Approaches for targeted proteomics and its potential applications in neuroscience

Abstract

An extensive guide on practicable and significant quantitative proteomic approaches in neuroscience research is important not only because of the existing overwhelming limitations but also for gaining valuable understanding into brain function and deciphering proteomics from the workbench to the bedside. Early methodologies to understand the functioning of biological systems are now improving with high-throughput technologies, which allow analysis of various samples concurrently, or of thousand of analytes in a particular sample. Quantitative proteomic approaches include both gel-based and non-gel-based methods that can be further divided into different labelling approaches. This review will emphasize the role of existing technologies, their advantages and disadvantages, as well as their applications in neuroscience. This review will also discuss advanced approaches for targeted proteomics using isotope-coded affinity tag (ICAT) coupled with laser capture microdissection (LCM) followed by liquid chromatography tandem mass spectrometric (LC-MS/MS) analysis. This technology can further be extended to single cell proteomics in other areas of biological sciences and can be combined with other 'omics' approaches to reveal the mechanism of a cellular alterations. This approach may lead to further investigation in basic biology, disease analysis and surveillance, as well as drug discovery. Although numerous challenges still exist, we are confident that this approach will increase the understanding of pathological mechanisms involved in neuroendocrinology, neuropsychiatric and neurodegenerative disorders by delivering protein biomarker signatures for brain dysfunction.

Current Status of Two-Dimensional Gel Electrophoresis and Multi-Dimensional Liquid Chromatography as Proteomic Separation Techniques

“…The basic procedure of that strategy includes enzymatic peptides from a complex proteome, followed by separation in the first-dimension with any one of several different chromatography's, and in the second-dimension with reversed-phase chromatography; the separated enzymatic peptides are on-line input into mass spectrometer for MS/MS analysis, followed by protein identification with database search. MDLC-MS/ MS-based proteomics techniques were developed rapidly, mainly including stable isotope-labeled MDLC-MS/MS [25] such as ICAT (Isotope-Coded Affinity Tags) [26][27][28][29], 18 O [30,31], ICPL (Isotope-Coded Protein Labeling) [32,33], IPTL (Isobaric Peptide Termini Labeling) [34,35], iTRAQ (Isobaric Tags For Relative And Absolute Quantification) [36,37], TMT (peptide Tandem Mass Tag) [38,39], and SILAC (Stable Isotope Labeling Of Amino Acids In Cell Culture) [40,41], and non-labeled MDLC-MS/MS such as label-free [42,43], SRM/MRM (Selected or Multiple Reaction Monitoring) [44,45], SWATH (Sequential Window Acquisition Of All Theoretical Spectra) [46,47], and AQUA (Absolute Quantification) [25,48], according to whether the sample is isotope-labeled or not. Those MDLC-MS/MS techniques have extensively used in the field of proteomics because of their high-throughput, high-accuracy, and high-sensitivity in analysis of a proteome, and that they easily overcome the disadvantages of 2DGE and 2D DIGE.…”

Quantitative proteomic analysis of intracerebral hemorrhage in rats with a focus on brain energy metabolism

“…Furthermore, these two studies have some limitations: One of the studies employed a traditional 2D gel combined with mass spectrometry (MS) (Chiu et al, 2012). This approach still suffers from low recognition sensitivity and linearity and a relatively lowthroughput, and it cannot analyze highly basic/hydrophobic proteins (Aggarwal, Choe, & Lee, 2006;Sethi, Chourasia, & Parhar, 2015). Moreover, the major disadvantage of two-dimensional gel electrophoresis (2-DE) is poor reproducibility or gel to gel variability (Sethi et al, 2015).…”

“…This approach still suffers from low recognition sensitivity and linearity and a relatively lowthroughput, and it cannot analyze highly basic/hydrophobic proteins (Aggarwal, Choe, & Lee, 2006;Sethi, Chourasia, & Parhar, 2015). Moreover, the major disadvantage of two-dimensional gel electrophoresis (2-DE) is poor reproducibility or gel to gel variability (Sethi et al, 2015). Another study utilized a label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based relative quantification approach to evaluate rat brains with ICH (Ren et al, 2014).…”

Abordagem para análise proteômica de neurônios contendo neuromelanina na substância negra, isolados por microdissecção a laser

“…A MCL tem sido cada vez mais utilizada em diversas áreas de pesquisa biomédica e suas aplicações nestas áreas são baseadas principalmente em biologia molecular, como a genética e proteômica (Kerk et al, 2003;Chung e Shen, 2015;Sethi et al, 2015). A combinação da MCL e técnicas proteômicas podem fornecem uma visão valiosa sobre possíveis caminhos e mecanismos envolvidos na patogênese de diversas doenças neurodegenerativas (Standaert, 2005;Chung e Shen, 2015;Sethi et al, 2015).…”

“…A combinação da MCL e técnicas proteômicas podem fornecem uma visão valiosa sobre possíveis caminhos e mecanismos envolvidos na patogênese de diversas doenças neurodegenerativas (Standaert, 2005;Chung e Shen, 2015;Sethi et al, 2015). No entanto, apesar das inúmeras vantagens oferecidas pela MCL, este método ainda não foi amplamente incorporado no campo da neurociência.…”