Proteomic Analysis of In Vitro Plant Tissues (Coffea canephora): A Case of Study.

Since the term proteomics was coined by Marc Wilkins in 1994, there has been an explosion in the number of articles reporting the use of the proteomics technique. As the layers of biological organization and their regulation increase, the complexity of living beings increases. Thus, we go from the genome to tissues, cells, cellular compartments, and phenotypes and the complexity of the tools used to study this complexity also increases. Unlike the genome study, in the case of the proteome, we have a more complex panorama. We have a spatial and temporal proteome. Proteomics helps to answer complex biological questions since proteins' function depends on their molecular structure, subcellular localization, and posttranslational modifications. In this protocol, we describe a methodology to extract proteins using different methods, separating proteins by electrophoresis in double-dimensional gels and analyzing the gels using specialized software that allows obtaining information on the number and abundance of the proteins from the gels.

Proteomic profile in plasma as a biomarker of pregnancy in cows.

Pregnancy has its own protein dynamics, reflecting the hormonal profile. Quantitative and qualitative changes in plasma protein profile may provide useful information about this condition. Any alterations may be a signal heralding clinical or subclinical pathology. The objective of our study was to compare the plasma protein profile between selected months of pregnancy in cows for a better understanding gestation course. For this purpose, we collected blood from healthy pregnant (n = 30; n = 6 for each pregnancy stage) and non-pregnant (C; n = 6) Holstein-Friesian cows during a routine veterinary examination. Collected samples were selected according to pregnancy month (first, second, third, sixth, and ninth), prepared, and separated by two-dimensional electrophoresis. The Delta-2D program compared and statistically evaluated scanned gel images from the appropriate months. The mean volume of the spots was considered. The MALDI TOF/TOF spectrometer was used to identify statistically significant proteins. There were 11 distinct proteins found, including peptidyl-prolyl cis-trans isomerase F, oligoribonuclease, and PRELI domain-containing protein 3B (all of them have the lowest abundance in the C group), alpha-1B-glycoprotein, L-gulonolactone oxidase, hemopexin (first month with higher abundance than control), alpha-2-HS-glycoprotein (significantly higher abundance in the first month than in remaining groups), ermin (absent in the first month and lower abundance in the third and sixth months than in the remaining groups and control), endophilin-A2 (significant differences between the control and the second, third, sixth, and ninth months), apolipoprotein A-I (significant difference between control and the first and sixth months), alpha-1-antiproteinase (significant difference between control and the ninth month). The study demonstrated the distinctions between plasma protein composition and alterations during the pregnancy course which may potentially serve as diagnostic tools.

Quality evaluation of highly purified human menopausal gonadotropin preparations by means of gel electrophoresis and mass spectrometry.

Human gonadotropins are glycoprotein hormones with a highly complex structure, which demands the application of sophisticated analytical methodologies to assess their quality. The principal objective of this study was a comparative evaluation of gel electrophoretic techniques and mass spectrometry-based methods for the quality study of the two urinary-derived, highly purified, human menopausal gonadotropin preparations, Menopur 75/75 I. U. and Meriofert 75 I. U. Molecular mass (Mr), isoelectric point (pI), and isoform pattern of studied compounds were estimated via SDS-PAGE and 2D gel electrophoresis, whereas matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used for the downstream characterization of peptides obtained after in-gel tryptic digestion of selected protein spots. Additionally, for the estimation of the glycosylation pattern of these biologics, the enzymatic release of oligosaccharides was performed, and the isoform pattern was studied. Gel electrophoresis showed a typical electrophoretic behaviour for protein biotherapeutics medicines consisting of extremely complex spot patterns migrating at different masses and pIs. MS analysis proved to be a powerful tool for the identification and detailed characterization of the gonadotropins and the relevant peptides were identified with high sequence coverages. The results of this study are not only useful for the quality assessment of this class of complex biopharmaceuticals but may also serve as a supporting platform for further development of biopharmaceuticals based on modulation of the glycosylation pattern to enhance efficacy or reduce side effects.

