Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is widely used today for characterization of biological samples with high level of sensitivity and specificity. Different mass spectrometric methods are available for proteomic profiling and identification of biomarkers. One of the platforms is surface-enhanced laser desorption-ionization (SELDI), which has been used to obtain disease specific proteomic patterns.[22
] However, in this approach, only mass spectrometry peak patterns are obtained and the exact identity of the peaks are not determined (i.e. the proteins are not identified in this type of mass spectrometry).[23
] Other platforms such as tandem mass spectrometry permit actual identification of amino acid sequences of peptides and are preferable to SELDI for detecting biomarkers.
There are several labeling approaches for performing mass spectrometry-based quantitative proteomics analysis. These include labeling methods such as stable isotope labeling with amino acids in cell culture (SILAC)[12
] and isobaric tags for relative and absolute quantitation (iTRAQ),[24
] cysteine labeling using isotope-coded affinity tags,[14
] labeling with isotopically labeled acrylamide and C-terminal labeling using 18
In a SILAC experiment, cells representing different biological conditions are grown in media supplemented with either “light” or “heavy” isotope form of amino acids. In this method, labeled amino acids are metabolically incorporated into all peptides and subsequent pooling of differentially labeled samples in equal ratios provides quantification of peptides from each sample. This quantification of proteins is based on the relative intensities of corresponding differentially labeled peptides. SILAC has been used to study signaling in several systems including the phosphorylation dynamics of ion channels and to evaluate the brain derived neurotrophic factor (BDNF) induced change in neuronal phosphotyrosine proteome.[26
] However, the disadvantages of this method is that, it cannot be used for tissues or body fluids and might require further validation as the experiments are carried out in cell lines. Park et al.
used this method to quantitate the phosphorylation of Kv2.1 protein, which was transfected to HEK293 cell lines and activated by ionomycin to study calcineurin dependent dephosphorylation.[26
] It has also been used to identify BDNF-induced proteins, which includes tyrosine kinase receptor B, hepatocyte growth factor-regulated tyrosine kinase substrate
and signal transducing adaptor molecule. All these proteins are known to control the molecular trafficking of receptor tyrosine kinases.[27
2D-PAGE is widely used for protein separation for comparative proteomic profiling . DIGE is a vital part of the 2D-PAGE for protein quantitation,[28
] in spite of its limitations (the biggest limitation is that only the most abundant proteins can be analyzed).[29
] In DIGE, different samples are labeled with different fluorescent dyes prior to separation, facilitating analysis of diversity in protein abundance between samples. DIGE separates protein samples into spots, which could be subsequently identified by tandem mass spectrometry. Software is required to extract quantitative information; additional normalization[30
] and stringent statistical tests are often required for analyzing the data.[31
] The data obtained from the 2D gel analysis should be validated using immunohistochemical labeling or enzyme assays, as the protein spots in 2-D gel can co-migrate leading to confusion in the identity of proteins. Some of the neurological disease biomarkers revealed by DIGE approach are listed in . Gao et al.
performed DIGE to analyze the proteomic profile of cerebrospinal fluid in traumatic brain injury in infants, which is one of the common causes of mortality in infancy. The study identified candidate biomarkers such as prostaglandin D2 synthase and cystatin C,
further confirmed by Western blot.[19
] Maternal serum profiling was performed to identify candidate biomarkers for Down syndrome using 2D-DIGE coupled to MALDI-TOF MS analysis.[18
] This study proposed differential expression of molecules such as afamin, alpha-1-microglobulin, apolipoprotein E
in the maternal serum.
Figure 1 2D-DIGE based separation of proteins and identification by mass spectrometry. Proteins from two samples are labeled using Cy3 or Cy5 prior to pooling. First dimension separation of proteins is carried out using immobilized pI gradient strip and second (more ...)
Published studies using quantitative proteomic methods for identification of biomarkers in neurological disorders
iTRAQ reagents are a set of isobaric tags that bind to primary amines by covalent bond leading to the labeling of peptides. Measuring the relative intensity of these reporter ions in MS/MS spectra allows the relative quantitation of the proteins in samples. Choe et al.
used 8-plex iTRAQ quantitation of proteins in cerebrospinal fluid of the patients with Alzheimer's disease. The study observed a decrease in some proteins, which include albumin, clusterin
] The proteins with the increased expression include apolipoprotein E
and cystatin C
. Since eight types of the reporter molecules are now available, this method allows multiplexing of up to eight sets of samples in a single mass spectrometry experiment. provides the list of some of proteomic studies in neurological diseases. Hergenroeder et al.
investigated for candidate serum biomarkers for brain injury using iTRAQ method coupled with LC-MS/MS. The study identified serum amyloid A, C-reactive protein
and retinol binding protein 4
and these candidate biomarkers were verified by enzyme-linked immunosorbent assay (ELISA) in an independent set of serum samples from cases of traumatic brain injury and healthy volunteers.[21