By Dr. Megha Agrawal, Dr. Shyamasri Biswas, Dr.

Kim Van Vliet, Contributing Editors

Vacuum MALDI-TOF Technology

for Advanced Applications
in Pharmaceutics and Medicine

dvances in vacuum technology have seen unprecedented

growth of new characterization
techniques for design and development
of therapeutics, drug delivery systems
and novel medical solutions to battle
complex diseases. Characterization of
clinically important materials and devices are of paramount importance to gain
insight into the functioning of medical
devices that will eventually pave the way
to deliver solutions for complex medical
problems. For example, accurate characterization and analysis of different biological macromolecules such as proteins,
DNA, RNA, carbohydrates and lipids
directly from complex samples such as
cells, tissues and blood can help develop
new medical pathways for development
of advanced therapeutics for numerous
medical applications.
Precise characterization of delicate
biosamples that have biomedical relevance requires a contamination-free
environment that is possible to achieve
with low pressure technologies. Stateof-art vacuum pumps and hardware that
constitute a characterization and analysis
chamber are being routinely employed in
2

various clinically important characterization techniques for biomaterials and

macromolecules. Vacuum technology is
expected to play a very important role
in development of better functional and
more advanced bio-characterization techniques in the future.
MALDI-TOF (Matrix Assisted Laser
Desorption/Ionization Time of Flight) is
one such technique that has gained importance for its precise characterization
abilities for a range of biological samples.
MALDI-TOF has emerged as a valuable
characterization tool for biotechnologists
and medical professionals to analyze clinically important biological samples and
molecules. MALDI-TOF is an automated
mass spectrometry technique that is carried out in a high vacuum environment to
characterize and analyze proteins, small
molecules and for rapid identification of
microorganisms such as fungi and bacteria. Some of the key benefits of using
MALDI-TOF are (1) its fast turnaround
time (in minutes) (2) ability to analyze
small samples (3) excellent workflow,
and (4) specific, reproducible, reliable
and accurate results. We describe in this
column important recent examples of the

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application of MALDI-TOF technology

to analyze important biological samples
for a wide range of applications.
In MALDI-TOF characterization, a
sample is placed on a target plate and
mixed with an appropriate matrix. The
mixture of sample and matrix crystallizes
when the sample slide is introduced into a
low pressure (high vacuum) environment.
The next step involves a precise laser burst
that ionizes the protein. The sample molecule and the matrix now enter the gas
phase and this leads to release of the matrix, sample molecules and ions from the
target plate. These ions are subjected to
an electric field and then accelerate in the
time of flight (TOF) analyzer as shown in
Figure 1. In the time of flight (TOF) tube,
the ions travel to the detector, and then the
mass to charge ratio (m/z) of the sample
ions can be calculated. In the time of flight
tube, lighter ions travel faster reaching the
detector first and heavier ions travel slower, requiring more time to reach the detector. The ions are finally detected with a
sensor (Figure 2). The resulting spectrum
represents the protein makeup of each
sample. This spectrum is then compared
against a large database of spectra of the
April 2015 Vacuum Technology & Coating

Figure 1. MALDI-TOF process in a high vacuum chamber [Source: Sigma-aldrich, http://

www.sigmaaldrich.com/technical-documents/articles/biology/custom-dna-oligos-qc-analysis-by-mass-spectrometry.html].

organism that the sample was isolated

from, for example, bacteria or fungi. The
identification can be determined at the
family, genus and species level.
For early investigation of targeted antimicrobial therapy rapid and reliable
identification of bacterial and fungal microorganisms is critical [1]. The delay in
appropriate antibiotic treatment against
these microorganisms is a major risk
factor for infection in children. It is very
important to initiate the antibiotic treatment as fast as possible. For that, the rapid identification of the correct species is
important to help in identifying potential
pathogens. Conventional methods of bacterial species identification are time consuming and labor intensive. MALDI-TOF
MS based identification has started a revolution in microbiology laboratories and
reduced the time of microbiological identification from a single bacterial colony to
minutes (Figure 3) [2, 3].
Novel applications of MALDI-TOF
MS include the identification of species
directly from complex clinical specimens
such as urine or blood and the determination of antibiotic resistance. Using MALDI-TOF MS, a urinary pathogen has been
identified directly from a urine sample and
microorganisms causing bacterial meningitis have also been detected directly from
patient samples [4]. Identification directly from positive blood cultures through
MALDI-TOF MS significantly reduces
the turnaround time in comparison to conventional identification (35 min vs. 3036
hours) and shows a critical impact on an-

tibiotic tailoring [5].