Comparative Proteomic Analysis of Capsule Proteins in Aminoglycoside-Resistant and Sensitive Mycobacterium tuberculosis Clinical Isolates: Unraveling Potential Drug Targets.

BACKGROUND: Tuberculosis (TB), a global infectious threat, has seen a concerning rise in aminoglycoside-resistant Mycobacterium tuberculosis (M.tb) strains. The potential role of capsule proteins remains largely unexplored. This layer acts as the primary barrier for tubercle bacilli, attempting to infiltrate host cells and subsequent disease development. METHODS: The study aims to bridge this gap by investigating the differentially expressed capsule proteins in aminoglycoside-resistant M.tb clinical isolates compared with drug-sensitive isolates employing two-dimensional gel electrophoresis, mass spectrometry, and bioinformatic approaches. RESULTS: We identified eight proteins that exhibited significant upregulation in aminoglycoside-resistant isolates. Protein Rv3029c and Rv2110c were associated with intermediary metabolism and respiration; Rv2462c with cell wall and cell processes; Rv3804c with lipid metabolism; Rv2416c and Rv2623 with virulence and detoxification/adaptation; Rv0020c with regulatory functions; and Rv0639 with information pathways. Notably, the Group-based Prediction System for Prokaryotic Ubiquitin-like Protein (GPS-PUP) algorithm identified potential pupylation sites within all proteins except Rv3804c. Interactome analysis using the STRING 12.0 database revealed potential interactive partners for these proteins, suggesting their involvement in aminoglycoside resistance. Molecular docking studies revealed suitable binding between amikacin and kanamycin drugs with Rv2462c, Rv3804c, and Rv2623 proteins. CONCLUSION: As a result, our findings illustrate the multifaceted nature of aminoglycoside resistance in M.tb and the importance of understanding how capsule proteins play a role in counteracting drug efficacy. Identifying the role of these proteins in drug resistance is crucial for developing more effective treatments and diagnostics for TB.

Variability of haptoglobin beta-chain proteoforms.

Existing knowledge on changes of the haptoglobin (Hp) molecule suggests that it may exist in multiple proteoforms, which obviously exhibit different functions. Using two-dimensional electrophoresis (2DE) in combination with mass spectrometry and immunodetection, we have analyzed blood plasma samples from both healthy donors and patients with primary grade IV glioblastoma (GBM), and obtained a detailed composite 2DE distribution map of ß-chain proteoforms, as well as the full-length form of Hp (zonulin). Although the total level of plasma Hp exceeded normal values in cancer patients (especially patients with GBM), the presence of particuar proteoforms, detected by their position on the 2DE map, was very individual. Variability was found in both zonulin and the Hp ß-chain. The presence of an alkaline form of zonulin in plasma can be considered a conditional, but insufficient, GBM biomarker. In other words, we found that at the level of minor proteoforms of Hp, even in normal conditions, there was a high individual variability. On the one hand, this raises questions about the reasons for such variability, if it is present not only in Hp, but also in other proteins. On the other hand, this may explain the discrepancy between the number of experimentally detected proteoforms and the theoretically possible ones not only in Hp, but also in other proteins.

Two-Dimensional Gel Electrophoresis for Protein Separation of Plant Samples.

This chapter provides a description of the procedure for two-dimensional electrophoresis that can be performed for any given gel size and isoelectric focusing range. This will enable the operator to recognize critical steps and gain sufficient information to generate 2D images suitable for computer-assisted analysis of 2D-gel, as well as mass spectrometry analysis for protein identification and characterization.

Phenol-Based Protein Extraction Method for Plant Proteomic Studies.

The protein extraction method based on the phenol solution and combined with protein precipitation with ammonium acetate in methanol and purification in the same solution, and additionally in acetone and ethanol, is recommended for proteomic studies of plant tissues. The obtained protein samples do not require additional nucleic acid digestion and removal of interfering contaminations. The presented protocol was used to analyze the proteome of common buckwheat flowers and leaves.

Two-Dimensional Gel Electrophoresis in Studies of Flower and Leaf Proteome of Common Buckwheat.

