Study on interaction with high-abundant blood proteins and identification of low-abundant proteins to 5-phenyl-1-(p-tolyl)-1 H-1,2,3-triazole by serum proteomics.

In this study, a comprehensive investigation was undertaken to elucidate a simple triazole compound, 5-phenyl-1-(p-tolyl)-1 H-1,2,3-triazole (PPTT), its interactions with high-abundant proteins and identification of low-abundant proteins by serum proteomics. Employing a combination of spectroscopic techniques and computational chemistry, the interactions between PPTT and three high-abundance blood globular proteins, namely human serum albumin (HSA), human immunoglobulin G (HIgG), and hemoglobin (BHb), were explored, thereby ascertaining their binding constants and thermodynamic parameters at the molecular level. Subsequently, based on the differential proteomics, utilizing two-dimensional gel electrophoresis (2-DE) in conjunction with matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS), the research team isolated and identified differentially expressed low-abundance proteins in human blood serum samples following exposure to PPTT. The results showed that there were twenty highly expressed proteins identified from blood serum samples intervened by PPTT. Combining bioinformatics techniques, these proteins were classified, providing preliminary insights like preproprotein or precursors inhibiting the activity of elastase, defending and regulating the immune system, carrying lipid, and other functions into their biological functionalities. One of the differential proteins, apolipoprotein A-1 (ApoA-1) protein, was selected as a possible target to explore the mechanism of action of PPTT intervention on the related signaling pathways involved in human hepatocellular carcinomas(Hep G2) cells. These research findings offer scientifically sound guidance for further in-depth exploration, development, and application of the 1,2,3-triazole compound.

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.

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.

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.

DIGE-Based Biomarker Discovery in Blood Cancers.

Cancer of blood or bone marrow-derived cells dysregulates normal hematopoiesis and accounts for over 6% of all cancer cases annually. Proteomic analyses of blood cancers have improved our understanding of disease mechanisms and identified numerous proteins of clinical relevance. For many years, gel-based proteomic studies have aided in the discovery of novel diagnostic, prognostic, and predictive biomarkers, as well as therapeutic targets, in various diseases, including blood cancer. Fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) facilitates comparative proteomic research to identify differential protein expression in a simple and reproducible manner. The versatility of 2D-DIGE as a quantitative proteomic technique has provided insight into various aspects of blood cancer pathology, including disease development, prognostic subtypes, and drug resistance. The ability to couple 2D-DIGE with additional downstream mass spectrometry-based techniques yields comprehensive workflows capable of identifying proteins of biological and clinical significance. The application of 2D-DIGE in blood cancer research has significantly contributed to the increasingly important initiative of precision medicine. This chapter will focus on the influential role of 2D-DIGE as a tool in blood cancer research.

2D-DIGE Analysis of Liver Disease in Mice.

Hepatocellular carcinoma (HCC) is the major type of primary liver cancer. In this chapter, we describe our routine two-dimensional difference gel electrophoresis (2D-DIGE) workflow for analysis of mouse liver tissue in physiological conditions, as well as of mouse HCC. 2D-DIGE still constitutes a valuable comparative proteomics technique, not only providing information on global protein expression in a sample but also on potential posttranslational protein modifications, occurrence of protein degradation fragments, and the existence of protein isoforms. Thus, 2D-DIGE analysis provides highly complementary data to non-gel-based shotgun mass spectrometry (MS) methods (e.g., liquid chromatography (LC)-MS/MS)-allowing, for example, identification of novel protein biomarkers for HCC or increasing insights into the molecular mechanisms underlying hepatocarcinogenesis.

Two-CyDye-Based 2D-DIGE Analysis of Aged Human Muscle Biopsy Specimens.

