Page 198 - Veterinary Toxicology, Basic and Clinical Principles, 3rd Edition

Toxicoproteomics in Diagnostic Toxicology Chapter | 10 165

VetBooks.ir parent protein can be determined by the relative peak entail either label-free or labeled probe methods (Espina
Detection strategies for protein or peptide microarrays
areas of UV-detected peptides observed from that protein
et al., 2004; Cretich et al., 2006). The label-free strategy
(Chelius and Bondarenko, 2002). Another HPLC-based
approach for relative protein quantitation involves the use is a direct detection method that includes MS, surface
of internal protein standards (Bondarenko et al., 2002). In plasmon resonance, or atomic force microscopy. SELDI
this technique, biological samples are spiked with an microarray chips commonly use MS-based detection of
internal standard protein prior to enzymatic digestion of proteins or peptides. Labeled probe methods of detection
the proteins into peptides. The chromatographic peaks include utilizing chromagens (similar to ELISA proto-
observed can be normalized to the peak area of the inter- cols), chemiluminescence, fluorescence, or radioactive
nal standard and the relative concentrations of the proteins decay-based detection techniques.
are inferred. Microarray technology is progressively becoming a
Unlike 2-DGE/MS tools, multidimensional HPLC/MS versatile platform for its potential use in diagnostic toxi-
is amenable to high-throughput analyses and has the ability cology. Although efforts to standardize array analyses
to resolve peptide mixtures regardless of molecular mass or have been challenging, microarrays make it possible to
hydrophobicity. This is important because resolving peptide simultaneously screen thousands of samples and profile
mixtures by 2-DGE is impractical due to their narrow large numbers of proteins from biological samples.
isoelectric points and molecular weight ranges and because
2-DGE tools cannot be directly interfaced with MS instru- Mass Spectrometry
mentation. Even though 2-DGE is superior when separating
intact proteins, methods based on pairing multidimensional Recent developments in hybrid mass spectrometers have
HPLC and MS technology are becoming more refined and revolutionized the ability to analyze proteins and peptides,
continue to advance proteomics research. providing high-throughput automation combined with
high sensitivity and resolving power. In general, mass
spectrometers have three components, an ion source, a
Protein and Peptide Microarrays
mass analyzer, and a detector. The ion source is the com-
Protein and peptide microarray technologies offer a com- ponent in which protein and peptide ions are produced;
plimentary approach to traditional separation methodolo- the mass analyzer separates or resolves these ions based
gies and mass spectrometry. This technology incorporates on their mass-to-charge (m/z); and the detector detects the
the use of a variety of high-throughput microarray plat- selected ions from the mass analyzer. One stage mass
forms to probe protein function, abundance and to glob- analysis is commonly used to measure the molecular
ally analyze protein expression in biological systems. The weights of intact proteins or peptides. However, in order
platforms commonly used in proteomics include prote- to obtain peptide sequence information, hybrid mass spec-
ome, antibody, reverse-phase protein, and lectin microar- trometers must be employed. MS/MS, or tandem mass
rays (Lina et al., 2011). spectrometry, detects intact peptide ions in the first mass
Protein function microarrays use immobilized capture analyzer. Once selected, the peptide ion enters the colli-
ligands to screen for protein drug, protein enzyme, sion cell where it is fragmented into secondary ions by
protein protein interactions (Kawahashi et al., 2003; collision-induced dissociation (CID). These ions are then
Nielsen et al., 2003; Zhu et al., 2003; Cretich et al., separated in the second mass analyzer, their m/z ratios
2006). Analytical microarrays, such as those used in detected, and MS/MS spectra are generated.
surface-enhanced laser desorption (SELDI)/TOF MS, use Bioinformatics database tools use the MS/MS data to gen-
retentate chromatography for protein profiling and detec- erate peptide amino acid sequence information and com-
tion (Merchant and Weinberger, 2000; Cretich et al., pare them with theoretical sequences in protein or
2006). This technique is capable of on-chip sample genome databases to identify proteins.
fractionation utilizing various chromatographic surface There are two major types of hybrid mass analyzers
chemistries and can probe for chemical properties in used in proteomics research, matrix-assisted laser desorp-
proteins such as phosphorylation, glycosylation, hydro- tion ionization/time-of-flight (MALDI-TOF/TOF) instru-
phobicity, or anionic cationic properties. ments and electrospray ionization/tandem MS instruments
Microarrays require immobilization of a capture ligand (ESI/MS/MS) (Karas and Hillenkamp, 1988; Fenn et al.,
or chromatographic media onto a planar solid support, 1989; Hillenkamp et al., 1991). The MS technology cho-
often resulting in thousands of spots per slide (MacBeath sen depends on the type of MS data that needs to be gen-
and Schreiber, 2000; Kumble, 2003). Samples of interest erated from toxicoproteomic experiments. For example,
are added to the microarray and the entire array is MALDI-TOF/TOF instruments are fast, robust mass ana-
scanned to detect proteins or peptides that interact with lyzers, have a large dynamic range and do not require
the ligand or chromatographic media. labor-intensive sample preparation. MALDI-TOF/TOF