Two-dimensional gel electrophoresis (2-DE) is a proteomic tool used for the separation of protein mixtures according to protein isoelectric point and molecular mass. Although gel-free quantitative and qualitative proteomic study techniques are now available, 2-DE remains a useful analytical tool. The presented protocol was performed to analyze the flower and leaf proteome of common buckwheat using 24 cm immobilized pH gradient strips (pH 4-7) and visualization of proteins on gels via colloidal Coomassie G-250 staining.

Applications of Proteomics in Probiotics Having Anticancer and Chemopreventive Properties.

Proteomics has grown in importance in molecular sciences because it gives vital information on protein identification, expression levels, and alteration. Cancer is one of the world's major causes of death and is the major focus of much research. Cancer risk is determined by hereditary variables as well as the body's immunological condition. Probiotics have increasing medical importance due to their therapeutic influence on the human body in the prevention and treatment of numerous chronic illnesses, including cancer, with no adverse effects. Several anticancer, anti-inflammatory, and chemopreventive probiotics are studied using different proteomic approaches like two-dimensional gel electrophoresis, liquid chromatography-mass spectrometry, and matrix-assisted laser desorption/ionization mass spectrometry. To gain relevant information about probiotic characteristics, data from the proteomic analysis are evaluated and processed using bioinformatics pipelines. Proteomic studies showed the significance of different proteomic approaches in characterization, comparing strains, and determination of oxidative stress of different probiotics. Moreover, proteomic approaches identified different proteins that are involved in glucose metabolism and the formation of cell walls or cell membranes, and the differences in the expression of critical enzymes in the HIF-1 signaling pathway, starch, and sucrose metabolism, and other critical metabolic pathways.

Deciphering a proteomic signature for the early detection of breast cancer from breast milk: the role of quantitative proteomics.

INTRODUCTION: Breast cancer is one of the most prevalent cancers among women in the United States. Current research regarding breast milk has been focused on the composition and its role in infant growth and development. There is little information about the proteins, immune cells, and epithelial cells present in breast milk which can be indicative of the emergence of BC cells and tumors. AREAS COVERED: We summarize all breast milk studies previously done in our group using proteomics. These studies include 1D-PAGE and 2D-PAGE analysis of breast milk samples, which include within woman and across woman comparisons to identify dysregulated proteins in breast milk and the roles of these proteins in both the development of BC and its diagnosis. Our projected outlook for the use of milk for cancer detection is also discussed. EXPERT OPINION: Analyzing the samples by multiple methods allows one to interrogate a set of samples with various biochemical methods that complement each other, thus providing a more comprehensive proteome. Complementing methods like 1D-PAGE, 2D-PAGE, in-solution digestion and proteomics analysis with PTM-omics, peptidomics, degradomics, or interactomics will provide a better understanding of the dysregulated proteins, but also the modifications or interactions between these proteins.

Recognition of Immunoreactive Proteins in Leishmania infantum Amastigote-Like and Promastigote Using Sera of Visceral Leishmaniasis Patients: a Preliminary Study.

PURPOSE: Visceral leishmaniasis (VL) is a systemic and parasitic disease that is usually fatal if left untreated. VL is endemic in different parts of Iran and is caused mainly by Leishmania infantum. This study aimed to recognition immunoreactive proteins in amastigote-like and promastigote stages of L. infantum (Iranian strain) by antibodies present in the sera of VL patients. METHODS: Total protein extract from amastigote-like and promastigote cells was separated by two-dimensional electrophoresis (2DE). To detect the immunoreactive proteins, 2DE immunoblotting method was performed using different pools of VL patients' sera. RESULTS: Approximately 390 and 430 protein spots could be separated in 2DE profiles of L. infantum amastigote-like and promastigote stages, respectively. In immunoblotting method, approximately 295 and 135 immunoreactive proteins of amastigotes-like reacted with high antibody titer serum pool and low antibody titer serum pool, respectively. Approximately 120 and 85 immunoreactive proteins of promastigote extract were recognized using the high antibody titer sera pool and low antibody titer sera, respectively. CONCLUSION: The present study has recognized a number of antigenic diversity proteins based on the molecular weight and pH in amastigote-like and promastigote stages of L. infantum. These results provide us a new concept for further analysis development in the field of diagnosis biomarkers and vaccine targets.

