Proteomic Analysis Techniques and Methodology

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Table of Contents

Introduction
Summary of the Overall Objective
The Rationale for Using 2D-PAGE MALDI-TOF-MS/MS
Methodology and Results
Potential Usefulness and Application of Findings
Citation and Advancement of the Study
Conclusion
Reference List

Introduction

Proteomic analysis plays a central role in the identification and characterization of proteins expressed by various genes in biological systems. Techniques employed in the proteomic analysis include molecular approaches, separation methods, protein identification procedures, and protein structure techniques (Murphy, Dowling, & Ohlendieck 2016). Gel-based and mass spectroscopy-based techniques constitute separation methods and identification procedures of proteins.

The exposition of the application of these techniques requires a review of an experimental study, which reported its finding in a peer-reviewed journal. In this view, the coursework selected the peer-reviewed article by Liu et al. (2018) named ‘Comparative proteomic analysis of chicken, duck, and quail egg yolks.’ Therefore, to enhance the understanding of proteomic analysis, this coursework summarises the objective of the study, evaluates the rationale of applying proteomic techniques, recaps proteomic methodology and results obtained, assesses the significance of findings, and provides details regarding citation and advancement of the study.

Summary of the Overall Objective

The objective of the study was to compare the nature of proteins in egg yolks of chicken, duck, and quail, which are three common species of poultry. In comparing proteins in egg yolks, the study utilized two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) coupled with matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) (Liu et al. 2018). Phylogenetic analysis of the mitochondrial genome shows that duck and quail have a close evolutionary relationship with chicken.

According to Liu et al. (2018), the comparison of protein sequences and structure obtained from these three species of poultry shows a high level of homology. Moreover, the study aimed to undertake a comparative analysis of amino acid sequences to establish the expression pattern of proteins in egg yolks in three types of eggs. The establishment of expression patterns and protein-specific regions formed the basis of ascertaining biomarkers, which act as reference residues in the identification of proteins (Liu et al. 2018). Accurate identification of proteins increases food safety because it would single out adulterated eggs and prevent health risks, such as egg allergy and protein hypersensitivity.

The Rationale for Using 2D-PAGE MALDI-TOF-MS/MS

Since the study focused on identifying the nature of proteins in different egg yolks, 2D-PAGE is an appropriate method of separating these proteins according to their isoelectric points and molecular masses. Murphy, Dowling, and Ohlendieck (2016) report that the inventor of 2D-PAGE, Patrick O’Farrell, describes it as a method with a high-resolution of separating proteins based on two physicochemical properties.

Negative and positive charges of amino acid residues aid in the separation of proteins because contribute to the overall charge (Paulo 2016). In the first dimension, 2D-PAGE separates proteins in egg yolks based on their isoelectric points. Ampholytes create a pH gradient that allows proteins to migrate through the gel until they attain their respective isoelectric points, where they contain no charge to allow them to migrate (Marengo & Robotti 2016).

In the second dimension, 2D-PAGE separates proteins in egg yolks based on their molecular masses. PAGE acts as a molecular sieve in the second dimension, which allows small proteins to migrate further than large ones. Therefore, 2D-PAGE permits the identification and characterization of proteins in egg yolks according to their charges and molecular masses.

The 2D-PAGE coupled with MALDI-TOF-MS/MS is also appropriate to allow accurate quantification of proteins separated according to their charges and molecular masses using mass spectrometry. The use of MALDI as the ionization strategy prevents the fragmentation of delicate protein molecules. Calderaro et al. (2014) describe MALDI as a ‘soft ionization technique’ because it does not cause fragmentation of biomolecules, such as proteins and nucleic acids, and it can discriminate biopolymers in their complex mixtures. TOF is a relevant component of spectrometry because it enhances the separation of a wide range of ionized proteins based on their mass-to-charge ratios.

TOF promotes the resolution of proteins by accelerating them at different speeds in a tandem spectrometer (Shah & Gharbia 2017). Tandem mass spectrometry (MS/MS) employs two mass analyzers in assessing precursor ions (MS1) and evaluating productions using mass-to-charge ratios. The advantage of tandem mass spectrometry is that it has a high resolution and a wide mass range (Murphy 2015). Hence, the use of MS/MS allowed quantification of a diverse range of proteins with a high level of resolution.

Methodology and Results

The study sampled 15 fresh eggs, 5 of each chicken, duck, and quail, from the Poultry Research Centre in the Huazhong Agricultural University. Egg yolks were extracted from eggs by following established procedure, which entailed separation of yolks, dilution nine times with cold deionized water with acidic pH of 5, centrifugation at 4 degrees Celsius, dialysis to obtain proteins as pellet, precipitation with absolute ethanol, extraction with cold hexane, and vacuum evaporation to obtain globular proteins (Liu et al. 2018).

