Proteomics - Biomedical Informatics in Translational Research - page 93

The aggressive cytokeratin isoforms that are associated with poor clinical outcomes
in the lung adenocarcinoma were identified by a combination of 2DGE and 2D
Western blots workflows [24]. Several reports have stressed the importance of pro-
filing host humoral immune responses to developing cancer in the human body and
2D Western blots of proteins displayed on a 2D gel facilitate this type of screening
to identify novel cancer antigens from established cancer cell lines or directly using
the cancer tissue specimens. This type of screening was demonstrated with breast
cancer cell lines using sera from breast cancer patients [25] for identifying tumor
antigens.
Despite the advantages offered by 2DGE in profiling clinical samples to associ-
ate distinct protein patterns or altered protein expressions with pathologic condi-
tions, this method suffers from several shortcomings. Problems detecting
low-abundance proteins; underrepresentation of extremely acidic, basic, and
hydrophobic proteins; and problems detecting proteins with high and low molecu-
lar weights are among these shortcomings. In addition the method is laborious and
plagued with run-to-run variation, which confounds matching spots between runs.
Nonetheless, 2DGE is the only technique that has a high-resolution capacity to pro-
vide visualization of more than 1,000 intact protein spots in one experiment. The
2DGE technique alone and its multiplexed DIGE version have been used to profile
various types of clinical samples, including serum/plasma, CSF, urine, and several
types of cancer tissue specimens in order to identify markers associated with disease
conditions by differential proteomics [26-30]. To give an example, the 2DGE pro-
tein patterns from 24 patients with B-cell chronic lymphocytic leukemia (B-CLL)
discriminated the patients based on the clinical variables and identified the altered
expression of several classes of proteins: redox enzymes, heat shock protein, and
disulfide isomerase associated with the shorter survival [31]. The recently intro-
duced saturation labeling technique using fluorescent dyes improved the profiling of
scarce tissue samples using very minute amounts of protein ( g) and recent

reviews anticipate further improvements to the 2DGE workflows [32].

< 5 μ

6.3.2 MALDI-TOF
The ability to ionize intact large nonvolatile biopolymers using the MALDI tech-
nique has impacted biology and biomedicine in addressing the structural analysis of
proteins [5]. Unlike ESI, MALDI is a static technique in which the analyte of interest
is mixed with an UV absorbing organic compound and spotted on a target for laser
irradiation to generate the ions from an analyte. The suitability or compatibility of a
time-of-flight (TOF) analyzer in recording the ions associated with each laser pulse
exploited this combination further during the last decade. That, along with
improvements in the speed of electronics, have helped MALDI evolve into a most
robust, sensitive, and very high throughput platform for proteomic applications.
The MALDI-TOF technique has been widely used in conjunction with the
2DGE workflows to identify the proteins mainly by peptide mass fingerprinting
(PMF). The front-end NanoLC separation process, which is the key for online
ESI/LCMS shotgun proteomics, has been adapted to collect fractions directly onto
the MALDI target plates and has demonstrated the capabilities of LC/MALDI in
proteomic applications such as biomarker discovery [33]. The added advantage of