Blue Native Gel Electrophoresis

This technique is a valuable tool in studying membrane protein complexes.

Introduction:
Blue-Native Polyacrylamide Gel Electrophoresis (Blue Native PAGE) was originally described as a
technique for the separation of enzymatically active membrane protein complexes under mild conditions.
Principle: In this variation of gel electrophoresis, the anionic dye Coomassie Brilliant Blue is added to the
sample prior to loading and binds to protein complexes during electrophoresis under physiological
conditions.
The technique has gained interest from researchers focused on functional proteomics in recent years, as it
allows the study of protein-protein interactions, and the separation and analysis of very hydrophobic
proteins, such as membrane proteins, their complexes, and even super-complexes.
In a further development, Blue-Native PAGE is a fully complementary method to high resolution two-
dimensional electrophoresis and also liquid chromatography of proteins, the most frequently used
separation methods in proteomics. The technique is also useful for prefractionation of mg amounts of
sample for subsequent analysis of smaller protein subsets. In most cases Blue-Native PAGE is combined
with a second dimension, which is either a second Blue-Native PAGE after equilibration with a medium-
mild detergent, or SDS PAGE for mapping of the related subunits.

Method: Hydrophobic proteins and complexes are first solubilized with a mild nonionic detergent, like
Triton X-100 or digitonin. Digitonin, the preferred detergent as it is the mildest, allows the separation of
intact super-complexes. Coomassie Blue is added to the sample and cathodal running buffer and remains
bound to all hydrophobic proteins and to many water-soluble proteins by hydrophobic interactions even
when an electric field is applied. Coomassie Blue is anionic so all protein-dye complexes become
negatively charged in the pH 7.5 buffer used, and the complexes migrate towards the anode. Separation of
protein complexes occurs according to size in the range 10 kDa to 10 MDa. These protein-dye-complexes
are soluble in the absence of detergent, which minimizes the risk of denaturation. Aggregation of the
proteins is also prevented because of their overall negative charge. Detection of the proteins and complexes
is straightforward as the attached blue dye makes them visible. Porosity gradient gels from 4 to 16% T are
employed; this allows large super complexes to enter the gel, prevents small complexes and single proteins
from migrating out of the gel, and applies a band-sharpening effect.
After the first dimension electrophoresis is complete, the lanes containing the separated complexes are cut
out with a sharp knife or ruler edge, equilibrated in SDS solution, and embedded into a stacking gel layer
of a second dimension discontinuous SDS gel. During this process the complexes fall apart into their
components (subunits) to form protein-SDS micelles that separate in the SDS gel according their molecular
sizes. A Tris-tricine buffer system is preferred over the conventional Tris-glycine gel, because it offers an
improved resolution of low molecular weight proteins. Gels containing non-labeled proteins can be stained
after the separation with Coomassie Blue, silver stain, or with a fluorescent stains such as Deep Purple or
Sypro Ruby.

Blue native PAGE gels are interpreted in a different manner then conventional 2-D gels. Vertically ligned
spots indicate the protein composition of a protein complex. Larger complexes and super complexes are
located on the left hand side of the image. Blue Native DIGE is a useful additional method for studying
membrane proteins and protein complexes and offers some distinct advantages over conventional 2-D
electrophoresis or liquid chromatography techniques.
SALDI

Surface-assisted laser desorption/ionization (SALDI) is a soft laser desorption technique used for mass
spectrometry analysis of biomolecules, polymers, and small organic molecules.[1][2] In its first
embodiment it used graphite matrix. At present laser desorption/ionization methods using other inorganic
matrices such as nanomaterials are often regarded as SALDI variants. As an example, Titania nanotube
arrays (NTA) as a substrate can be used to detect small molecules.[3] SALDI is used to detect proteins and
protein-protein complexes.[4] A related method named "ambient SALDI" - which is a combination of
conventional SALDI with ambient mass spectrometry incorporating the direct analysis real time (DART)
ion source has also been demonstrated.[5] SALDI is considered one of the most important techniques in
MS and has many applications.[6]

Principle:
The main principle of SALDI relies on a medium that absorbs energy from a laser and then transfers the
energy to the target sample. This class of techniques where the bulk of energy goes to the substrate instead
of the sample molecules is known as soft ionization techniques. The development of SALDI started as a
modification of matrix-assisted laser desorption/ionization (MALDI). The former technique suffered from
ionization interference from the matrix molecules of MALDI. SALDI substituted an active surface of
specific substrates, usually made of inorganic components, for the organic matrix of MALDI.[9]
SALDI is a three-stage process. The first stage is mainly concerned with mixing the samples with the
substrate. In the second stage, the laser pulses are applied to the mix where the substrate absorbs the laser
energy and transfers it to the sample molecules. In the final stage desorption and ionization occur and the
potential difference accelerates produced ions into the mass analyzer.
Instrument:
SALDI as an improvement of MALDI, used the very similar instrument to that of MALDI. It employs a
laser source for pulsed laser generation which is responsible for excitation of the sample mixture, which
consists of the analyte and substrate materials.On the other side of the instrument, the mass analyzer which
separates the analytes according to their mass to charge ratio (m/z) and the detector are located. Analytes
are accelerated to the analyzer by applying potential difference.
Applications