Proteomic-Based Discovery of Predictive Biomarkers for Drug Therapy Response and Personalized Medicine in Chronic Immune Thrombocytopenia.

Purpose: ITP is the most prevalent autoimmune blood disorder. The lack of predictive biomarkers for therapeutic response is a major challenge for physicians caring of chronic ITP patients. This study is aimed at identifying predictive biomarkers for drug therapy responses. Methods: 2D gel electrophoresis (2-DE) was performed to find differentially expressed proteins. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS) analysis was performed to identify protein spots. The Cytoscape software was employed to visualize and analyze the protein-protein interaction (PPI) network. Then, enzyme-linked immunosorbent assays (ELISA) were used to confirm the results of the proteins detected in the blood. The DAVID online software was used to explore the Gene Ontology and pathways involved in the disease. Results: Three proteins, including APOA1, GC, and TF, were identified as hub-bottlenecks and confirmed by ELISA. Enrichment analysis results showed the importance of several biological processes and pathway, such as the PPAR signaling pathway, complement and coagulation cascades, platelet activation, vitamin digestion and absorption, fat digestion and absorption, cell adhesion molecule binding, and receptor binding. Conclusion and Clinical Relevance. Our results indicate that plasma proteins (APOA1, GC, and TF) can be suitable biomarkers for the prognosis of the response to drug therapy in ITP patients.

Two-Dimensional Polyacrylamide Gel Electrophoresis Coupled with Nanoliquid Chromatography-Tandem Mass Spectrometry-Based Identification of Differentially Expressed Proteins and Tumorigenic Pathways in the MCF7 Breast Cancer Cell Line Transfected for Jumping Translocation Breakpoint Protein Overexpression.

The identification of new genes/proteins involved in breast cancer (BC) occurrence is widely used to discover novel biomarkers and understand the molecular mechanisms of BC initiation and progression. The jumping translocation breakpoint (JTB) gene may act both as a tumor suppressor or oncogene in various types of tumors, including BC. Thus, the JTB protein could have the potential to be used as a biomarker in BC, but its neoplastic mechanisms still remain unknown or controversial. We previously analyzed the interacting partners of JTBhigh protein extracted from transfected MCF7 BC cell line using SDS-PAGE complemented with in-solution digestion, respectively. The previous results suggested the JTB contributed to the development of a more aggressive phenotype and behavior for the MCF7 BC cell line through synergistic upregulation of epithelial-mesenchymal transition (EMT), mitotic spindle, and fatty acid metabolism-related pathways. In this work, we aim to complement the previously reported JTB proteomics-based experiments by investigating differentially expressed proteins (DEPs) and tumorigenic pathways associated with JTB overexpression using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Statistically different gel spots were picked for protein digestion, followed by nanoliquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis. We identified six DEPs related to the JTBhigh condition vs. control that emphasize a pro-tumorigenic (PT) role. Twenty-one proteins, which are known to be usually overexpressed in cancer cells, emphasize an anti-tumorigenic (AT) role when low expression occurs. According to our previous results, proteins that have a PT role are mainly involved in the activation of the EMT process. Interestingly, JTB overexpression has been correlated here with a plethora of significant upregulated and downregulated proteins that sustain JTB tumor suppressive functions. Our present and previous results sustain the necessity of the complementary use of different proteomics-based methods (SDS-PAGE, 2D-PAGE, and in-solution digestion) followed by tandem mass spectrometry to avoid their limitations, with each method leading to the delineation of specific clusters of DEPs that may be merged for a better understanding of molecular pathways and neoplastic mechanisms related to the JTB's role in BC initiation and progression.

Proteomics Investigation of the Impact of the Enterococcus faecalis Secretome on MCF-7 Tumor Cells.

