Clinical Biochemistry of Domestic Animals (Sixth Edition) - UMK ...

130
Chapter | 5 Proteins, Proteomics, and the Dysproteinemias
of proteomics. Mass spectrometry actually measures the
mass-to-charge ratio (m/z) of ions under vacuum. To perform
this process, a means of generating the necessary ions,
a mass analyzer and a detector are required ( Patterson and
Aebersold, 1995 ). In proteomic investigation, the most widespread
approach is the MALDI time-of-flight (TOF) mass
spectrometer, which is used in combination with database
searches to identify the protein in a specific spot on 2DE
protein maps.
Protein identification by MALDI-TOF mass spectrometry
is based on some prior knowledge of the amino acid
sequences of likely proteins to be identified or of equivalent
proteins in other species. It is dependent on enzymic
cleavage of the protein into shorter peptides, the size of
which is defined by their component amino acids. Before
mass spectrometry, the protein spot to be identified is cut
out of the polyacrylamide gel and subjected to hydrolysis
by a specific protease, usually trypsin. The site of action
of trypsin is at the peptide bond of the basic amino acids
lysine and arginine. The trypsin digest products of any
proteins whose primary structure is held on international
protein databases such as UniProt ( www.ebi.uniprot.org )
or NCBI ( www.ncbi.nlm.nih.gov ) can be predicted from
the position along the protein sequence of the arginine
or lysine residues. The tryptic digest product of any protein
whose primary structure can be derived from genetic
(DNA) databases can also be determined. These tryptic
digest “ fingerprints ” are characteristic of each protein and
identify protein spots after 2DE.
The trypsin-digested sample is mixed with a matrix compound
and dried on a metal slide, which is inserted into the
mass spectrometer. Bombardment of the slide by a laser
results in the ionization of the peptides and application of a
high voltage causes the ions to travel rapidly to the detector
with smaller ions having a greater velocity. Thus, small peptides
have a shorter time of flight than larger peptides, and
from this the mass of each peptide is determined to a high
degree of accuracy. The data generated by the MALDI-TOF
are thus the mass of each of the peptides produced by the
trypsin digest of the protein excised from the 2DE gel.
Identification of the protein is completed by comparison
of the masses of all the peptides produced by trypsin digest
to the protein and gene databases. This is especially useful in
species where the whole genome has been sequenced such
as human or mouse. There are means to identify proteins by
this peptide fingerprint approach even where the genome
has not been determined ( Wait et al. , 2002 ), though a number
of genomes of the domestic species (cow, dog, chicken)
are close to being fully sequenced, which will simplify the
proteomic investigation of samples from these species. More
advanced mass spectrometry using ESI in tandem MS with
quadruple instrumentation can directly determine sequences
of peptides, but these methods are more time consuming. An
advantage of MALDI-TOF is that it can be used in robotic
systems where computer-controlled workstations can excise
multiple spots from a 2DE gel and automatically perform
the trypsin digest, transfer the hydrolysate to the mass spectrometer,
and identify the protein as a probability score of
the most likely candidate protein.
c . Non-Gel-Based Proteomic Analysis
When first developed, proteomics combined protein separation
on 2DE and mass spectrometry. However, for use
in diagnostic investigations, 2DE is an expensive, timeconsuming,
and difficult technique to reproduce precisely.
It is likely to remain a research tool unless major advances
are made in the robustness of the methodology. Interest
is shifting to non-gel-based approaches to proteomics in
which alternatives to 2DE are used for protein and peptide
separation but with mass spectrometry still being used for
identification. These methods have a greater potential for
automation, throughput, precision, and accuracy, which
may in the future allow their use in disease diagnosis.
One such approach is surface enhanced laser desorption
ionization mass spectrometry (SELDI-MS). In this
method, a sample such as serum is preincubated with a
“ protein chip, ” which has one of a variety of surfaces to
which proteins bind with differing affinity. These surfaces
are designed to bind with protein by ion-exchange, hydrophobic,
or metal chelate interaction. After washing away
unbound protein, the protein chip is placed in the SELDI-
MS instrument and subjected to mass spectrometry. The
system is optimized to identify biomarkers for disease by
contrasting samples from healthy and diseased animals.
This approach can identify peptide or protein peaks in
the mass spectrogram that have potential as biomarkers
and has been used to identify biomarkers for ovarian cancer
and other diseases in humans ( Petricoin et al. , 2002 ).
A drawback of the current SELDI-TOF system is that identification
of the protein biomarkers requires further investigation
by traditional protein biochemistry methods.
Another approach to non-gel-based proteomics is nano
liquid chromatography coupled to mass spectrometry.
Native proteins are in general too large for mass spectrometry,
so before separation the sample is treated with trypsin,
producing shorter peptides. The peptides are separated
by high-pressure liquid chromatography (HPLC) with
the output coupled to an ESI mass spectrometer ( Gaskell,
1997 ; Mehlis and Kertscher, 1997 ). The results can be
plotted with elution volume from the HPLC against the
mass/charge ( m/z ratio) of the peptide and the size of the
peptides compared to protein and gene databases.
An interesting finding from a number of proteomic
investigations using gel and nongel approaches designed
to identify cancer biomarkers has been that many of the
identified candidate biomarkers have already been identified
with many of them being APP ( Diamandis and van
der Merwe, 2005 ). It will be fascinating over the next few
years to see if these advanced techniques can earn a role in
the veterinary diagnostic laboratory.