1. INTRODUCTION:

‘Protein’ is derived from the greek term proteios, which means ‘first rank’. Proteins have major role in growth and maintenance of human body with structural, metabolite, transport, immune, signaling and regulatory functions. Each protein consists linear sequence of amino acids attached by peptide bonds. There are nearly 20 amino acids.

They are naturally occurring and each having particular characteristics defined by it’s side chain known as R group which imparts chemical individuality to the molecule. The polypeptide folds into particular conformation depending on the sequence of amino acids and the interaction between side chains, giving 3D structure.¹‘Proteome’ refers to all the proteins that an organism, genome, cell, tissue can express at a certain time. Each species has its own unique proteome. Proteome characterize the functional information of different genes. ‘Proteomics’ is the whole study of proteome Proteomics is the large-scale study of the structure and function of proteins in complex biological sample. The biological function of proteins and their expression levels are dependent on mRNA levels, translation control and regulation on host cell. Proteomics study focuses on Protein identification, Protein quantification, Protein localization, Post transitional modifications, Functional proteomics, Structural proteomics, Protein-protein interactions.²

2. Protein Analysis Techniques:

Figure 1: An overview of proteomics techniques.³

2.1 Conventional Techniques:

2.1.1 Chromatography-based techniques:

2.1.1.1 Ion Exchange Chromatography:

The IEC is a common and versatile tool for the purification of proteins based on charged groups on its surface. The proteins vary from each other in their amino-acid sequence; certain amino acids are anionic and the others are cationic. The net charge contained by a protein at physiological pH is evaluated by equilibrium between these charges. Initially, it separates the protein on the basis of their charge nature (anionic and cationic), further on the basis of comparative charge strength. The IEC technique is highly valuable due to it’s low cost and it’s capacity to persist in buffer conditions.³

2.1.1.2 Size Exclusion Chromatography:

SEC separates the proteins through a porous carrier matrix with distinct pore size on the premise of permeation. therefore, the proteins are separated on the basis of molecular size. The SEC is robust technique capable of handling proteins in numerous physiological circumstances in the presence of detergents, ions and co-factors or at various temperatures. The SEC is used to separate low molecular weight proteins and is an effective tool for the purification of non-covalent multi-meric protein complexes under biological conditions.^4,31

2.1.1.3 Affinity Chromatography:

Affinity chromatography is the most commonly used method for antibody purification. AC has many advantages such as characteristic selection, high stability, low cost and good repeatability. Affinity chromatography is a method of purification using the specific biological properties of biological macromolecules, which can be specifically and reversibly bound to ligands. The ligand is bound to the matrix, and the target protein is separated by the specificity of the interaction between the substance to be purified and the ligand. shixiang Liu et al. used immobilized metal affinity chromatography (IMAC) to purify angiotensin-converting enzyme inhibitory peptides from casein hydrolysates IMAC separates proteins by immobilized metal ions on substrates to form coordination bonds with electron donors such as N, S, and O. Immunoaffinity chromatography uses antigen or antibody as a ligand to adsorb and purify the other by their highly selective reversible binding.⁴

2.1.2 Enzyme-Linked Immunosorbent Assay:

The ELISA is a plate based highly sensitive immunoassay technique designed for detecting and quantifying soluble substances like peptides, proteins, antibodies and hormones. ELISA is mainly used for diagnostic purpose. This assay utilizes the antigen or antibodies on the solid surface and addition of enzyme-conjugated antibodies to it and measure the fluctuations in enzyme activities that are proportional to antibody and antigen concentration in the biological specimen. ⁵

