A proteomic analysis comprises two steps: (i) separation of the protein mixture in order to allow the efficient detection of the particular proteins included in the mixture and (ii) identification of the separated proteins by various analytical methods, mainly by mass spectrometry (MS). Separation is usually performed by two-dimensional (2-D) gel electrophoresis or liquid chromatography (LC) or more often by combination of the two approaches.82–84
Two-dimensional electrophoresis is the principal separation method in a proteomic analysis and has the advantage that it allows the simultaneous detection of thousands of proteins. It comprises two steps (dimensions), separation of the proteins on the basis of differences in their net charge by isoelectric focusing (IEF), and separation of the focused proteins on the basis of differences in their molecular masses by sodium dodecyl sulfate (SDS) polyacrylamide gels.85;86
When liquid chromatography is applied for protein separation, the intact proteins can be first fractionated by ion exchange and/or hydrophobic interaction chromatography or by a combination of various binding principles. In a next step, the proteins of the fractions collected are digested and the peptides are separated by reversed phase HPLC and analyzed by MS.
The most efficient and most widely used protein identification method in proteomics is the peptide mass fingerprint (PMF) approach which is mainly performed by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF-MS).87
For MS analysis, protein spots are excised from 2-D gels, the proteins are in-gel digested, the digests together with the matrix are applied on the sample target and are measured in the mass spectrometer.88
In a high-throughput proteomic analysis, most of the operations are performed in automated mode and several thousands of protein spots can be analyzed by one person per day. The analysis of certain groups of proteins is more favorably performed by LC/MS, e.g. very acidic/basic, small proteins/peptides, and hydrophobic proteins. However compared to 2-D gels, isoforms, post-translational modifications, and processing products are very difficult to discern via LC/MS.89
shows the workflow for the proteomic analysis of the brain, involving preparation of protein subfractions, protein separation by 2-D electrophoresis or LC and identification by mass spectrometry.
Proteomics is routinely used in clinical diagnosis today, and it has been applied in the investigation of infectious diseases, cancer, heart, and neurological disorders. The applications are summarized in several review articles.81;89–94
In the study of AD, proteomics has been mainly used for the analysis of human brain, of the brain of animals serving as models of AD and of cerebrospinal fluid (CSF) from patients with AD with the goal to collect information about gene products involved in the diseases i.e., alterations in protein levels and post-translational modifications.89
Proteomics has also been used in the investigation of Down syndrome (DS) and other neurodegenerative disorders. Almost all subjects with DS over 40 years show neuropathological and neurochemical abnormalities on post-mortem brain examinations that are indistinguishable from those seen in Alzheimer’s disease. Thus, results from the study of the DS brain may be useful in the AD studies.95–98
IIIa. Proteins with deranged levels and modifications in AD
Most neuroproteomics analyses involve 2-D electrophoresis and MALDI-TOF-MS steps, as this approach allows for a reliable quantification of changes in protein levels. shows the proteins of total brain extract separated on a 2-D gel. The most abundant proteins detected in 2-D gels carrying total brain extract are structural proteins, like tubulin chains and actin, heat shock proteins, dihydropyrimidinase-related proteins-2 and -3, and house-keeping enzymes. The less abundant proteins are mainly hypothetical proteins, enzymes, as well as structural proteins. The relative levels of the low-abundance brain proteins vary in the range 0.005–0.01% and of the high-abundance about 3% in comparison with the levels of total proteins detected in the corresponding gel.89
In one study, the LC-MS/MS approach was used for the detection of proteins enriched in amyloid plaques in the AD brain.99
Proteins of total brain extract separated on a 2-D gel.
