advances-in-protein-chemistry

technique has been applied to the investigation of protein secretion within bodily fluids, constituents of protein complexes, changes in
proteins within subcellular fractions and overall changes in the expression of proteins [3].
Inserting isotopes within proteins by developing mammalian cells in media using a stable isotope of an amino acid describes SILAC
[13]. Quantification of protein expression is determined by comparing cells produced using SILAC against cells untreated by SILAC.
Both of these samples can be analyzed within the same assay because of the m/z change triggered by the stable isotope label. SILAC
is, however, constrained to applications of tissue culture [3]. A specific example of SILAC used for protein quantification is metabolic
labeling using 15 N in vivo. Amino acids labeled with 15 N isotopes are used in the synthesis of a protein. Both the 14 N and the 15 N protein
isotopes are mixed before sample preparation to act as an internal standard for quantification especially when using LC-MS. In LC-MS,
an internal standard that is separated from the compound of interest can ionize at different rates using the soft ionization techniques of
ESI or MALDI, especially if using a gradient elution method. Using an isotopically labeled compound enables both the compound of
interest and the internal standard to be measured at the same ionization conditions [3].
A primary issue when performing proteomics is quantification. There are two approaches for quantification in proteomics: labelfree
methods and label methods. Label methods involve using stable isotope labels to act as an internal standard without quenching
the signal of the analyte. As a result, the reproducibility of the data is enhanced. In contrast, there are some label methods that are
cheaper and easier to use along with the fact that they provide acceptable reproducibility. By using a linear ion trap MS with a label-free
methodology, Higgs et al. was able to achieve less than 10 % relative standard deviation (RSD) for their bottom-up proteomic application
[14]. Differential MS is another label-free methodology used by Wiener and coworkers in which ions that demonstrate differences
in intensity that are statistically significant are additionally probed by tandem MS for sequencing [15]. Since the results that show a
statistical difference are targeted for identification, this methodology enables efficient calculations [3].
Matrix-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry
The primary advantage of MS over other analytical detectors is its specificity to molecules. The ionization techniques discussed earlier
described preparing a liquid sample of MS analysis. Utilization of matrix-assisted laser desorption/ionization or MALDI enables the
scientist to study intact tissue and the distribution of the proteins within this tissue. This ionization technique needs to be matched with
imaging mass spectrometry or IMS to analyze tissue specimens. The direct analysis of tissue specimens entails minimal effort and time in
sample preparation and enables direct comparison between different biological species or samples [16].
With a lateral image resolution 300 to 500 Å, IMS can detect a variety of molecules such as proteins, fatty acids, and pharmaceutics
from sections of frozen fresh tissue. The analyst first cuts sections of the tissue down to 100 to 150 Å thick and then mounts them on
target plates where they can equilibrate to room temperature. After these steps, the analyst applies an energy-absorbing compound
or a matrix to the tissue section. A UV laser then ablates an area of the tissue that is approximately 500 Å wide. This process results in
molecular ions that are detected via the MS detector. Each area that is ablated in the section corresponds to a single MS. Extraction of
the relative abundances of an ion for a single m/z ratio can then provide a contour plot with the ion intensities projecting outward and
the location of the section given on the x and y axes. It is critical to deposit the energy-absorbing compound in a homogeneous manner
to obtain a high-resolution image. Either using a robot to spray the matrix with a continuous coating or automatically printing groupings
of droplets are techniques to accomplish this task. MALDI MS then probes each pixel coordinate or micro spot. Provided that signals
are above the limit of quantification, a contour plot can be obtained at any m/z ratio. Ultimately, MALDI is operational for identification
and quantification of both infected and healthy biological tissue. In addition, it can provide insight into describing changes in a biological
system. Applications of this technique include reaction of a tissue to administration of a drug, reconstructions of a biological organ, and
three-dimensional images of the brain and its proteins [16].
The primary advantage of IMS is that it can automatically image dozens upon dozens of compounds in tissue sections with targeting
compounds, antibodies or even prior knowledge of the tissue being analyzed. IMS allows for imaging of proteolytic catalysis or posttranslational
modifications unlike most bottom-up proteomic assays. While not effective for proteomic analysis, secondary ionization
mass spectrometry or SIMS enables high-resolution imaging, on the order of 500 to 1000 Å, for molecules less than 1000 m/z down to
elemental species [16].
Imaging technology
There are two general methodologies to analyzing biological tissue via IMS: imaging and profiling. Probing distinct regions (200 to
1000 µm in diameter) of tissue slices and performing computational analysis of the resulting mass spectra (typically, 5 to 20 in quantity) is
a general characterization of the profiling methodology. This method compares two different tissue specimens or two significant regions,
i.e., diseased vs. healthy, within a tissue. As a result, it is not necessary to have a high degree of spatial resolution [16].
Imaging involves analyzing the entirety of the tissue section by systematically sampling spots on the tissue. These spots, or raster,
are a constant distance away from each other both vertically and horizontally. This distance defines the image resolution. The ion
abundance, perhaps of a certain m/z, is plotted against the xy coordinates. This plot can then be used to assess the differences of protein
localization among samples [16].
To reduce degradation of proteins via proteolysis and to preserve morphology, the tissue sections must be submerged in liquid N2
immediately. Typically, the tissue is sliced into 100 Å to 120 Å sections within a cryostat and subsequently mounted on a sample plate
that is constructed of stainless steel or coated with gold to conduct electricity. Ethyl alcohol is used to wash the tissue to help mount it
and to rinse away excess salts and lipids thereby preventing ion suppression. Another option is to conduct protocols that stain tissue with
compounds that specifically facilitate IMS detection [16].
The use of a matrix that absorbs energy is necessary for MALDI IMS. Typically, it is an organic molecule of small size that
crystallizes simultaneously with your compound of interest on the surface of the tissue. It absorbs the energy from a laser enabling your
compound of interest to desorb and then ionize. Typical matrices used for MALDI are α-cyano-4-hydroxy-cinnamic acid (HCCA), 2,
5-dihydroxybenzoic acid (DHB), and 3, 5-dimethoxy-4-hydroxy-cinnamic acid (SA). Typical solvents used for analysis include 50:50
(v/v) water/ethyl alcohols or 50:50 (v/v) water/acetonitrile. HCCA and DHB are typically used for peptides and smaller compounds,
while SA is typically used for protein molecules [16].
Deposition of the matrix should be homogeneous to facilitate high-resolution imaging and to preclude substantial lateral migration
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