The gradual loss of skeletal muscle mass during aging and associated decline in contractile strength can result in reduced fitness, frailty, and loss of independence. In order to better understand the molecular and cellular mechanisms that underlie sarcopenia of old age and the frailty syndrome, as well as identify novel therapeutic targets to treat age-related fiber wasting, it is crucial to develop a comprehensive biomarker signature of muscle aging. Fluorescence two-dimensional gel electrophoresis (2D-DIGE) in combination with sensitive mass spectrometry presents an ideal bioanalytical tool for biomarker discovery in biogerontology. This chapter outlines the application of the 2D-DIGE method for the comparative analysis of human biopsy specimens from middle-aged versus senescent individuals using a two-CyDye-based method.

Protein Digestion for 2D-DIGE Analysis.

In-gel digestion of protein spots derived from two-dimensional gels and their subsequent identification by mass spectrometry is involved in a multitude of mass spectrometry-driven proteomic experiments, including fluorescence two-dimensional difference gel electrophoresis (2D-DIGE). This type of proteomic methodology has been involved in the establishment of comparative proteome maps and in the identification of differentially expressed proteins and their isoforms in health and disease. Most in-gel digestion protocols follow a number of common steps including excision of the protein spots of interest, destaining, reduction and alkylation (for silver-stained gels), and dehydration and overnight digestion with the proteolytic enzyme of choice. While trypsin has been a mainstay of peptide digestion for many years, it does have its shortcomings, particularly related to incomplete peptide digestion, and this has led to a rise in popularity for other proteolytic enzymes either used alone or in combination. This chapter discusses the alternative enzymes available and describes the process of in-gel digestion using the enzyme trypsin.

Construction of 2DE Patterns of Plasma Proteins: Aspect of Potential Tumor Markers.

The use of tumor markers aids in the early detection of cancer recurrence and prognosis. There is a hope that they might also be useful in screening tests for the early detection of cancer. Here, the question of finding ideal tumor markers, which should be sensitive, specific, and reliable, is an acute issue. Human plasma is one of the most popular samples as it is commonly collected in the clinic and provides noninvasive, rapid analysis for any type of disease including cancer. Many efforts have been applied in searching for "ideal" tumor markers, digging very deep into plasma proteomes. The situation in this area can be improved in two ways-by attempting to find an ideal single tumor marker or by generating panels of different markers. In both cases, proteomics certainly plays a major role. There is a line of evidence that the most abundant, so-called "classical plasma proteins", may be used to generate a tumor biomarker profile. To be comprehensive these profiles should have information not only about protein levels but also proteoform distribution for each protein. Initially, the profile of these proteins in norm should be generated. In our work, we collected bibliographic information about the connection of cancers with levels of "classical plasma proteins". Additionally, we presented the proteoform profiles (2DE patterns) of these proteins in norm generated by two-dimensional electrophoresis with mass spectrometry and immunodetection. As a next step, similar profiles representing protein perturbations in plasma produced in the case of different cancers will be generated. Additionally, based on this information, different test systems can be developed.

Reprogrammable Gel Electrophoresis Detection Assay Using CRISPR-Cas12a and Hybridization Chain Reaction.

Hybridization chain reaction (HCR) is a DNA-based target-induced cascade reaction. Due to its unique enzyme-free amplification feature, HCR is often employed for sensing applications. Much like DNA nanostructures that have been designed to respond to a specific stimulus, HCR employs nucleic acids that reconfigure and assemble in the presence of a specific trigger. Despite its standalone capabilities, HCR is highly modular; therefore, it can be advanced and repurposed when coupled with latest discoveries. To this effect, we have developed a gel electrophoresis-based detection approach which combines the signal amplification feature of HCR with the programmability and sensitivity of the CRISPR-Cas12a system. By incorporating CRISPR-Cas12a, we have achieved greater sensitivity and reversed the signal output from TURN OFF to TURN ON. CRISPR-Cas12a also enabled us to rapidly reprogram the assay for the detection of both ssDNA and dsDNA target sequences by replacing a single reaction component in the detection kit. Detection of conserved, both ssDNA and dsDNA, regions of tobacco curly shoot virus (TCSV) and hepatitis B virus (HepBV) genomes is demonstrated with this methodology. This low-cost gel electrophoresis assay can detect as little as 1.5 fmol of the target without any additional target amplification steps and is about 100-fold more sensitive than HCR-alone approach.

