Medical Design Briefs - October 2023 - 19

nents of the sample are identified by
correlating known masses of substances
to the masses identified in the sample or
through a fragmentation pattern.
There are many ways to desorb and
ionize molecules. One of the traditional
methods involves bombarding the sample
with a highly energetic source, such
as a corona discharge or a stream of
highly charged ions. While such sources
are highly efficient, they can easily destroy
large, labile biomolecules such as
peptides and proteins that are important
to medical diagnostic testing. In
some cases, fragmenting the molecules
is the goal as such fragments can provide
structural information if broken in
a reproducible manner. However, there
are many times when it is necessary to
retain the structure of the molecules,
which requires a different approach.
Two soft, gentler desorption/ionization
techniques that allow the intact detection
of biomolecules were awarded
the 2002 Nobel prize in chemistry - the
UV MALDI (ultraviolet matrix-assisted
laser desorption and ionization) technique
and electrospray ionization (ESI).
UV MALDI utilizes a pulsed UV laser operating
in the 330-360 nm range that
strikes a matrix of molecules with ultraviolet
light to vaporize, desorb, and ionize
the molecules before introducing them
into the mass spectrometer for analysis.
The UV MALDI technique was one of
the first to involve fast-pulsed lasers for
mass spectrometry. ESI involves dissolving
sample material into an injectable
liquid that forms an aerosol when subjected
to a high voltage. Often, complex
samples require separating out components
before ESI using liquid chromatography
due to the formation of multiple
charges resulting in a complex mass
spectra. However, major benefits to this
technique include high ionization efficiency
and the ability to work at atmospheric
pressure.
With the UV MALDI technique, pulsed
UV laser light alone can still be too energetic
and fragment larger molecules. So, it
was discovered that adding a co-absorbing
matrix in solution - a separate molecule
added with a larger molecule like a protein
- and allow them to air dry into a
thin, crystalline film, enables large biomolecules
to be desorbed and ionized intact.
While the UV MALDI technique was a
significant advancement that is still used
today, it is not without its drawbacks.
Medical Design Briefs, October 2023
Samples must be mixed with a matrix
material to be analyzed, which complicates
sample preparation to such a degree
that expensive matrix sprayers were
developed to achieve more reproducible
thin films. The matrix material also gets
desorbed, ionized, and detected, which
can interfere with the analysis of lower
mass molecules. This process is further
complicated because sample preparations
are typically loaded into a high vacuum
chamber through an interlock system
and remain under vacuum to
achieve the proper result.
To address the limitations of the UV
MALDI process, researchers have sought
ways to remove the matrix requirement
and analyze samples under atmosphere
pressure. After the invention of the UV
MALDI process, longer wavelength lasers
were tested in the mid-IR region. It
was discovered that IR MALDI offered
enough energy to ionize the molecules
without fragmentation, while eliminating
the need to add a comatrix. To accomplish
this, a pure protein compound
is deposited on an IR transparent substrate
to avoid local heating effects and
directly hit with a pulsed, mid-IR laser. If
the sample has high absorption at the
mid-IR laser wavelength, it acts as its own
matrix. The molecules are desorbed and
ionized without significant fragmentation.
Unfortunately, larger biomolecule
concentrations are needed due to the
lower ionization efficiency of using matrix-free,
IR MALDI versus UV MALDI.
Atmospheric pressure (AP) UV MALDI
sources have been developed to alleviate
the need to load samples into high vacuum
but ionized sample material can be lost
during transport into the mass spectrometer
resulting in lower detection efficiency.
Lower ionization efficiency of IR MALDI
combined with sample material loss
during transport from AP sources required
a new solution. A solution presented itself
in the form of a hybrid technique where
the efficient and soft (gentle) desorption
of pulsed, mid-IR lasers could be combined
with the efficient ionization of atmospheric
pressure ESI source. The results of
this new technique have been presented
under a variety of names (ELDI, LAESI,
MALDESI), and commercial product attempts
but a final, inexpensive MS source
and ideal application remain elusive.
Enter Real-Time Medical Diagnostics
All biological samples such as tissue
contain a large amount of water, which is
the medium under which most biological
processes take place. Water has the
highest mid-IR absorption around 3 µm
and, therefore, mid-IR lasers operating
in this region allow endogenous sample
water to act as a light absorbing matrix.
The most common lasers operating
in the 3 µm range include the Er:YAG
and optical parametric oscillator (OPO)
lasers. Due to electron to photon inefficiencies
required large power supplies
inherent in YAG products, Er:YAG lasers
proved too large and cost prohibitive to
OPO lasers are compact, affordable, and easily integrated into mass spectrometers for use in clinical settings.
(Credit: OPOTEK)
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Medical Design Briefs - October 2023

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