The levels of about 100 proteins were found to be changed in the AD brain and cerebrospinal fluid in comparison with the control brain and CSF, respectively. The observed changes in the protein levels usually vary in the order of 20–100% in comparison with the average levels of the control group. The largest changes were observed for the glial fibrillary acidic protein (GFAP), which distinguishes astrocytes from other glial cells during the development of the brain. For this protein, ten-fold or even higher levels were observed in individual AD brains, compared to the average levels of the control group.100
shows an example of a protein, synaptosomal protein 25 kDa (snap25), which shows reduced levels in the AD brain in comparison with the control brain. The proteins detected by proteomics approaches to show changed levels or modifications in AD have various functions, mainly involved in conformational changes, neurotransmission, guidance, signal transduction, metabolism and detoxification. The CSF proteins with deranged levels are plasma proteins. The proteins detected by proteomics methods to have deranged levels in the AD brain and the CSF are listed in .
Example of a protein, in this case the synaptosomal protein 25 kDa (snap25), which shows reduced levels in the AD brain in comparison with the control brain.
Heat shock proteins (HSP) are involved in conformational changes of the brain proteins, i.e. in protein folding and assembly into oligomeric structures.101;102
Several heat shock proteins showed deranged levels in the AD brain. Increased expression was observed for alpha crystallin B, glucose regulated protein 94 (GRP 94) and HSP70 RY, and decreased expression for heat shock cognate (HSC71), GRP 75, HSP60 and T-complex protein-1.103
The inconsistency in the direction of change of the heat shock proteins in AD may be explained by the different response of the individual heat shock proteins to stress conditions. Increased expression of heat shock proteins in AD may represent a defensive mechanism of response to amyloid fibril formation, as chaperone proteins are capable of preventing aggregation of other proteins and in particular inhibiting self-assembly of polyglutamine proteins into amyloid-like fibrils. Reduced levels of the HSP70 proteins, in particular of the HSC71, might be linked to their protective role in neuronal death associated with AD. HSC71 may be involved in the structural maintenance of the proteasome and conformational recognition of misfolded proteins by proteases.104;105
Thus, decreased activity of HSC71 which is attributed to either reduced levels103;106
or increased oxidative modification of the protein104
may be responsible for impaired protein clearance and deposition of amyloid-β peptide in the AD brain.
Synaptosomal proteins, soluble N-ethylmaleimide sensitive factor attachment proteins (SNAPs) forms α, β, γ, synaptososmal associated protein 25 (SNAP 25), synaptotagmin and vesicular proteins, are involved in synaptic transmission which is deranged in AD. Proteomics studies showed reduced expression for SNAP 25100
(), β-SNAP and synaptotagmin I107
in the AD brain and suggest impaired neurotransmission in AD.
Guidance is facilitated by proteins signaling the correct path to the growing axon. Dihydropyrimidinase-related protein 2 (DRP-2), also known as collapsin response mediator protein-2, is a cytosolic protein that shows a high homology to the rat turned on after division 64 kDa protein and the chicken collapsin response mediator protein-62, which are involved in path finding and migration of neurons. The functions of DRP-2 are not well known. It is possibly involved in neuronal repair and interacts with and modulates the activity of collapsin, a protein that elongates dendrites and directs them to adjacent neurons. Proteomics studies revealed deranged expression levels108
and oxidative modification104
of DRP-2 in the AD brain. The reduced levels of DRP-2 in the AD brain may be responsible for synaptic loss in AD as it can not respond to the formation of new synapses. Moreover, DRP-2 is immediately modified by oxidation and looses activity, which results in shortened dendrites that would cause decreased interneuronal communication and memory impairment.109
The levels of several signal transduction proteins have been found to be deranged in the AD brain. Thus, reduced levels were found for 2′, 3′-cyclic nucleotide-3′phosphodiesterase (CNPase), 110
nucleoside diphosphate kinase (NDK)-A111
and increased expression for 14-3-3 gamma and epsilon proteins.113
CNPase is associated with oligodendroglia and myelination. NDKs are oligomeric enzymes, involved in the maintenance of the intracellular pool of deoxynucleotide triphophates and nucleotide triphosphates, playing regulatory roles in the activation of G-proteins.114
Stathmin, a major substrate of cyclin-dependent kinases and protein kinase A, is involved in multiple signal transduction pathways. Stathmin possibly plays a role in neurodegeneration and impaired signaling because of its negative correlation with neurofibrillary tangles, a hallmark of AD neuropathology, and interaction with tubulin to cause microtuble destabilization.112
14-3-3 proteins form homo- or heterodimers and are primarily, but not exclusively expressed in neurons and bind to and modulate the function of a wide variety of cellular proteins. They are involved in neuronal development, signal transduction, cell growth and cell death. They show increased levels in AD which may be rather linked to apoptosis than to other cellular activities, as they are highly enriched in areas of massive neuronal death caused by pathological processes.115
The increased levels of 14-3-3 proteins in AD may represent a protective response against apoptosis.