The extracted egg yolk proteins were analyzed using 2D-PAGE to separate them according to their charges and molecular masses. Liu et al. (2018) state that they used the Ettan IPGphor 3 System and the Ettan DALT Six System in performing the first and the second dimensions of 2D-PAGE. The gels generated from 2D-PAGE were replicated 3 times and visualized using silver staining. Using SPSS version 13, the ANOVA and Tukey’s tests were used to compare means and post hoc analysis respectively.

Liu et al. (2018) identified protein spots of quail, duck, and chicken, excised them from 2D-PAGE, washed to remove silver stains, and digested using trypsin. The isolated proteins were mixed with matrix solution, applied to MADI-TOF-MS/MS spectrometer, and then analyzed. The identification of proteins entailed a homology search on the non-redundant database of the National Centre for Biotechnology Information using the MASCOT program and Basic Local Alignment Search Tool.

Results of 2D-PAGE demonstrated that the distribution of protein spots exhibited five distinct patterns. Three patterns were analogous in all types of eggs, but two patterns were similar in egg yolks of duck and quail. According to Liu et al. (2018), the five patterns constitute potential biomarkers that could be used in the identification of eggs. Respectively, the analysis of 32, 35, and 32 protein spots of chicken, quail, and duck using MALDI-TOF-MS/MS revealed the existence of 12 dissimilar proteins.

Specific proteins identified are vitellogenin-3 and β-2-glycoprotein-1 in chicken, riboflavin-binding protein, and hemopexin in duck and quail (Liu et al. 2018). Phylogenetic analysis of vitellogenin-related proteins revealed that chicken and quail have a closer evolutionary relationship than a duck. The apparent differences in proteins reflect the evolutionary environments of chicken, quail, and duck.

Potential Usefulness and Application of Findings

Findings of the study are beneficial to proteomic analysis for they demonstrate that 2D-PAGE linked to MALDI-TOF-MS/MS is an effective technique of identifying and characterizing different proteins in egg yolks of poultry. 2D-PAGE successfully separates proteins according to their physicochemical properties, whereas MALDI offers a soft ionization method of proteins for a highly resolved analysis in tandem spectrometry (Paulo 2016; Shah & Gharbia 2017).

The identification and characterization of proteins in egg yolks provides mechanisms of classifying poultry according to their evolutionary relationships. Since the findings identified potential biomarkers, they form the basis of characterizing proteins in egg yolks and recognizing those that cause allergy and hypersensitivity, as well as avert adulteration. Overall, the recognition of the protein spot distribution, types of proteins, and evolutionary relationship aid in the identification of eggs in the food industry for human safety and health.

Citation and Advancement of the Study

The analysis of citations shows that one research article has cited and advanced the work in the study because it was published recently in June 2018. In their study, Geng et al. (2018) established that there are 86 glycoproteins out of 217 sites of glycosylation in chicken egg yolk, which play a significant role in metabolic processes, binding, catalysis, and regulation of expressions. Some of these glycoproteins are protease inhibitors, proteases, complement proteins, and antibodies.

Conclusion

The analysis of the article shows that the study employed 2D-PAGE coupled with MALDI-TOF-MS/MS in the identification and characterization of proteins in egg yolks of duck, chicken, and quail. This proteomic analysis technique is not only useful but also relevant in the characterization of fragile biomolecules, such as proteins and nucleic acids. The establishment of the evolutionary relationship and biomarkers formed the basis of characterizing eggs in the food industry and ensuring safety due to increasing cases of adulteration. The advancement of this work focused on the role of glycosylation in influencing the structure and functions of identified proteins in egg yolks.

Flowchart of the Approach.

Reference List

Calderaro, A, Arcangeletti, M, Rodighiero, I, Buttrini, M, Gorrini, C, Motta, F, Germini, D, Medici, M, Chezzi, C & Conto, F 2014, ‘Matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry applied to virus identification’, Scientific Reports, vol. 4, pp. 1-10.

Geng, F, Xie, Y, Wang, J, Majumder, K, Qiu, N & Ma, M 2018, ‘N-glycoproteomic analysis of chicken egg yolk’, Journal of Agricultural and Food Chemistry, vol. 66, no. 43, pp. 11510-11516.

Liu, Y, Qiu, N, Gao, D & Ma, M 2018, ‘Comparative proteomic analysis of chicken, duck, and quail egg yolks,’ International Journal of Food Properties, vol. 21, no. 1, pp. 1311-1321.

Marengo, E & Robotti, E 2016, 2D page map analysis: methods and protocols, Humana Press, New York.

Murphy, RC 2015, Tandem mass spectrometry of lipids: analysis of complex lipids, Royal Society of Chemists, Cambridge.

Murphy, S, Dowling, P & Ohlendieck, K 2016, ‘Comparative skeletal muscle proteomics using two-dimensional gel electrophoresis’, Proteomes, vol. 4, no. 27, pp. 1-28.

Paulo, JA 2016, ‘Sample preparation for proteomic analysis using a GeLC-MS/MS strategy’, Journal of Biological Methods, vol. 3, no. 3, pp. 1-16.

Shah, HN & Gharbia, SE 2017, MALDI-TOF and tandem MS for clinical microbiology, Wiley, Chichester.

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