Optimizing fungal extracellular vesicle proteomic profiling through combined analysis of in-solution and in-gel digestion.

Proteomics offers a powerful tool to identify proteins within diverse microbial organisms, environments, and organelles, including extracellular vesicles (EVs). Fungal EVs are of particular interest due to their roles in cellular development and communication. While several methods exist to isolate EVs from cells, a universally accepted approach for EV protein characterization is lacking. This study investigated in-solution digestion (SD) and in-gel digestion (GD), for characterizing proteins from Neurospora crassa EVs, followed by LC-MS/MS analysis. GD identified three to four-times more proteins than SD while using the same number of unique peptides. Although GD requires a higher amount of starting sample, it offers a more comprehensive protein identification for fungal EVs, potentially preventing the omission of crucial data.

Proteomic Analyses of the Mouse Brain.

Proteomics is a scientific field that aims to identify and characterize all proteins within a biological system, including their posttranslational modifications (PTMs), quantitative changes, and protein-protein interactions. Over the last two decades, proteomic approaches have been widely used in neuroscience research, providing multidimensional insights into the biology and pathology of the brain.Here, we present a basic protocol for profiling protein expression in the mouse brain, which involves total protein extraction, fractionation, digestion, and identification through liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). This method is compatible with many prevalent techniques used for protein quantitation, PTM analysis, and protein-protein interaction mapping.

Proteomics and phosphoproteomics of C3 to CAM transition in the common ice plant.

Among all post-translational modifications of proteins, phosphorylation is one of the most common and most studied. Since plants are sessile organisms, many physiological processes on which their survival depends are regulated by phosphorylation and dephosphorylation. Understanding the extent to which a plant proteome is phosphorylated at specific developmental stages and/or under certain environmental conditions is essential for identifying molecular switches that regulate physiological processes and responses. While most phosphoproteomic workflows proposed in the literature provide tools to exclusively analyze phosphorylated proteins, it is imperative to examine both the proteome and the phosphoproteome to reveal the true complexity of a biological process. Here we describe a mass spectrometry-based phosphoproteomics workflow to analyze both total and phosphorylated proteins. Our method includes phenol-based protein extraction, as well as techniques to measure the quantity and quality of protein extracts. In addition, we compare in detail the efficiency and suitability of in-gel and in-solution trypsin digestion methods. A metal oxide affinity chromatography technique for rapid and efficient enrichment of phosphorylated peptides and an LC-MS/MS method for analysis of the phosphorylated peptides are described. Finally, we present and discuss the results generated by applying this workflow to our study of the C3 to CAM transition in the common ice plant (Mesembryanthemum crystallinum). Overall, our workflow provides robust methods for the identification of phosphoproteins and total proteins. It can be broadly applied to many other organisms and sample types, and the results provide a more accurate picture of the molecular switches that regulate different biological processes.

Affinity Purification Followed by Liquid Chromatography-Tandem Mass Spectrometry to Identify Proteins Interacting with ABA Signaling Components.

Abscisic acid (ABA) is a key phytohormone involved in plant development, seed germination and responses to osmotic stresses, such as drought and high salinity. SNF1-related protein kinases (SnRK2s) play important roles in ABA-dependent and ABA-independent osmotic stress signaling. SnRK2s phosphorylate transcription factors and ion channels in response to ABA or osmotic stress to induce the expression of stress-responsive genes and stomatal closure, respectively, to confer osmotic stress tolerance. The activity of SnRK2s is directly or indirectly regulated by several protein factors. Identification of downstream substrates or upstream regulators of SnRK2s is very useful for elucidating protein components that regulate ABA and osmotic stress signaling. Here, we describe the use of affinity purification by coimmunoprecipitation and liquid chromatography-tandem mass spectrometry to identify protein complexes involved in ABA and osmotic stress signaling in plants. We previously identified several protein factors that regulate ABA and osmotic stress signaling by using this method.

Mass spectrometry analysis of human tear fluid biomarkers specific for ocular and systemic diseases in the context of 3P medicine.