Oxidative stress, either detected as protein oxidation, lipid peroxidation, DNA oxidation and 3-nitrotyrosine formation, is considered to be a common mechanism for different age-related neurodegenerative pathologies. The levels of antioxidant proteins in the brain were studied using proteomics technologies. Superoxide dismuatase (CuZn, SOD1) catalyzes the dismutation of superoxide anion into hydrogen peroxide. The protein showed decreased levels in AD and the decrease may reflect cell loss in AD.116
The expression of two other proteins, carbonyl reductase and alcohol dehydrogenase, involved in oxidative stress, were found to be increased in AD.117
These two cytosolic enzymes catalyze reduction of carbonyls, which are toxic metabolic intermediates, increased in the AD brain104;105
and serve as markers for oxidative stress, suggesting a possible role of oxidation-related decrease in protein function in the process of neurodegeneration. The increased levels of these enzymes may represent an adaptive mechanism to detoxify the toxic intermediates. Peroxiredoxin I and II showed increased and peroxiredoxin III decreased levels in AD brain.118;119
Peroxiredoxins are a family of peroxidases that protect biomolecules by reducing hydrogen peroxide and alkyl hydroperoxides. There is evidence that over-expression of peroxiredoxins, in particular of peroxiredoxins I and II, in various cell lines abrogates the effects of agents that positively modulate apoptosis, including hydrogen peroxide.120
Enhanced apoptosis has been demonstrated in AD121
and up-regulation of these enzymes could represent a cellular response against apoptosis. In other neurodegenerative disorders, like DS and Pick’s disease, deranged levels for peroxiredoxins were observed as well.118;119
Oxidative stress is observed in the AD brain in regions where amyloid β-peptide 1-42 is present, but not in cerebellum where AD pathology is not observed.122
Several oxidized proteins were detected by immunoblots in the AD brain and identified by MALDI-TOF-MS (). One of the oxidized proteins is creatine kinase (BB isoform). Amyloid β-peptide inhibits creatine kinase and causes lipid peroxidation leading to decreased energy utilization, alterations in cytoskeletal proteins and increased excitotoxicity, all reported for AD. The other oxidized proteins are glutamine synthase, which is related to increased excitotoxicity, ubiquitin C-terminal hydrolase L-1, associated to aberrant proteasomal degradation of damaged or aggregated proteins, α-enolase, linked to altered energy production and dihydropyrimidinase-related protein 2, related to reduced growth cone elongation.122;123
The ubiquitin C-terminal hydrolases are suggested to hydrolyze small adducts of ubiquitin and generate free monomeric ubiquitins, thereby allowing recycling of ubiquitin, which is essential to the function of the proteolytic machinery. Thus, putative dysfunction of this oxidized deubiquitinating enzyme may be one way by which oxidative stress promotes ubiquitin-positive inclusion biogenesis, a pathological characteristic of the AD brain, which leads to neurodegeneration.
Using LC-MS/MS, Liao et al., identified 26 brain proteins enriched in amyloid plaques excised with laser capture microdissection from AD brains.99
The list includes cytoskeletal proteins (coronin, tau), membrane trafficking proteins (clathrin, dynamin 1, dynein), proteases (ATPases, cathepsin), signal transduction proteins (14-3-3 proteins), chaperons and others ().