MALDI-TOF MS can be an ideal tool
for biomedical diagnostics as well as the
molecular diagnosis of clinical specimens, or for obtaining histological clues
from an endoscopic biopsy. Besides these,
imaging mass spectrometry MALDI-IMS
is also a powerful tool for the detection
and localization of drugs, proteins, and

Figure 2. Analysis and detection of a protein sample by MALDI-TOF [Source: The

VITEK MS technology, http://www.vitekms.
com/technology.html]

Figure 3. Workflow of MALDI-TOF MS based species identification. The bacterial single colony is added to the target plate and the resulting characteristic mass peak profile is compared to
a reference database. [Source: Dierig et al 2015]

Vacuum Technology & Coating April 2015 www.vactechmag.com or www.vtcmag.com 

lipids in tissue. A huge development in

the MALDI-IMS technique has been observed during the last decade, with it being considered one of the most promising
innovative measurement techniques in
biochemistry, and a powerful and versatile
tool for spatially resolved chemical analysis of diverse sample types ranging from
biological and plant tissues to biological
and polymer thin films, geared toward future clinical applications.
The combination of MALDI-TOF MS
with gel electrophoretic separation using
protein visualization by staining procedures like Coomassie Brilliant Blue or
silver salts or fluorescent dyes of high
detection sensitivity has been established
as a widely used approach in proteomics.
After gel separation (1D or 2D) proteins
are visualized and protein spots are excised from gels, digested with trypsin, and
digestion mixtures are analyzed by MALDI-TOF MS (Figure 4) [6].
Links between people, evidence, and
crime scenes are often established by
comparing DNA profile information obtained from biological evidence with reference samples. In certain cases, identification of the tissue of origin of the DNA
profiles can reveal significant insights into
the crime scene and contribute toward
solving them. The discovery of messenger RNA (mRNA) biomarkers that are
differentially expressed in bodily fluids
commonly found at crime scenes (e.g.,
venous blood, saliva, and semen) has contributed to the prospect of using mRNA
in forensic investigations [7]. To date, the
most frequently described mRNA profiling methods for forensic applications is
comprised of quantitative real-time PCR
(qPCR) methods but these have technical constraints like the limited availability of fluorescent dyes/tags. To address
these constraints, some research has
been done in forensic genetics, in which
MALDI-TOF MS has successfully been
used for a variety of analytical studies
involving nucleic acids. MALDI-TOF
MS has been employed for single nucleotide polymorphism (SNP) typing, DNA
profiling, gene expression analyses, and
DNA/RNA sequencing [8]. Some advantages offered by MALDI-TOF MS
over these methods include short analysis
time, fluorescent dye free reagents, no re4

Figure 4. Scheme of the steps involved in a proteomics experiment. [Source: Susnea et al 2013]

quirement for internal standards, and the

analysis of high-level multiplex PCR. As
a proof-of-principle application, a recent
study by Donfack and Wiley explored for
the first time the high multiplex and fluorescence-free attributes of MALDI-TOF
MS for the identification of venous blood,
saliva, and semen via cDNA profiling [9].
Polyethylene glycol (PEG) is one of
the most prominent polymer groups utilized for drug conjugation. The accurate
characterization of PEG is essential since
its molecular mass distribution (MMD)
and polydispersity can result in undesired
nonhomogeneous final products. Therefore evaluation of PEG before applying
it to drug conjugation is important. Four
monomethoxylated PEGsuccinimidyl
succinate (mPEGSS) derivatives were
investigated in terms of polydispersity
and MMD [10]. The formation and nature of polyphenols from tyrosinase catalyzed reactions using methylparaben as
a substrate was explored with the help
of MALDI-TOF MS. In a recent study,
Gonalves et al used MALDI-TOF MS
analysis and showed that the enzymatic
oxidation of methylparaben alone leads to
the polyhydroxybenzene formation [11].
Clinically relevant concentrations of
pharmaceutical drugs, like lopinavir, an
HIV protease inhibitor, have also been
quantified in peripheral blood mononuclear cells using MALDI-TOF mass

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spectrometry [12]. MALDI-TOF MS has

also been used to identify rapidly new bioactive ingredients in Traditional Chinese
Medicines (TCMs). In a study by Liu, et
al, they purified and identified three novel
peptides with antioxidant properties from
Cornu Bubali (water buffalo horn, WBH),
using MALDI-TOF MS [13]. WBH is
a traditional Chinese medicine that was
used for dispelling heat, counteracting
toxins, and relieving convulsions. Sample preparation procedures, including
extraction, purification, and isolation, are
usually required prior to the analysis (Figure 5) [14].
Direct analysis of tissue by MALDI-TOF MS has become applicable with
the development of many new matrices
for analysis of small molecular compounds and also does not require tedious
extraction and purification procedures
prior to MS analysis. Protein profiles and
images were obtained directly from thin
tissue sections cut on a cryostat from
fresh-frozen tissue blocks. Tissue biopsies or other relevant tissue samples were
frozen immediately after acquisition in
liquid nitrogen or isopentane to preserve
the samples morphology and minimize
protein degradation through enzymatic
proteolysis (Figure 6) [15].
In a recent study, MALDI-TOF MS has
been also found to be used as a fast and
sensitive tool to monitor the crosslinking
April 2015 Vacuum Technology & Coating