1. Forensics
Forensic investigation owes DIOS the favor of producing evidence in contraceptive polymers in an alleged
sexual assault case that could've never been made by any other analytical technique.[15][16] Moreover,
Pihlainen K. et al. reported that the technique showed great promise in the forensic analysis of illicit
drugs.They also reported that the interference was diminished by using this technique.[17][18]
In addition, another report stated that DIOS identified 11 impurities.[19] Profiling the impurities was
expected to lead to their origin. Eight years later, the authors published another report and mentioned that
the technique identified the catecholamines in a human peripheral blood lymphocyte extract.[20] Also the
quantitation of salicylate in human serum was proved using the DIOS-MS in negative ion mode.[21]

2. Biomedical
Thomas et al. worked on a group of enzyme systems, were able to achieve monitoring and direct analysis
of enzyme-catalyzed reaction by DIOS-MS . One famous result was the reaction of acetylcholineesterase
(AChE) with acetylcholine producing choline.This approach gained more fame when showed the ability to
detect the selectivity of different enzyme inhibitors. The study started with hyperzine A, tacrine, and 2,6-
dimethoxyphenyl-N-butylcarbamate, which are all inhibitors of AChE. The Inhibitor constant (ki) value of
each of the inhibitors was found to be an important factor of their inhibition potentials. DIOS-MS has
another advantage over MALDI, it can detect additional information in the low-mass region of mass
spectrum as it can detect peptide peaks, and also identify post-translation modifications. These capabilities
have great applications in protein identification with more confidence.[6]

3. Clinical diagnosis
The DIOS-MS technique was employed as a novel technique for patient diagnosis by examining the
patients' plasma. The study focused on patients with polycystic ovarian syndrome (POS) by comparing the
DIOS metabolic profiles generated with those of healthy subjects .The information obtained can be used to
estimate disease progression and the effect of medical treatment.[6]

4. Pharmaceutical
An interesting area for researchers is to mobilize or immobilize some target proteins. It's a requirement in
drug development mechanisms as some proteins' binding partners are not discovered yet. This was
achieved by Zou et al. by using the DIOS technique. They employed a Psi surface as a probe to immobilize
a targeted protein .
Next, the test drugs with the trial binding partners were introduced and incubated the probe. The
immobilized proteins were able to capture the drug molecules that had a high affinity for the targeted
protein. The ones with low affinity were washed off. The next step was to identify the captured drug
molecules, and this was done by the SALDI analysis. The process offers great selectivity in testing drug
candidates; it filters out the weak candidates and picks the most effective ones. It's not limited to proteins, it
can work with macro biological molecules like DNA and RNA.[6]
Another famous test was done with hemoglobin. In this test, a hemoglobin-modified surface was
employed. The target was to identify the non-covalent binding between hemoglobin and relevant
chemicals. Among 13 different chemicals that included antimicrobials, insecticides, fungicides and
herbicides, only triphenyltin chloride succeeded in binding hemoglobin strongly. This was a practical
warning of the high toxicity of this material relative to other tested compounds.

5. Metabolic profiling
With increasing work and research in metabolomics, new techniques were needed for introducing novel
study approaches. SALDI-MS and the family of direct analysis mass spectrometry (DAMS) were
introduced as novel approaches in metabolomics. Goodacre et al. employed the DIOS-MS technique to
study the yeast. They portrayed the yeast metabolites secreted by yeast showing that the metabolic
"footprinting" of yeast is achievable. In a prior research work, the same group employed direct infusion
mass spectrometry (DIMS) with electrospray ionization. They studied the metabolic profiles of a large
number of wild types and mutants; and mathematical techniques were employed for data analysis to
determine potential biomarkers.[6]

6. Imaging mass spectrometry

SALDI has been employed for imaging of a mouse heart and brain tissues.[22] This was the first achieved
SALDI-MS. As in SALDI, laser has to penetrate through the tissue and be absorbed by the layer
underneath, thickness would be a limiting factor, and researchers were able to overcome this factor by
introducing an organic matrix onto the tissue section. This was named matrix enhanced surface-assisted
laser desorption/ionization mass spectrometry (ME-SALDI-MS) to account for the different processes
employed in the technique and refer to the modification that enhanced the technique.

More work was done for imaging of drug molecule distribution in brain tissue, then the cholesterol
distribution in brain tissue and the sucrose distribution in Gerbera jamesonii flower stem. Also biofluids for
direct analysis of drug molecules and their metabolites has been investigated.[6]