall material due to their ability to catalyze oxidative cleavage of glycosidic bonds. LPMOs may release different products, often in small amounts, with various oxidation patterns (C1 or C4) and with varying stabilities, making accurate analysis of product profiles a major challenge. So far, HPAEC has been the method of choice but it has limitations with respect to analysis of C4-oxidized products. Here, we compare various HPLC methods and present procedures that allow efficient separation of intact C1- and C4-oxidized products. We demonstrate that both PGC and HILIC (in WAX-mode) can separate C1- and C4-oxidized products and that PGC gives superior chromatographic performance. In contrast to HPAEC, these methods are directly compatible with mass spectroscopy and charged aerosol detection (CAD), which enables online peak validation and quantification with LOD levels in the low ng range. While the novel methods show lower resolution than HPAEC, this is compensated by easy peak identification, allowing, for example, discrimination between chromatographically highly similar native and C4-oxidized cello-oligomers. HPAEC-MS studies revealed chemical oxidation of C4-geminal diol products, which implies that peaks commonly believed to be C4-oxidized cello-oligomers, in fact are on-column generated derivatives. Non-destructive separation of C4-oxidized cello-oligosaccharides on the PGC column allowed us, for the first time, to isolate C4-oxidized standards. HPAEC fractionation of a purified C4-oxidized tetramer revealed that on-column decomposition leads to formation of the native trimer, which may explain why product mixtures generated by C4-oxidizing LPMOs seem to be rich in native oligosaccharides when analyzed by HPAEC. The findings and methods described here will aid in future studies in the emerging LPMO field.
    No preview · Article · Mar 2016 · Journal of Chromatography A
    • "Quantitative analysis is challenge because ion suppression phenomena and variation of signal over different shots of the same spot. The signals depend on the co-crystal morphology that can substantially affect ionization efficiency and cause variation of the sample density within a short distance113114115. However, there are many methodologies for quantitative analysis such as internal standard protocol. "
    [Show abstract] [Hide abstract] ABSTRACT: Over the last several decades, considerable interest has been burgeoned into synthesizing ionic liquid (IL) for wide application in mass spectrometry (MS). Our primary motivation for this article is to provide the researchers for in-depth knowledge and gain practical experience in the application of the ILs for MS. The research on ILs is still in its infancy, and high potential for new applications is possible. The article provides the principles of using ILs applied in MS techniques for important applications on the hot fields such as proteomics, pathogenic bacteria, quantification, and biomolecules analysis. The cited examples can provide typical features of the potential of the ILs to be used. IL matrices are more frequently used as both solvent and matrices because of their extremely high sensitivity to the solvation and laser energy. Scopes and problems encountered with these matrices are critically evaluated.
    No preview · Chapter · Sep 2014
  • Source
    • "Like all other ionization methods used with mass spectrometry there are drawbacks to MALDI. The most discussed are matrix background ions below m/z 1000 limiting the sensitivity of MALDI-TOF in this mass range [129], hot-spot issues in which analyte ion abundance varies within the matrix crystals [130] , and difficulty in quantita- tion [131]. MALDI was extended to atmospheric pressure MALDI by Liako et al. [132,133]. "
    [Show abstract] [Hide abstract] ABSTRACT: During the past 50 years mass spectrometry has advanced from a small molecule method to one critical to understanding biological processes. The ability to analyze compounds as large as protein complexes did not happen with any single invention, but occurred in steps. Thus, electrospray ionization can be traced to Zeleny (1917) and Dole (1968), but also to Colby and Evans (1973) with the demonstration of electrohydrodynamic ionization of organic compounds. Likewise, matrix-assisted laser desorption/ionization can be traced to early work with laser desorption by Mumma and Vastola (1972), and the importance of a matrix in analysis of nonvolatile compounds can be attributed to Barber et al. with the introduction of fast atom bombardment (1981). Here, we look back over the past 50 years at the development of desorption methods for the analysis of nonvolatile compounds and the associated attempts at understanding the mechanism by which solid or liquid phase compounds are converted to gas-phase ions.
    Full-text · Article · Jul 2014 · International Journal of Mass Spectrometry
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