Urinary proteomic profiles of prostate cancer with different risk of progression and correlation with histopathological features.

Prostate cancer (PCa) is the most common tumor in men with extremely variable outcome, varying from latent or indolent form to very aggressive behavior. High grade tumors, expansions exceeding the prostatic capsule into the surrounding soft tissues and spreading through lymph vascular channels, represent the most consistent unfavorable prognostic factors. However, accuracy in the prediction of the disease progression is sometimes difficult. Along with new molecular diagnostic techniques and more accurate histopathological approaches, proteomic studies challenge to identify potential biomarkers predictive of PCa progression. In our study we analyzed the urinary proteomes of 42 patients affected by PCa through two-dimensional electrophoresis associated with mass spectrometry. Proteomic profiles were correlated to histopathological features including pTNM stage and tumor differentiation in order to provide new promising markers able to define more accurately the PCa aggressiveness and driving new therapeutic approaches.

The Analysis of Recombination-Dependent Processing of Blocked Replication Forks by Bidimensional Gel Electrophoresis.

The perturbation of the DNA replication process is a threat to genome stability and is an underlying cause of cancer development and numerous human diseases. It has become central to understanding how stressed replication forks are processed to avoid their conversion into fragile and pathological DNA structures. The engineering of replication fork barriers (RFBs) to conditionally induce the arrest of a single replisome at a defined locus has made a tremendous impact in our understanding of replication fork processing. Applying the bidimensional gel electrophoresis (2DGE) technique to those site-specific RFBs allows the visualization of replication intermediates formed in response to replication fork arrest to investigate the mechanisms ensuring replication fork integrity. Here, we describe the 2DGE technique applied to the site-specific RTS1-RFB in Schizosaccharomyces pombe and explain how this approach allows the detection of arrested forks undergoing nascent strands resection.

Optimization of protein extraction for proteomic analyses of fresh and frozen "Musang King" durian pulps.

Four different methods were evaluated to extract proteins from "Musang King" durian pulps and subsequently proteins with different abundance between fresh and long term frozen storage were identified using two-dimensional polyacrylamide gel electrophoresis coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analyses. The acetone-phenol method was found to produce good protein yields and gave the highest gel resolution and reproducibility. Differential protein analyses of the durian pulp revealed that 15 proteins were down-regulated and three other proteins were up-regulated after a year of frozen storage. Isoflavone reductase-like protein, S-adenosyl methionine synthase, and cysteine synthase isoform were up-regulated during frozen storage. The down-regulation of proteins in frozen durian pulps indicated that frozen storage has affected proteins in many ways, especially in their functions related to carbohydrate and energy metabolisms, cellular components, and transport processes. This study will enable future detailed investigations of proteins associated with quality attributes of durians to be studied.

Proteomics and Neurodegenerative Disorders: Advancements in the Diagnostic Analysis.

Changes in protein structure and function, alteration in protein-protein interaction, and significant difference in protein concentration inside the body could play an important role in indicating the pathological evidence of abnormalities before the development of clinical symptoms and act as a critical detection and diagnostic tool commonly known as biomarkers. Biomarkers play important roles in the diagnosis of various chronic diseases, including cancer. Neurodegenerative disorders, including Parkinson's, Alzheimer's, Huntington's, prion, and multiple sclerosis, are well characterized by neuronal deterioration, resulting in precise modifications of neuronal proteins. Nowadays, the diagnosis of neurological disorders is based on proteins or biomarkers. These biomarkers may be found in the cerebrospinal fluid, blood, serum, plasma, saliva, or urine sample. Early diagnosis is urgently needed to prevent further damage. For early diagnosis, identifying the changes in novel protein levels and their functions under the disease conditions is necessary. These can be used as specific proteomic biomarkers for diseases, and they can be possibly identified using neuroproteomics. Neuroproteomics is an emerging tool to corroborate disease-associated protein profiles. It also gives an idea about how these proteins interact with other proteins and undergo post-translational modifications. Neuroproteomics is based on bioinformatics, which provides functional characteristics and advances in technology such as mass spectroscopy, and can help in the discovery of various disease-specific biomarkers. This review gives a complete idea about the types of biomarkers, sources of biomarkers, and techniques involved in the discovery of biomarkers for early diagnosis of neurodegenerative diseases.

