Clinical Chemistry 62:1
20–23 (2016)
Evolution of Quantitative MALDI-TOF Mass
Spectrometry for Clinical Applications
Marvin L. Vestal1*
The nearly simultaneous discoveries of electrospray ion-
ization (ESI)2 (1 ) and MALDI (2 ) 27 years ago removed
the volatility barrier for mass spectrometry (MS).
ESI-MS developed very rapidly, at least partly due to the
ease of interfacing with commercially available quadru-
pole and trap instruments widely used for analytical ap-
plications. TOF is particularly well suited to MALDI
with pulsed ion sources compatible with pulsed laser de-
sorption, but applications developed more slowly, at least
in part because higher-performance instruments were not
initially available. The potential of MALDI has stimu-
lated development of improved TOF instrumentation
particularly tailored for this ionization technique.
Laser ionization MS has been extensively studied for
⬎60 years, but until the introduction of an appropriate
matrix for absorbing the laser energy was introduced,
biological applications were very limited (3 ). Karas and
Hillenkamp (2 ) found that the use of a suitable matrix
could reduce the required photon intensity for desorp-
tion by an order of magnitude and coined the description
of the process as “matrix-assisted laser desorption ioniza-
tion.” Further development of the time-of-flight instru-
ment enabled the observation of proteins of masses ⬎100
kDa, such as ␤-D-galactosidase at approximately 117
kDa reported by Hillenkamp (4 ) at the Bordeaux Inter-
national Mass Spectrometry meeting in 1988. This sig-
naled the beginning of a new era in biological MS. The
resolving power obtained in this initial work was rather
low, and it was quickly realized that a major limitation
was due to ion fragmentation in flight. The first practical
MALDI-TOF mass spectrometer for high m/z was built
by Beavis and Chait within a few months of the discovery
of MALDI (5 ). This was a simple linear TOF with a
static single field source, drift tube, and detector. A major
advantage of the linear analyzer is that ions dissociating in
flight arrive at nearly the same time. Beavis and Chait also
conducted an extensive study of matrices for MALDI and
1
SimulTOF Systems, Sudbury, MA.
* Address correspondence to this author at: SimulTOF Systems, 60 Union Ave, Suite 1R,
Sudbury, MA 01776. Fax 978-783-2512; e-mail marvin.vestal@simultof.com.
Received June 8, 2015; accepted June 30, 2015.
Previously published online at DOI: 10.1373/clinchem.2015.239467
© 2015 American Association for Clinical Chemistry
2
Nonstandard abbreviations: ESI, electrospray ionization; MS, mass spectrometry; FDA,
US Food and Drug Administration; Hb A1c, glycated hemoglobin.
20
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developed the cinnamic acid derivatives that enabled
many practical applications of MALDI (6, 7 ).
The further development of TOF-MS has been
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driven by advances in hardware and electronics that im-
proved resolving power and allowed high-speed data ac-
quisition over a broad mass range. These include appli-
cation of pulsed ion extraction, improved ion mirrors,
tandem TOF-TOF analyzers, fast digitizers, and kilo-
hertz lasers. The general conditions for “time focusing”
with pulsed acceleration were first described by Wiley
and McLaren (8 ), and these conditions apply to MALDI
(9 ) as well as other ionization techniques. During the
first 2 decades after the discovery of the TOF-MS, these
instruments were generally considered a useful tool for
exotic studies of ion properties but were not widely used
to solve analytical problems. Cotter (10 ) has provided an
excellent description of the history, development, and
applications of TOF-MS in biological research before
1997, and Standing and Vestal (11 ) recently summarized
the development of TOF-MS from the beginning in
1946 to the present.
Samples for analysis by MALDI are embedded in
matrix crystals deposited on the surface of a sample plate
made up of 1 electrode of an ion accelerator. A laser pulse
impinges on the crystals and produces a pulse of desorbed
material, including ions within a plume of neutrals.
Pulsed and static electric fields may be applied to accel-
erate and focus the ions in both space and time. Early
MALDI-TOF instruments were limited to laser rates of a
few hertz by both the laser technology available and the
speed of available digitizers developed originally for dig-
ital oscilloscopes. More recent work has focused on im-
proved TOF instruments and software that allow the full
potential mass resolution of MALDI to be applied to
difficult biological analysis problems (12 ). Laser pulse
rates have increased to 1 kHz in many commercial instru-
ments, and some are now available with a laser rate of 5
kHz. The advantages of higher laser rates include higher
sample throughput, with increased sensitivity and dy-
namic range and better sample utilization.
Current Applications
MALDI-TOF MS has become an established technique
for analyzing a variety of nonvolatile molecules including
proteins, peptides, oligonucleotides, lipids, glycans, and
other molecules of biological importance. This technol-