2.1.3 Western Blotting:

Western blotting is an important and powerful technique for detection of low abundance proteins that involve the separation of proteins using electrophoresis technique by transferring onto nitrocellulose membrane and involves the precise detection of a target protein by enzyme-conjugated antibodies.³⁵ In this procedure, a large well is used to separate the sample by PAGE and lanes are created on the membrane containing immobilized protein with the employment of a manifold.Different combinations of primary antibodies are added to each well, with appropriate dilutions of each primary antibody so that expressed proteins are detected in a single condition. this approach is very flexible and can be focused to particular sets of proteins or protein function. the foundation of this approach is the large amount of data on individual antibodies, which are already characterized.Western blotting is a powerful tool for antigen detection from various microorganisms and is kind of helpful in diagnosis of communicable disease.⁶

2.2 Advanced Techniques:

2.2.1 Protein Microarray Technology:

The development of high throughput human protein manufacturing utilizing various techniques will make protein array approach more helpful.Protein-based microarrays have the advantage of enabling the global observation of biochemical activities at wide scale. Thousands of proteins can be screened simultaneously for post-translational modifications, protein-protein, protein-nucleic acid and protein-small molecule interactions.^7,29

Functional protein microarrays:

functional protein arrays are composed of arrays containing full-length functional proteins or protein domains. These protein chips are used to study the biochemical activities of an entire proteome in a single experiment. They are used to study numerous protein interactions, like protein-protein, protein-DNA, protein-RNA, protein-phospholipid and protein-small molecule interactions. Protein chips dissent from previously described methods; whereas screening by 2DE or LC MS/MS can potentially detect any protein, and protein chips can only provide data on set of proteins selected by the investigator.⁸

Antibody Microarray:

broad categories of antibody microarray experimental formats have been developed. Direct labelling, single antibody experiments, dual antibody, sandwich immunoassays are there. Various signal generation and signal enhancement strategies have been utilized in antibody arrays, which includes colorimetry, radioactivity, fluorescence, chemiluminescence, quantum dots and other nanoparticles, enzyme-linked assays, resonance light scattering, tyramide signal amplification, and rolling circle amplification.^9,34

2.2.2 Gel-based approaches:

2.2.2.1 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis:

SDS-PAGE is a high resolving technique for the separation of proteins according to their size, thus facilitates the approximation of molecular weight. Proteins are capable of moving with electric field in a medium having a pH dissimilar from their isoelectric point. Different proteins in mixture migrate with different velocities according to the ratio between its charge and mass. However, addition of sodium dodecyl sulfate denatures the proteins, protein becomes unfolded and coated with SDS detergent molecules, acquiring a high net negative charge that is proportional to the length of the polypeptide chain. When loaded onto a gel matrix and placed in electric field, the negatively charged protein migrate towards the positively charged electrode and are separated by molecular sieving effect. After visualization by staining technique, the size of protein can be estimated by comparison of its migration distance with that of known molecular weight.¹⁰

2.2.2.2 Two-Dimensional Gel Electrophoresis:

The two-dimensional polyacrylamide gel electrophoreses (2D-PAGE) is an efficient and reliable method for separation of proteins based on their mass and charge. 2DE analysis provides several types of information about the hundreds of proteins investigated simultaneously, including molecular weight, quantity, post-translational modifications. 2DE is widely used however principally for qualitative experiments and this method falls short in it’s reproducibility, inability to detect low abundant and hydrophobic proteins, low sensitivity in identifying proteins with pH values too low (pH <3) or too high (pH >10) and molecular masses too small (Mr<10 kD) or too large (Mr>150 kD) [2–5]. Poor separations of basic proteins due to “streaking” of spots and membrane proteins resolution are limiting factors in 2DE.⁵The challenge for protein visualization in 2DE is the compatibility of sensitive protein staining methods with mass spectrometric analysis. Therefore, several fluorescent staining methods have been developed for the visualization of 2DE patterns, along with sypro stainings and Cy-dyes.^11,12

