1. Tick species proteomic identification using MALDI-TOF mass spectrometry
Robert Hwang and May Dong
Department of Biology, Swarthmore College, Swarthmore, PA
Introduction
Ticks (Acari: Ixodidae) are hematophagous arthropods
found on every continent except Antarctica, and more than
twenty species in five genera can be found in Panamá. Ticks
cause irritating bites and are disease vectors in humans, live-
stock, and wildlife. Identification of tick species is vital to de-
termine whether a given tick could be a vector for a specific
pathogen.
Automation of the identification process may diminish
the risk of misidentifications. Molecular means are accurate
but have higher costs per sample. Matrix assisted laser de-
sorption/ionization time-of-flight (MALDI-TOF) represents
a unique tool for rapid identification of species. Minimal
amounts of tissue are needed to generate a protein spectrum,
which is ideal when working with archival specimens. MAL-
DI-TOF has been used successfully to identify fresh ticks1
as
well as preserved fleas2
, but no studies have been done testing
the feasibility of using MALDI to identify preserved ticks.
Question
• Can tick specimens be identified down to the genera level by
analyzing proteins found in leg pairs using a mass spectrom-
eter?
• Can tick specimens be identified down to the species level
by using the same methodology?
Results Discussion
This study has several important implications in science to-
day. In today’s rapidly changing global climate, conservation
and species preservation efforts have become more dire. Being
able to create a database of species down to the molecular lev-
el allows us to understand organismal and population biolo-
gy at a level that can keep up with accelerating anthropogenic
change.
Similarly, with genetics playing a larger role in many areas of
biological research today, it is even more important that taxo-
nomic identification can be assessed down to the molecular
level.
Conclusions
• Tick specimens were able to be successfully identified down
to the genus level with logarithmic similarity scores greater
than 2.7 for all specimens
• Tick specimens within the same genus were able to be suc-
cessfully identified down to the species level with logarithmic
similarity scores greater than 2.7 for all specimens
Methods
Preparation
• Fresh tick samples or ticks stored in 70% ethanol for up to
five years were set up under a microscope
• A pair of legs was removed using scalpel and placed inside a
molecular centrifuge tube
• 900 uL of 70% EtOH diluted in ultrapure water added to
each tube
• Each tube vortexed for 30 seconds before being placed in a
molecular centrifuge for 2 minutes at 13000 RPM
• EtOH solution was decanted from each tube and 20 uL of
70% formic acid + 20 uL of 100% acetonitrile was added
• Tick legs were macerated using a steel pestle for 20 seconds
before being centrifuged for 2 minutes at 13000 RPM
Analysis
• 1 uL of supernatant was spotted onto a steel plate and al-
lowed to dry
• Following supernatant, 1 uL of HCCA matrix was overlayed
over each sample and allowed to dry for five minutes before
plate was inserted into the MALDI for analysis
Acknowledgements
We would like to thank Dr. Jose Loaiza and Dr. Rolando
Gittens for advising and guiding us through research method-
ology. We would also like to thank Vincent Formica for mak-
ing this unique opportunity to engage in overseas research
possible. Funding for this project was provided by INDICA-
SAT AIP, and a Lang Research stipend from the Swarthmore
Department of Biology.
Figure 1. Comparing average protein spectra between species of different genera. Each peak represents a
protein of a specific mass. Averages were obtained by combining spectra from one fourth leg from two or
more individuals, with each individual leg reproduced in triplicate. Some peaks are conserved between gen-
era while others are unique or appear to be shifted.
Figure 2. Comparing average protein spectra between species of the same genus Amblyomma. Averages
were obtained as in Fig. 1 from preserved specimens collected as early as five years prior to this study. Many
peaks, such as 4536.01 m/z in A. calcarattum and 4536.06 in A. varium appear to be conserved within Am-
blyomma species.
Citations
1
Yssouf A, Flaudrops C, Drali R, Kernif T, Socolovschi C, Berenger
JM, Raoult D, Parola P. 2013. Matrix-assisted laser desorption ion-
ization–time of flight mass spectrometry for rapid identification of
tick vectors. J Clin Micro. 51(2):522-528.
2
Yssouf A, Socolovschi C, Leulmi H, Kernif T, Bitam I, Audoly G,
Almeras L, Raoult D, Parola P. 2014. Identification of flea species
using MALDI-TOF/MS. Comp Immuno, Micro, and Infectious
Disease. 37:153-157.