ogy has been applied to many analytical applications, but
widespread acceptance has been limited by many factors,
including, for example, the cost and complexity of these
instruments, relatively poor reliability, and insufficient
speed, sensitivity, resolution, and mass accuracy. Accep-
tance has also been limited by the widespread belief that
MALDI-TOF is not quantitative.
More than 25 years after the advent of the enabling
approaches of MALDI and ESI, there has been only mar-
ginal success in the implementation of MS in routine
clinical determinations (13 ). Linear MALDI-TOF in-
struments have recently been approved by the US Food
and Drug Administration (FDA) for clinical application
to pathogen identification. In these instruments, the laser
rate is limited to ⱕ50 Hz, and a small number of laser
shots (typically 50 –500) is summed to produce a spec-
trum. These spectra are acquired by looking for “sweet
spots” on the MALDI samples, and with samples depos-
ited on a spot with a nominal diameter of approximately
3 mm, only a small fraction (typically ⬍1%) of the sam-
ple molecules are ionized and analyzed. The databases
used with these instruments were developed to identify
pathogens from mass spectra in which neither the mass
nor the intensity were reproducible.
Recent work (14 ) has focused on developing
MALDI-TOF mass spectrometers that generate accurate
quantitative data by providing reproducible spectra on
complex samples. In these instruments, the laser rate is in
the 1- to 5-kHz range, and the laser spot is rastered over
the sample spot to ionize and analyze a large fraction
of the sample on a sample spot. At least 10 000 laser shots
are typically summed to obtain a spectrum, and as many
as 200 000 laser shots can be used if necessary to com-
pletely ionize a sample. This provides reproducible mass
spectra with no significant noise that are consistent for
multiple instruments and multiple users preparing sam-
ples. It also minimizes effects due to variations in the
amount and distribution of samples on the sample plate.
These instruments effectively reduce the variability of the
results due to instrument imperfections to the point that
this effect is negligible in the quality of the results ob-
tained. The remaining sources of uncontrolled variability
are sample preparation and deposition on the sample
plate. These effects are the dominant reasons for variabil-
ity in resolving power and measured masses and intensi-
ties of the peaks in the spectrum.
These instruments designed for routine clinical ap-
plications are fully automated and require little or no
expertise in MS. The user prepares the samples according
to a protocol established for the application, loads the
sample plates into the instrument, and receives and inter-
prets the results. The instrument parameters are deter-
mined from data provided with the samples, and data
acquisition, processing, and database searching are fully
Reflection
automated. No other interaction between the user and
the instrument is required.
Determination of Glycated Hemoglobin by
Quantitative Maldi-TOF
Diagnosis and monitoring of diabetes mellitus relies on
measurements of glycated hemoglobin (Hb A1c), the rel-
ative amount of glucose attached to the N-terminal valine
of the ␤-subunit of hemoglobin (15 ). Application of
quantitative MALDI-TOF to this measurement illus-
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trates the power of this technology for an important clin-
ical application (16 –17 ). Blood samples are collected in
a phlebotomy protocol and analyzed at a 1:2000 final
dilution as 5⫻ replicates spotted in 5 g/L sinapinic acid
MALDI matrix. Positive ion mass spectra in the 5- to
20-kDa mass range are acquired on a linear instrument.
Spectra acquired from each sample spot are averaged and
calibrated using the M1⫹ and M2⫹ ions of hemoglobin
␣- and ␤-subunits. Hb A1c is calculated from glycated ␣-
or ␤-subunit by comparing the signal from the unmodi-
fied species to that of the unmodified species, 162 Da
(glucose). Results are presented as a ratio of the percent
total glycation of each chain using the following
formulas:
100{I(␤⫹162)/[I␤ ⫹ I(␤⫹162)]} and
100{I(␣⫹162)/[I␣⫹I(␣⫹162)]}.
An illustration of the accuracy and reproducibility of the
technique is shown in Fig. 1. In this example, the same
sample was deposited on all 80 sample spots on the
MALDI plate. In this case, the final sample spectrum for
each spot results from the compilation of approximately
160 individual spectra, averaged in 100 laser-shot bun-
dles. These are acquired by rastering the laser beam over
the sample spot in a systematic pattern that evenly spans
the spot. Overlaying the spectra from all 80 spots illus-
trates the excellent reproducibility. As shown in the fig-
ure, the average CV for each set of 5 spots is ⬍1%, and
the SD for the Hb A1c determination for the whole plate
is 1.2%.
Results obtained by MALDI-TOF assay can be
compared with those obtained in a clinical laboratory
using clinically accepted HPLC methodology. Measure-
ments on analytical standards and clinical patient sam-
ples for quantification of total glycation on the ␤-subunit
of hemoglobin exhibit linearity over the range from 1.4%
to 18% with CVs ⬍1.66%. MALDI-TOF MS detects all
glycation of the ␣- and ␤-subunits of hemoglobin, but a
direct relationship has been established between the
MALDI-TOF method for the determination of the gly-
cated ␤ chain and the HPLC method for determination
of Hb A1c. Additionally, the strong correlation between
glycation of the ␣- and ␤-subunits allows their ratios to
Clinical Chemistry 62:1 (2016) 21
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Fig. 1. Illustration of the accuracy and reproducibility of quantitative MALDI for determination of Hb A1c.
The same sample was deposited on all 80 sample spots on the MALDI plate. The spectrum shown is an overlay of the spectra from all 80 spots;
each is the summation of ⬃16 000 laser shots. Sample plate is μFocus Plate from Hudson Surface Technology.