2.2.2.3 Two-Dimensional Differential Gel Electrophoresis:

Proteins may also be fluorescently tagged with Cy2, Cy3, or Cy5 prior to 2DE. CyDyes are the cyanine dyes carrying an N-hydroxysuccinimidyl ester reactive group that covalently binds the e-amino group of lysine residues in proteins. throughout DIGE, proteins in each of up to three samples can be labelled with one of these fluorescent dyes, and the differentially tagged samples can be mixed and loaded along onto a single gel, permitting the quantitative comparative analysis of three samples using a single gel. The DIGE technique has exhibited higher sensitivity and improved reproducibility by directly comparing samples under similar electrophoretic conditions.The resulting images are analyzed by software such as De-Cyder which are specifically designed for 2D-DIGE analysis.however, its labeling chemistry has some limitations; proteins without lysine cannot be tagged, and they require special equipment for visualization, and fluorophores are terribly costly.^13,14

2.3 Quantitative Techniques:

2.3.1 Isotope-Coded Affinity Tag Labeling:

Gel-free or MS based, proteomics techniques are rising as the methods of choice for quantitatively comparing protein levels among biological proteomes. ICAT is one of the most employed chemical isotope labeling methods and the first quantitative proteomic method to be based on MS.Each ICAT reagent consists three essential groups: a thiol-reactive group, an isotope-coded light or heavy linker, and a biotin segment to facilitate peptide enrichment. In an ICAT experiment, protein samples are first labeled with either light or heavy ICAT reagents on cysteine thiols. The mixtures of labeled proteins are then digested by trypsin and separated through a multistep chromatographic separation procedure. ICAT is very helpful to detect peptides with low expression levels, which is one of the bottleneck problems in analytic protein techniques.However, major limitations of this technique include selective detection of proteins with high cysteine content and difficulties within the detection of acidic proteins.^15,16

2.3.2 Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)

While the ICAT reagent solely interacts with the free sulfhydryl of homocysteine, 8% protein is noncysteine, the SILAC has emerged as an crucial proteomic technique. Compared with the ICAT, a distinguished in vitro labeling, SILAC is an example of in vivo coding requires no chemical manipulation, and there is very little chemical difference between the isotopically labeled amino acid and it’s naturally occurring counterpart.In addition, the amount of labeled proteins requires for analysis using SILAC technique is very less than that with ICAT. Therefore, the SILAC-based method is broadly applied in several areas of cell biology and proteomics. A significant drawback of SILAC is that it can’t be applied to tissue protein analysis directly. To beat this limitation, SILAC has been successfully applied to tissue proteome based on 15N isotope labeling.^17,27

2.3.3 Isobaric Tag for Relative and Absolute Quantitation (iTRAQ):

The iTRAQ reagent is standard for relative and absolute quantitation of proteins. This technology uses an NHS ester derivative to modify primary amino groups by linking a mass balance group (carbonyl group) and a reporter group (based on N-methylpiperazine) to proteolytic peptides through the formation of amide bond. Due to the isobaric mass design of the iTRAQ reagents, differentially labelled peptides appear only one peak in MS scans, reducing the probability of peak overlapping. When iTRAQ-tagged peptides are subjected to MS/MS analysis, the mass balancing carbonyl moietyis released as a neutral fragment, liberating the isotope encoded reporter ions which provides relative quantitative information on proteins. A drawback of the claimed iTRAQ technology is because of the enzymatic digestion of proteins prior to labelling, which artificially increases sample complexity and this approach desires a powerful multidimensional fractionation method of peptides before MS identification.¹⁸

2.4 High Throughput Technique:

2.4.1 X-Ray Crystallography:

X-ray crystallography is the most preferred technique for 3D structure determination of proteins. The extremely purified crystallized samples are exposed to X-rays and then subsequent diffraction patterns are processed to produce information about the size of the repeating unit that forms the crystal and crystal packing symmetry. X-ray crystallography has an extensive range of applications to study the virus system, protein-nucleic acid complexes and immune complexes. In addition, 3D protein structure provides detailed information relating the elucidation of enzyme mechanism, drug designing, site-directed mutagenesis and protein–ligand interaction. X-ray can reveal the precise 3D positions of most atoms in a protein molecule as a result of X-rays and covalent bonds have similar wavelength and therefore provides the best visualization of protein structure.¹⁹