Empowering Shotgun Mass Spectrometry with 2DE: A HepG2 Study.

One of the major goals of the Chromosome-Centric Human Proteome Project (C-HPP) is to catalog and annotate a myriad of heterogeneous proteoforms, produced by ca. 20 thousand genes. To achieve a detailed and personalized understanding into proteomes, we suggest using a customized RNA-seq library of potential proteoforms, which includes aberrant variants specific to certain biological samples. Two-dimensional electrophoresis coupled with high-performance liquid chromatography allowed us to downgrade the difficulty of biological mixing following shotgun mass spectrometry. To benchmark the proposed pipeline, we examined heterogeneity of the HepG2 hepatoblastoma cell line proteome. Data are available via ProteomeXchange with identifier PXD018450.

Identification of a Small Compound Targeting PKM2-Regulated Signaling Using 2D Gel Electrophoresis-Based Proteome-wide CETSA.

The cellular thermal shift assay (CETSA) has recently been devised as a label-free method for target validation of small compounds and monitoring the thermal stabilization or destabilization of proteins due to binding with the compound. Herein, we developed a modified method by combining the CETSA and proteomics analysis based on 2D gel electrophoresis, namely 2DE-CETSA, to identify the thermal stability-shifted proteins by binding with a new compound. We applied the 2DE-CETSA for analysis of a target-unknown compound, NPD10084, which exerts anti-proliferative activity against colorectal cancer cells in vitro and in vivo, and identified pyruvate kinase muscle isoform 2 (PKM2) as a candidate target protein. Interestingly, NPD10084 interrupted protein-protein interactions between PKM2 and ß-catenin or STAT3, with subsequent suppression of downstream signaling. We thus demonstrate that our 2DE-CETSA method is applicable for identification of target compounds discovered by phenotypic screening.

Differential Expression of MARK4 Protein and Related Perturbations in Females with Ovulatory PCOS.

BACKGROUND: Ovulatory PCOS (OPCOS) is the mildest form of the polycystic ovarian syndrome among all four determined phenotypes. Though the females with OPCOS are ovulating, hyperandrogenism and polycystic ovarian morphology increase the susceptibility of cardiovascular diseases, insulin resistance, hyperlipidemia and metabolic syndrome in these females. OBJECTIVES: The aim of the study was to identify the significance associated with OPCOS phenotype through serum proteomic profiling of OPCOS females and normal age-matched healthy ovulating females. METHODS: One and two-dimensional gel-based proteomic approaches were adopted to fractionate the complex serum proteome. Differential protein profiles generated were analyzed with PD-QUEST Software. Protein spots differing in intensity by >2-fold were selected and identified further by MALDI-TOF MS. Validation of identified protein was carried out by Biolayer Interferometry. RESULTS: One and two-dimensional gel profiles revealed a differential expression pattern of proteins. 10 selected spots were identified as GMP synthase [glutamine hydrolyzing], zinc finger protein 518A, pericentriolar material 1 protein, BCLAF1 and THRAP3 family member 3, MAP/microtubule affinityregulating kinase 4, H/ACA ribonucleoprotein complex subunit 1, Melanoma-associated antigen B3 and Zinc finger protein 658B. Expression of MAP/microtubule affinity-regulating kinase 4 (MARK4) was found to be downregulated in OPCOS females as compared to controls on validation. CONCLUSION: Reduced expression of MARK4 protein in OPCOS increases the associated risk of hyperlipidemia, hyperandrogenism and metabolic syndrome, thus the protein holds strong candidature as a drug target for the syndrome.