Breast cancer is the most prevalent form of cancer among women. The microenvironment of a cancer tumor is surrounded by various cells, including the microbiota. An imbalance between microbes and their host may contribute to the development and spread of breast cancer. Therefore, the objective of this study is to investigate the influence of Enterococcus faecalis on a breast cancer cell line (MCF-7) to mimic the luminal A subtype of breast cancer, using an untargeted proteomics approach to analyze the proteomic profiles of breast cancer cells after their treatment with E. faecalis in order to understand the microbiome and its role in the development of cancer. The breast cancer cell line MCF-7 was cultured and then treated with a 10% bacterial supernatant at two time points (24 h and 48 h) at 37 °C in a humidified incubator with 5% CO2. Proteins were then extracted and separated using two-dimensional difference (2D-DIGE) gel electrophoresis, and the statistically significant proteins (p-value < 0.05, fold change > 1.5) were identified via matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). The protein fingerprints showed a differential protein expression pattern in the cells treated with E. faecalis for 24 and 48 h compared with the control. We found 58 statistically significant proteins changes in the MCF-7 breast cancer cells affected by E. faecalis. Kilin and transgelin were upregulated after 24 h of treatment and could be used as diagnostic and prognostic markers for breast cancer. In addition, another protein involved in the inhibition of cell proliferation was coiled-coil domain-containing protein 154. The protein markers identified in this study may serve as possible biomarkers for breast cancer progression. This promotes their future uses as important therapeutic goals in the treatment and diagnosis of cancer and increases our understanding of the breast microbiome and its role in the development of cancer.

Use of Native-PAGE for the Identification of Epichaperomes in Cell Lines.

Epichaperomes are disease-associated pathologic scaffolds, composed of tightly bound chaperones, co-chaperones, and other factors. They mediate anomalous protein-protein interactions inside cells, which aberrantly affects the function of protein networks, and in turn, cellular phenotypes. Epichaperome study necessitates the implementation of methods that retain these protein complexes in their native cellular states for analysis. Here we describe a protocol for detection and composition analysis of epichaperomes in cell homogenates through native polyacrylamide gel electrophoresis.

"Conducted Properly, Published Incorrectly": The Evolving Status of Gel Electrophoresis Images Along Instrumental Transformations in Times of Reproducibility Crisis.

For the last ten years, within molecular life sciences, the reproducibility crisis discourse has been embodied as a crisis of trust in scientific images. Beyond the contentious perception of "questionable research practices" associated with a digital turn in the production of images, this paper highlights the transformations of gel electrophoresis as a family of experimental techniques. Our aim is to analyze the evolving epistemic status of generated images and its connection with a crisis of trust in images within that field. From the 1980s to the 2000s, we identify two key innovations (precast gels and gel docs) leading to a "two-tiered" gel electrophoresis with different standardization procedures, different epistemic statuses of the produced images and different ways of generating (dis)trust in images. The first tier, exemplified by differential gel electrophoresis (DIGE), is characterized by specialized devices processing images as quantitative data. The second tier, exemplified by polyacrylamide gel electrophoresis (PAGE), is described as a routine technique making use of image as qualitative "virtual witnessing." The difference between these two tiers is particularly apparent in the ways images are processed, even though both tiers involve image digitization. Our account thus highlights different views on reproducibility within the two tiers. Comparability of images is insisted upon in the first tier while traceability is expected in the second tier. It is striking that these differences occur not only within the same scientific field, but even within the same family of experimental techniques. In the second tier, digitization entails distrust, whereas it implies a collective sentiment of trust in the first tier.

Development of a novel two-dimensional gel electrophoresis protocol with agarose native gel electrophoresis.