2.4.2 Mass Spectroscopy:

A mass spectrometer is often the main tool for protein identification, regardless of the selection of a specific proteomic separation technology, be it gel-based or gel-free. In gel-free approaches like ICAT and MudPIT, samples are directly analyzed by MS. while, in gel-based proteomics (2DE and 2D-DIGE), the protein spots are first removed from the gel and then digested with trypsin. The resultant peptides are separated by LC or directly analyzed by MS. The experimentally derived peptide masses are correlated with the peptide fingerprints of known proteins in the databases using search engines (e.g., Mascot, Sequest). There are two main ionization sources which include matrix assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) as well four major mass analyzers, which are time-of-flight (TOF), ion trap, quadrupole, and Fourier Transform Ion Cyclotron (FTIC) which are currently in use for the protein identification and characterization. A combination of different mass analyzers in tandem such as quadrupole-TOF and quadrupole-ion trap has greatly enhanced their capabilities for proteome analysis. Simple mass spectrometers such as MALDI-TOF are used for only measurement of mass, whereas tandem mass spectrometers are used for amino acid sequence determination.^19,32

There has been a recent trend in proteomics toward the development and application of technologies for the targeted analysis of proteins within the complex mixtures. Selected Reaction Monitoring (SRM) is a powerful tandem mass spectrometry method that can be useful for monitoring target peptides within a complex protein digest. The specificity and sensitivity of the approach, as well as its capability to multiplex the measurement of many analytes in parallel, has made it a technology of particular promise for hypothesis driven proteomics.^20,28

2.4.3 NMR Spectroscopy:

The NMR is a leading tool for the investigation of molecular structure, folding and behavior of proteins. Structure determination through NMR spectroscopy usually involves various phases, each using a discrete set of extremely specific techniques. The samples are prepared and measurements are taken followed by interpretive approaches to verify the structure. The protein structure is fundamental in many research areas, involving structure-based drug design, homology modeling and functional genomics. The NMR can be coupled with various approaches like LC or UHPLC to boost the resolution and sensitivity for high throughput protein profiling. Further, the structural information can be generated, which is compared in relation to the identification of metabolites in complex mixtures.²⁰

Figure 2: Schematic representation of protein analysis³

2.5 Bioinformatics:

Bioinformatics is a vital component of proteomics. The use of Bioinformatics for proteomics has gain significantly affluent during the previous couple of years.³⁴ The development of new algorithm for the analysis of higher amount of info with increased specificity and accuracy aids in the identification and quantitation of proteins. The management of such a high quantity of data is the main issue associated with these kind of analysis. The largest proteome repositories including PRIDE proteomics identification database, Proteome Commons and Peptide Atlas project offer direct access to most of stored data and are valuable tools for data mining. The KEGG, Ingenuity, Pathway Knowledge Base Reactome and Bio Carta aresome of the pathway databases that embody a comprehensive data regarding metabolism, signaling and interactions. In addition to these comprehensive databases, the specific databases for signal transduction pathways like GenMAPP or PANTHER have been developed. Moreover, databases like BioGRID, IntAct, MINT and HRPD contain the information with relation to protein interactions in complexes.^21,22