Fluorescence Proteomic Technology to Analyze Peripheral Blood Mononuclear Cells in Chronic Chagas Disease.

The thiol moieties of cysteinyl residues in proteins undergo a number of modifications including nitrosylation, oxidation, persulfidation, sulfenylation, and others. These protein modifications may influence gain as well as loss of function in biological and disease conditions. Herein, we describe a quantitative approach that combines accurate, sensitive fluorescence modification of cysteinyl-S-nitrosyl (SNOFlo) groups that leaves electrophoretic mobility unaffected and offers the measurement of changes in S-nitrosylation (SNO) status relative to protein abundance. This approach has been useful in evaluating the global protein abundance and SNO profile of Chagas seropositive individuals that were categorized in clinically asymptomatic (C/A) and clinically symptomatic (C/S) subgroups and compared to normal healthy (N/H) controls. Through analyzing the proteome datasets with different bioinformatics and statistics tools, potential pathologic mechanisms in disease progression are identified. We also propose a panel of protein biomarkers that have a potential to identify the infected individuals at risk of developing clinical Chagas disease.

Prognostic significance of G6PD expression and localization in lung adenocarcinoma.

Abnormal expressions of extracellular matrix (ECM) proteins are correlated with increased tumor progression, an advanced histologic grade, and metastasis. LCN1 cells derived from a pulmonary large cell neuroendocrine carcinoma were grown to form an Aegagropila-shaped conglomeration on a suspension culture dish (LCN1-sus). In contrast, LCN1 cells cultured in a type I collagen dish were adherent and tended to grow as spindle-shaped individual cells (LCN1-co). In this study, aiming at the discovery of predictive markers for tumor invasion, we performed protein profiling between LCN1-sus and LCN1-co cells using two-dimensional gel electrophoresis. Twenty-six protein spots with >1.2-fold quantitative differences between LCN1-sus and LCN1-co cells were detected. Among the identified proteins, we focused on and immunohistochemically investigated G6PD in lung cancer. G6PD expression was significantly associated with a higher pathological TNM stage (p = 0.0024), lymph node metastasis (p = 0.0187), poorer differentiation (p = 0.0046), pleural invasion (p = 0.0197), vascular invasion (p < 0.0001), lymphatic invasion (p = 0.0200) and poorer prognosis (p = 0.0005) in adenocarcinoma. Especially, G6PD-positive patients with overexpression at the invasive front had significantly poorer survival than those without overexpression (p = 0.0058). Moreover, multivariable analysis revealed that G6PD expression was an independent adverse-prognostic factor. These results suggest that G6PD may be a novel predictive prognostic marker for lung adenocarcinoma.

Increase in constitutively active MEK1 species by introduction of MEK1 mutations identified in cancers.

The kinase MEK1 is an essential component of the mitogen-activated protein kinase cascades. Somatic mutations that have been identified in the MEK1-coding gene generally enhance kinase activity. Consequently, MEK1 has attracted much interest as a target for cancer therapy to block the aberrant activity. By using Phos-tag affinity electrophoresis, we found that the introduction of mutations detected in certain sporadic cancers or in MEK-inhibitor-resistant cancer cells produced constitutively active MEK1 species containing phosphorylated Ser-218 and Ser-222 residues; it also enhanced the constitutive activity of the kinase. Phosphorylation profiling of the mutants in the presence of inhibitors of RAF/MEK demonstrated that several mutations conferred resistance to multiple inhibitors as a result of an increase in the quantity of active MEK1 species containing the two phosphorylated Ser-218 and Ser-222 residues. Phos-tag-based phosphorylation profiling of MEK1 can therefore provide clinical insights into characteristics of individual mutations in the MEK1-coding gene.