Figure 5. scheme for identification of bioactive ingredients in TCMs by MALDI-TOF MS [Source: Lu and Cai 2013].

step and to optimize the reaction conditions prior to electrospray ionization (ESI)
analysis or as a supportive tool to complement ESI data [16].
Concluding Remarks
MALDI-TOF MS has been developed
considerably in recent years and now constitutes a quantum leap in the identification of pathogenic microbes and is also a

powerful tool for the detection and localization of drugs, proteins, and lipids in tissue. The technology provides a very rapid,
cost-effective, easy-to-use and reliable detection method. However, MALDI-TOF
MS applications can go far beyond their
current use. Identification directly from
complex clinical specimens such as urine
or cerebrospinal fluid can make a timely
difference in the management of patients

with severe infections whereas identification of the DNA profiles and expression
of biomarkers in bodily fluids such as venous blood, saliva, and semen has contributed greatly to forensic investigations. In
addition, the future potential of this technology has not been completely explored,
and novel applications are currently being
developing, such as rapid pathogen typing
for outbreak situations, early detection of

Figure 6. Scheme outlining the different steps involved for profiling and imaging mass spectrometry of mammalian tissue samples [Source:
Chaurand et al 2004].
Vacuum Technology & Coating April 2015 www.vactechmag.com or www.vtcmag.com 

antibiotic resistance and recognition of

mixed infections by employing advanced
software algorithms. Automation of the
microbiology workflow incorporating imaging algorithms to identify bacterial colonies and robotic-guided MALDI-TOF
MS identification may further position
this new technology as indispensable for
every state-of-the-art laboratory.
References for Further Reading
1. Perez, K.K., et al., Integrating rapid diagnostics and antimicrobial stewardship
improves outcomes in patients with antibiotic-resistant Gram-negative bacteremia.
J Infect. 69(3): p. 216-25.
2. El-Bouri, K., et al., Comparison of bacterial identification by MALDI-TOF mass
spectrometry and conventional diagnostic
microbiology methods: agreement, speed
and cost implications. Br J Biomed Sci.
69(2): p. 47-55.
3. Dierig, A., R. Frei, and A. Egli, The Fast
Route to Microbe Identification: Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry
(MALDI-TOF MS). Pediatr Infect Dis J.
34(1): p. 97-9.

4. 
Tadros, M. and A. Petrich, Evaluation
of MALDI-TOF mass spectrometry and
Sepsityper Kit for the direct identification
of organisms from sterile body fluids in a
Canadian pediatric hospital. Can J Infect
Dis Med Microbiol. 24(4): p. 191-4.
5. Jamal, W., R. Saleem, and V.O. Rotimi,
Rapid identification of pathogens directly
from blood culture bottles by Bruker matrix-assisted laser desorption laser ionization-time of flight mass spectrometry
versus routine methods. Diagn Microbiol
Infect Dis. 76(4): p. 404-8.
6. Susnea, I., et al., Application of MALDI-TOF-mass spectrometry to proteome
analysis using stain-free gel electrophoresis. Top Curr Chem. 331: p. 37-54.
7. An, J.H., et al., Body fluid identification in
forensics. BMB Rep. 45(10): p. 545-53.
8. Pusch, W., et al., MALDI-TOF mass spectrometry-based SNP genotyping. Pharmacogenomics, 2002. 3(4): p. 537-48.
9. Donfack, J. and A. Wiley, Mass spectrometry-based cDNA profiling as a potential
tool for human body fluid identification.
Forensic Sci Int Genet. 16C: p. 112-120.
10. Kemptner, J., et al., GEMMA and MALDI-TOF MS of reactive PEGs for pharmaceutical applications. J Pharm Biomed

vtcmag@vtcmag.com 

Anal. 52(4): p. 432-7.

11. Goncalves, I., et al., Antimicrobial lubricant formulations containing poly(hydroxybenzene)-trimethoprim conjugates
synthesized by tyrosinase. Appl Microbiol
Biotechnol.
12. van Kampen, J.J., et al., Qualitative and
quantitative analysis of pharmaceutical
compounds by MALDI-TOF mass spectrometry. Anal Chem, 2006. 78(15): p.
5403-11.
13. Liu, R., et al., Purification and identification of three novel antioxidant peptides
from Cornu Bubali (water buffalo horn).
Peptides. 31(5): p. 786-93.
14. Lu, M. and Z. Cai, Advances of MALDI-TOF MS in the analysis of traditional
Chinese medicines. Top Curr Chem. 331:
p. 143-64.
15. Chaurand, P., et al., Proteomics in diagnostic pathology: profiling and imaging
proteins directly in tissue sections. Am J
Pathol, 2004. 165(4): p. 1057-68.
16. Gomes, A.F. and F.C. Gozzo, Chemical
cross-linking with a diazirine photoactivatable cross-linker investigated by MALDI- and ESI-MS/MS. J Mass Spectrom.
45(8): p. 892-9.

April 2015 Vacuum Technology & Coating