be used as a QC check for the determination of the gly-
cated ␤ chain.
Future Clinical Applications Of Maldi-TOF
These data show the utility and value of the MALDI-
TOF MS method for performing a quantitative protein
assay. This application demonstrates the ability to mea-
sure and quantify proteins at micromole concentrations
with CVs of ⱕ2% from analyses of diluted, whole blood
samples. These methods for assaying an analyte in a
bodily fluid can be applied to any sample that is amenable
to analysis by MALDI-TOF MS. Steps in a method in-
clude sampling bodily fluids from subjects, processing
the samples of bodily fluids collected to produce a sample
extract suitable for analysis by MALDI-TOF MS, load-
ing the sample extract into the ion source of the MALDI-
TOF mass spectrometer, ionizing the sample extract,
generating a TOF mass spectrum, and analyzing and in-
terpreting the MALDI-TOF mass spectrum generated to
provide meaningful results. The components of the
method can be designed to meet the requirements im-
posed by the nature of the bodily fluid, the analyte, and
the various specifications of sensitivity, selectivity, and
accuracy of the assay. The methods have considerable
flexibility and are not “one-size-fits-all.” Instead, the
choices made for each step of a method for a specific assay
reflect the requirements imposed by these limitations.
Although there has been only marginal success in the
implementation of MS in routine clinical determina-
tions, and only 2 applications have been approved by the
FDA for use in pathogen identification, analytes could
include almost any nonvolatile molecules of biological
importance. The components of interest could be the

22 Clinical Chemistry 62:1 (2016)

 Reflection

intact analytes themselves; chemically or enzymatically
derived stable molecules (e.g., molecular fragments) de-
rived from the intact molecule, such as proteolytic pep-
tides or polysaccharides; or chemically modified forms of
the original analyte (e.g., methylated, acetylated, or oth-
erwise intentionally modified forms).
There is no reason MALDI methods could not be
used to analyze any bodily fluid containing an analyte of
interest, including blood and blood products, breast
milk, cerebrospinal fluid, lymph fluid, saliva, urine, gas-
tric and digestive fluid, tears, stool, semen, prostatic
ture, where standardized technologies will be routinely
applied to help define the pathobiology underlying dis-
ease, the clinical status of individuals with disease, and as
part of advanced work flows toward novel biomarkers
stemming from numerous protein-based discovery ef-
forts currently ongoing worldwide.
Author Contributions: All authors confirmed they have contributed to
the intellectual content of this paper and have met the following 3 require-

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fluid, vaginal fluid, amniotic fluid, and interstitial fluids
derived from tissue.
The following applications may soon be accepted for
routine clinical use in addition to expanded use for
pathogen identification: (a) cancer typing directly from
serum, tissue extracts, and other bodily fluids; (b) tissue
imaging; (c) proteins for cancer typing; (d) small mole-
cules for drug disposition; (e) biomarker identification
and validation; (f) MS immunoassay; (g) peptide quanti-
fication; and (h) clinical assays of biomarkers for diagno-
sis and treatment.
Improved ionization techniques and technical ad-
vances are driving MALDI-TOF toward this bright fu-
ments: (a) significant contributions to the conception and design, acquisi-
tion of data, or analysis and interpretation of data; (b) drafting or revising
the article for intellectual content; and (c) final approval of the published
article.
Authors’ Disclosures or Potential Conflicts of Interest: Upon man-
uscript submission, all authors completed the author disclosure form. Dis-
closures and/or potential conflicts of interest:
Employment or Leadership: M.L. Vestal, Virgin Instruments Corp.
Consultant or Advisory Role: None declared.
Stock Ownership: M.L. Vestal, Virgin Instruments Corp.
Honoraria: None declared.
Research Funding: None declared.
Expert Testimony: None declared.
Patents: None declared.

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