A new protocol for conducting two-dimensional (2D) electrophoresis was developed by combining the recently developed agarose native gel electrophoresis with either vertical sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) or flat SDS agarose gel electrophoresis. Our innovative technique utilizes His/MES buffer (pH 6.1) during the first-dimensional (1D) agarose native gel electrophoresis, which allows for the simultaneous and clear visualization of basic and acidic proteins in their native states or complex structures. Our agarose gel electrophoresis is a true native electrophoresis, unlike blue native-PAGE, which relies on the intrinsic charged states of the proteins and their complexes without the need for dye binding. In the 2D, the gel strip from the 1D agarose gel electrophoresis is soaked in SDS and placed on top of the vertical SDS-PAGE gels or the edge of the flat SDS-MetaPhor high-resolution agarose gels. This allows for customized operation using a single electrophoresis device at a low cost. This technique has been successfully applied to analyze various proteins, including five model proteins (BSA, factor Xa, ovotransferrin, IgG, and lysozyme), monoclonal antibodies with slightly different isoelectric points, polyclonal antibodies, and antigen-antibody complexes, as well as complex proteins such as IgM pentamer and ß-galactosidase tetramer. Our protocol can be completed within a day, taking approximately 5-6 h, and can be expanded further into Western blot analysis, mass spectrometry analysis, and other analytical methods.

Quantitative Aspects of the Human Cell Proteome.

The number and identity of proteins and proteoforms presented in a single human cell (a cellular proteome) are fundamental biological questions. The answers can be found with sophisticated and sensitive proteomics methods, including advanced mass spectrometry (MS) coupled with separation by gel electrophoresis and chromatography. So far, bioinformatics and experimental approaches have been applied to quantitate the complexity of the human proteome. This review analyzed the quantitative information obtained from several large-scale panoramic experiments in which high-resolution mass spectrometry-based proteomics in combination with liquid chromatography or two-dimensional gel electrophoresis (2DE) were used to evaluate the cellular proteome. It is important that even though all these experiments were performed in different labs using different equipment and calculation algorithms, the main conclusion about the distribution of proteome components (proteins or proteoforms) was basically the same for all human tissues or cells. It follows Zipf's law and has a formula N = A/x, where N is the number of proteoforms, A is a coefficient, and x is the limit of proteoform detection in terms of abundance.

FungiProteomeDB: a database for the molecular weight and isoelectric points of the fungal proteomes.

Proteins' molecular weight (MW) and isoelectric point (pI) are crucial for their subcellular localization and subsequent function. These are also useful in 2D gel electrophoresis, liquid chromatography-mass spectrometry and X-ray protein crystallography. Moreover, visualizations like a virtual 2D proteome map of pI vs. MW are worthwhile to discuss the proteome diversity among different species. Although the genome sequence data of the fungi kingdom improved enormously, the proteomic details have been poorly elaborated. Therefore, we have calculated the MW and pI of the fungi proteins and reported them in, FungiProteomeDB, an online database (DB) https://vision4research.com/fungidb/. We analyzed the proteome of 685 fungal species that contain 7 127 141 protein sequences. The DB provides an easy-to-use and efficient interface for various search options, summary statistics and virtual 2D proteome map visualizations. The MW and pI of a protein can be obtained by searching the name of a protein, a keyword or a list of accession numbers. It also allows querying protein sequences. The DB will be helpful in hypothesis formulation and in various biotechnological applications. Database URL https://vision4research.com/fungidb/.

Proteomics analysis of human breast milk by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) coupled with mass spectrometry to assess breast cancer risk.

Breast cancer (BC) is one of the most common cancers and one of the most common causes for cancer-related mortality. Discovery of protein biomarkers associated with cancer is considered important for early diagnosis and prediction of the cancer risk. Protein biomarkers could be investigated by large-scale protein investigation or proteomics, using mass spectrometry (MS)-based techniques. Our group applies MS-based proteomics to study the protein pattern in human breast milk from women with BC and controls and investigates the alterations and dysregulations of breast milk proteins in comparison pairs of BC versus control. These dysregulated proteins might be considered potential future biomarkers of BC. Identification of potential biomarkers in breast milk may benefit young women without BC, but who could collect the milk for future assessment of BC risk. Previously we identified several dysregulated proteins in different sets of human breast milk samples from BC patients and controls using gel-based protein separation coupled with MS. Here, we performed 2D-PAGE coupled with nano-liquid chromatography-tandem MS (nanoLC-MS/MS) in a small-scale study on a set of six human breast milk pairs (three BC samples vs. three controls) and we identified several dysregulated proteins that have potential roles in cancer progression and might be considered potential BC biomarkers in the future.