3. Challenges:

The growth of proteomics represents challenges and solutions both. Some of the challenges are poor stability of proteins making it tough to analyse. Few species which are less abundant, it difficult to carry out detection as amplification of proteins isn’t attainable similar to DNA. Proteins are more difficult to work with than DNA and RNA. They have secondary and tertiary structure that has to often be maintained during their analysis. Proteins can be denatured by the action of enzymes, heat, light, or by aggressive mixing as in beating egg whites. Approximately half of the whole protein content in plasma comes from albumin (∼55mg/ml) and along with another 10 proteins they make up 90% of the total content. Low abundant proteins like cytokines are normally present at 1–5pg/ml. Each proteomics technology can solely analyze proteins within 3–4 orders of magnitude, and mostly at the higher concentration end of the spectrum. The removal of high abundant proteins from plasma or serum is thus a prerequisite for conducting elaborated proteomic studies on low abundant proteins. However, several potentially important biomarkers may be lost in this process due to non-specific binding or the co-removal of proteins/ peptides intrinsically bound to the high abundant carrier proteins. The cost is also a precluding issue. Most proteomics technologies use advanced instrumentation, critical computing power, and expensive consumables.²

Several challenges associated with data storage, integration, robustness has to be addressed while understanding proteomics data relevant to specific disease and cell dynamics. Since there’s increase in accessibility to Proteomics techniques it will lead to increase in volume of data generated and storage capacity is lacking to retain the information generated in the form of output.^22,30 Although cloud storage offers solution. there are issues related with sensitivity and privacy of data. Raw data that is discarded will result in making more space for data storage but question is will it be a reality with proteomics, which is shown in the figure given below. (figure 3)

Figure 3: Challenges in proteomics analysis²

4. Recent Advances:

Advances in proteomics have focused on the identification of post-translational modifications of proteins, protein–protein interactions, and the locations of proteins within cells. Recent proteomic studies seek to determine the structures or folding of proteins on a large scale and in vivo Advances in cryogenic electron microscopy (cryo-EM) have resulted in a tremendous increase in the number of difficult structures that have been determined for proteins. However, so far, these studies are performed mostly in vitro, so it is important to determine how these structures conform to those in cells.⁴

Developments in mass spectrometers over the last decade have been numerous. The drive to increase confidence in the identification of peptides and posttranslational modifications pushed the development of high resolution and high-mass accuracy instruments, most notably Orbitrap and time-of-flight (TOF) mass analyzers. Additionally, Two methods in particular, electron transfer dissociation (ETD) and ultraviolet photo dissociation (UVPD)—have been used to achieve more efficient fragmentation of intact proteins, especially when used in combination. A common strategy to improve the performance of mass spectrometers has been to create hybrid instruments such as Orbitrap Fusion Lumos Tribrid mass spectrometer, which includes five different ion separation/storage device. The ion routing multipole can also be used for higher-energy collisional dissociation (HCD) ion fragmentation for routine analysis of digested protein mixtures which results in a larger number of peptide (and hence protein identifications). Over the last 20 years, there has been increasing interest in using ion mobility spectrometer (IMS) devices to add ion separation capabilities to mass spectrometers. A different type of ion mobility, differential mobility spectrometer or Field Asymmetric IMS (FAIMS) and Structures for Lossless Ion Manipulations (SLIMs) has been used.^24,25

Many of these advances come from improvements in MS technology that delivers new capabilities and measurement improvements. Researchers then are able to leverage these advances into measurements of new features of biological systems and to improve the diagnosis of medical conditions.^24,26

5. CONCLUSION:

In the previous several years, incredibly useful advances are made in the field of proteomics. The technologies offer rapid, sensitive and provide greater proteome coverage. Furthermore, combination of these technologies has been successful in purification, analysis, characterization, quantification, sequence and structural analysis and bioinformatics analysis of large number of proteins in all types of eukaryotic and prokaryotic organisms. All sectors related to biological sciences have been benefited with increasing use of proteomics techniques. Recent advances in proteomics technologies, together with bioinformatics tools facilitate the discovery of pathways and data analysis relevant to proteomics. However, further work is still required to enhance the reproducibility and performance of well-known proteomics tools.

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Received on 01.12.2022 Modified on 07.05.2023

Asian J. Pharm. Res. 2023; 13(4):249-255.

DOI: 10.52711/2231-5691.2023.00046