2012 EDUCATIONAL BOOK - American Society of Clinical Oncology

Table 1. Summary of Genomics Measurement Technologies and
Normalization Techniques.
Genomic Measurement Technology Platforms
DNA sequence Sequencing NGS
Gene expression microarray Affymetrix GeneChip
Agilent microarray
Illumina BeadChip
Gene expression RNA-seq NGS
ChIP-chip Roche NibleGen
DNA binding ChIP-seq NGS
Methylation array Illumina BeadChip
Bisulfite sequencing NGS
Methylation
Binding assays (MBDSeq, MeDiP) NGS or microarray
SNP-chip Affymetrix SNP array
Illumina SNP array
SNP
Sequencing NGS
Abbreviations: NGS, next generation sequencing (performed sequencers, including
Illumina Solexa, Applied Biosystems SOLiD and Roche 454); ChIP,
chromatin immunoprecipitation; MBDSeq, methyl-CpG binding domain protein
sequencing; MeDiP, methylated DNA immunoprecipitation; SNP, single nucleotide
polymorphism.
low dynamic range for quantification, and high-quality antibodies
may not be available for all targets of interest.
Reverse-phase protein microarrays (RPPM) are similar to
TMAs in that the protein mixture of interest is spotted onto
a glass slide and used for hybridization to an antibody. The
spots used for RPPM can be smaller than for TMA, and,
thus, a larger number of specimens can be surveyed simultaneously.
Again, the quality of the RPPM is dependent on
the availability and biochemical properties of the antibody.
Two-dimensional protein separation using 2D-DIGE is
achieved by isoelectric focusing and electrophoresis in a
polyacrylamide gel. Newly developed multicolor fluorophores
allow separate labeling of different protein mixtures,
which are then combined and analyzed together by 2D-
DIGE. However, reproducibility and sensitivity of the 2D
gels can be problematic, and it may miss some of the
smallest and largest proteins in the analysis.
KEY POINTS
● Although DNA sequencing data reveal a complex
genome with numerous mutations, the biologic and
clinical significance of genetic aberrations are largely
unknown.
● Microarray-based technologies for gene expression
analysis are being rapidly replaced by next generation–sequencing
technology.
● New advances in proteomic technologies have made
unbiased and quantitative analysis of thousands of
proteins possible, leading to novel insights into tumor
biology and clinical features.
● Although they are powerful discovery tools, each
finding must be vigorously validated before clinical
application.
● New genetic and proteomic analysis technology allows
comprehensive analyses of pathways rather
than individual genes or proteins, and they are promising
tools to identify biomarkers and potential therapeutic
targets.
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FERTIG, SLEBOS, AND CHUNG
Mass spectrometry-based applications have revolutionized
protein detection and quantification, and the advances
are now starting to be used for clinical applications. All MS
instruments work by first ionizing biomolecules into a gasphase,
separating these ions by their mass/charge properties
and detecting them after separation in the instrument. One
of the first MS applications uses was Matrix-Assisted Laser
Desorption Ionization (MALDI), where low molecularweight
proteins are ionized from a solid matrix and separated
by mass and charge to yield a detection profile unique
for a given mixture of proteins. MALDI-MS has the advantage
that it requires minimal sample processing and that
it can analyze a large number of samples in a short time.
However, it is limited to the smallest proteins, does not
provide protein identification, and is very prone to experimental
variation.
Since MS instruments are most sensitive and accurate
with small molecules, detection strategies that include digestion
of proteins to peptides have been highly successful in
recent years. Here, a complex mixture of proteins is first
digested into peptides by trypsin, which cleaves proteins at
lysine (K) and arginine (R) residues. These peptides are then
ionized using a solid matrix (MALDI) or, more commonly, by
electrospray, where a solution of peptides is forced through
a thin needle to form a continuous spray that creates peptide
ions for analysis. Additional biochemical separation can be
applied before MS analysis using isoelectric focusing- or
strong cation exchange columns for a multidimensional
peptide separation. After separation and ionization, peptides
may be analyzed and detected directly by MS, creating
a “fingerprint” of the original proteins; however, this only
works for relatively simple protein mixtures. For true identification
purposes, peptide ions collide with an inert gas to
form fragment ions that are analyzed in a second MS phase
(tandem-MS or MS/MS) to create fragment MS spectra that
are unique for each peptide. Modern instruments can collide
and analyze thousands of peptides per minute, and these
analyses can be performed on a large scale and typically
yield thousands of protein identifications with quantitative
information. Identification is achieved by creating a list of
spectra predicted from genome sequencing information to
include all possible human peptides that could potentially
match the obtained MS spectra. These theoretical spectra
are then matched to the observed MS spectra through
database-search algorithms (Sequest, X!Tandem, Myrimatch).
Knowledge of the full sequences of all proteins is
then used to reassemble the identified peptides into a list of
proteins, similar to the process used to create DNA sequencing
alignments from known genomic data. The number of
times spectra were observed can function as a measure of
quantity for each protein.
When only a limited number of proteins are of interest, a
more targeted approach is possible in which the MS instrument
is restricted to a narrow range of peptide masses,
which can then be measured with high sensitivity and over
a large dynamic range (selected or multiple reaction monitoring
[SRM or MRM]). These assays can be multiplexed to
create highly specific assays that are also capable of measuring
specific protein isoforms or modified protein forms.
Proteins can be detected even in lysates generated from
formalin-fixed paraffin-embedded tissues, since trypsin digestion
can free up sufficient amounts of peptides for SRM or
MRM detection.