advances-in-protein-chemistry

of the compounds of interest. High-resolution imaging is typically accomplished by using a homogenous spray coating or a spotted
array. The best spatial resolution is achieved by a spray coating that is homogeneous while improved spectral quality and precision is
achieved by using an array that is densely spotted. On the other hand, a heterogeneous coating provides locations on the specimen that
are either dilute or concentrated for ablation. Crystal formation, as a result, is random and images are both highly pixilated and poor in
quality. Robotic spotting instruments are commercially accessible and use a variety of techniques such as capillary deposition, ink-jet
printer, piezoelectric and acoustic methods. Robotic spray-coating instruments are also available that use either a thermally assisted
spray or a mist-nebulizer [16].
The best type of MS detector for proteomic analysis of biological tissue is the time-of-flight or TOF MS. The TOF detector
complements the pulsed laser used by MALDI. In addition, it is easy to maintain, has a simple design, is capable of detecting multiple
m/z ratios, is efficient in ion-transmission, and has a theoretically unlimited dynamic range [16]. Ions generated during the MALDI
process are accelerated through the field-free drift tube of the TOF detector. The ions collect at the multichannel plate detector where
the small m/z ions arrive first and the large m/z ions arrive last. If properly calibrated, then the arrival time is converted to m/z ratio via
m 2Vt
=
2
z L
2
Where V is the voltage potential applied to accelerate ions, t is the time the ion arrives at the detector, and L is the length of the fieldfree
drift tube [2].
MALDI/IMS quantification
To quantify proteins or peptides using MALDI IMS, the precision among pixels should be good. In other words, two neighboring
pixels with the same protein concentrations should provide the same mass spectra. These spectra typically do not differ from each
other by more than 15%. Some of the items to consider if there is a significant difference include extraction efficiency of the MALDI,
ionization efficiency of a particular compound, ion-suppression effects and the effect of post-acquisition processing. In particular,
sample preparation and matrix application are the two techniques that need to be mastered to obtain good precision among pixels.
A constant laser power and voltage should also be maintained within the assay to achieve maximum precision. To prevent variation
among operators, robotics is typically employed. It is important to note that even if these parameters are kept under control and high
reproducibility is achieved, estimation of relative concentrations of two different proteins is still difficult to obtain by comparing peak
areas or peak heights [16].
MS reproducibility should be accurately estimated when attempting to correlate MS signal intensities to values with biological
meaning. Statistical analysis and data preprocessing are tools a scientist can use to probe the biological meaning from these data. Data
preprocessing steps include background removal algorithms, signal intensity normalization and peak alignment [17]. The purpose of the
normalization of signal intensity to the total ion current is to minimize variations in the sample preparations. As a result of these data
preprocessing steps, a new ion image can be generated that better represents the biological system [16].
After the data preprocessing steps are completed, an average mass spectrum is either taken of each target area within a specimen
or of each tissue specimen itself. Statistical analyses are then performed on each of these spectra. Principal component analysis (PCA)
is a methodology used to rank the variance in the system to determine the number of significant components that are in the system
[17]. Scores can be assigned to components based on how it changes its signal intensity relative to the standard deviation of multiple
experiments. This methodology is called significance analysis of microarrays or SAM. The changes within multiple experiments help
to estimate the fraction of components that are seen by chance (potential false positives) when scores are greater than a predetermined
threshold. SAM specifically exposes components that demonstrate a significant change between two groups of tissues. A method that
blindly groups samples based on their profiles is hierarchical clustering analysis or HCA. HCA specifically computes the dissimilarity
between particular experiments [16]. Based on the dissimilarity values, a dendrogram is created, which clusters or groups similar
components and depicts these similar groups in a graph that looks similar to a tournament bracket [17].
Surface Characterization
A specific instrument used for the characterization of proteins is the atomic force microscope (AFM). The AFM is made out of a
cantilever with a tip where van der Waals forces are measured between the tip and the sample surface. These van der Waals forces cause
the tip to be redirected, and this movement is optically detected by the instrument. Constant force applied to tip enables a strict up-anddown
movement of the tip providing data on the topography of the sample surface. The flexibility of the AFM lies in the fact that it can
be used for non-conducting samples [2]. The three modes used for AFM are contact mode, non-contact mode, and tapping mode [2]. In
contact mode, the tip makes contact with the sample at all times. If the sample is a liquid, then surface tension forces brought about by
adsorbed gases or the surface layer of the liquid can pull the tip into the sample. As a result, the tip can potentially damage the sample
and dampen the tip. This mode is not the best setting for biological or protein samples. In tapping mode, the tip briefly makes contact
with the surface and is immediately retracted. The typical oscillation is on the order of hundreds of kHz. The least popular mode of AFM
is the non-contact mode where the tip is constantly a few nanometers away from the surface of the sample. Not surprisingly, the van der
Waals forces detected on the tip and cantilever are much smaller [2].
In AFM, the data provided is a force curve, which is a plot of the force applied onto the tip versus the displacement of the tip. The
location of the tip and movement of the AFM cantilever can be detected down to the nanometer thanks to the reproducible piezoelectric
detection units. The movement of the cantilever can be used to compute the force applied using Hooke’s law [18]. There are typically
two plots provided by a force curve. When the tip is coming toward the surface of the sample, this is the approaching curve. When the
tip is moving away from the surface of the sample, this is the retracting curve. With respect to noise, the two curves are overlaid on top
of each other if there is no sample present. There should be two regions to these curves. There should be a horizontal region called the
noncontact region that represents the baseline when a tip is not touching a surface. There also should be a region of the curve which rises
up from the noncontact region called the vertical contact region which accounts for the van der Waals forces measured by the tip. The
contact point is the spot on the force curve where the two regions meet and it is a critical portion of the force curve because it becomes a
reference as to where the tip made contact with the surface [19].
The “polyprotein strategy” has been used to investigate proteins using AFM, especially the globular proteins [19]. This technique
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