Over the last two decades, a large number of non-communicable/chronic disorders reached an epidemic level on a global scale such as diabetes mellitus type 2, cardio-vascular disease, several types of malignancies, neurological and eye pathologies-all exerted system's enormous socio-economic burden to primary, secondary, and tertiary healthcare. The paradigm change from reactive to predictive, preventive, and personalized medicine (3PM/PPPM) has been declared as an essential transformation of the overall healthcare approach to benefit the patient and society at large. To this end, specific biomarker panels are instrumental for a cost-effective predictive approach of individualized prevention and treatments tailored to the person. The source of biomarkers is crucial for specificity and reliability of diagnostic tests and treatment targets. Furthermore, any diagnostic approach preferentially should be noninvasive to increase availability of the biomaterial, and to decrease risks of potential complications as well as concomitant costs. These requirements are clearly fulfilled by tear fluid, which represents a precious source of biomarker panels. The well-justified principle of a "sick eye in a sick body" makes comprehensive tear fluid biomarker profiling highly relevant not only for diagnostics of eye pathologies but also for prediction, prognosis, and treatment monitoring of systemic diseases. One prominent example is the Sicca syndrome linked to a cascade of severe complications that include dry eye, neurologic, and oncologic diseases. In this review, protein profiles in tear fluid are highlighted and corresponding biomarkers are exemplified for several relevant pathologies, including dry eye disease, diabetic retinopathy, cancers, and neurological disorders. Corresponding analytical approaches such as sample pre-processing, differential proteomics, electrophoretic techniques, high-performance liquid chromatography (HPLC), enzyme-linked immuno-sorbent assay (ELISA), microarrays, and mass spectrometry (MS) methodology are detailed. Consequently, we proposed the overall strategies based on the tear fluid biomarkers application for 3P medicine practice. In the context of 3P medicine, tear fluid analytical pathways are considered to predict disease development, to target preventive measures, and to create treatment algorithms tailored to individual patient profiles.

Analysis of Murein Hydrolases and Proteases Through Zymography.

Zymography has been used to analyze enzymatic activity and processing of enzymes for many years. We have used bacterial cells copolymerized into the acrylamide gel to analyze specific activity of murein hydrolases of interest. In addition, this method has been widely used to examine and distinguish protease activities using different substrates. This chapter provides instruction for zymography of both extracellular murein hydrolases and proteases produced by Staphylococcus aureus.

Two-Dimensional Gel Electrophoresis and Pro-Q Diamond Phosphoprotein Stain-Based Plant Phosphoproteomics.

Pro-Q diamond phosphoprotein gel stain is a fluorescent stain to detect phosphorylated proteins in polyacrylamide gels with high sensitivity. Here, we describe an entire procedure for phosphoproteomics analysis of Arabidopsis seedlings by a combination of Pro-Q diamond stain and two-dimensional gel electrophoresis (2-DE). The workflow involves total protein preparation, protein separation by 2-DE, the second-dimensional gel staining, phosphoproteins detection, and peptides preparation for matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Approximately 300 phosphoproteins can be detected using this method.

How to effectively prepare a sample for bottom-up proteomic analysis of nanoparticle protein corona? A critical review.

Since the interest in the biomedical applications of inorganic nanoparticles (NPs) has rapidly grown over the last decades, there is a need for a thorough characterization of bio-nano interactions. NPs introduced to the body (mostly intravenously) encounter plasma proteins, that instantly create a so-called "protein corona" on the NPs surface, giving the nanomaterial a new biological identity. Type of the proteins that interact with NPs may affect the in vivo fate of NPs. For that reason, it is particularly important to establish analytical methods capable of corona protein identification. Bottom-up proteomics is most often used for that purpose. A crucial part of the experiment is sample preparation, as it is already proven that different protocols may lead to distinct results. This review is aimed at providing a characterization of two main stages of sample preparation: separation of NPs with protein corona from the unbound proteins and the digestion of corona proteins. Separation techniques such as centrifugation, magnetic separation, and chromatography and three digestion methods (in-gel, in-solution, and on-particle) are described with special emphasis paid on their advantages and disadvantages as well as their influence on the result of identification. This paper also indicates the need for standardization of protein corona identification protocols, as some of the proteins may be preferentially detected while applying a particular digestion procedure.

Purification and Characterization of Recombinant Expressed Apple Allergen Mal d 1.

Mal d 1 is the primary apple allergen in northern Europe. To explain the differences in the allergenicity of apple varieties, it is essential to study its properties and interaction with other phytochemicals, which might modulate the allergenic potential. Therefore, an optimized production route followed by an unsophisticated purification step for Mal d 1 and respective mutants is desired to produce sufficient amounts. We describe a procedure for the transformation of the plasmid in competent E. coli cells, protein expression and rapid one-step purification. r-Mal d 1 with and without a polyhistidine-tag are purified by immobilized metal ion affinity chromatography (IMAC) and fast-protein liquid chromatography (FPLC) using a high-resolution anion-exchange column, respectively. Purity is estimated by SDS-PAGE using an image-processing program (Fiji). For both mutants an appropriate yield of r-Mal d 1 with purity higher than 85% is achieved. The allergen is characterized after tryptic in gel digestion by peptide analyses using HPLC-MS/MS. Secondary structure elements are calculated based on CD-spectroscopy and the negligible impact of the polyhistidine-tag on the folding is confirmed. The formation of dimers is proved by mass spectrometry and reduction by DTT prior to SDS-PAGE. Furthermore, the impact of the freeze and thawing process, freeze drying and storage on dimer formation is investigated.

[An in-gel digestion method of chymotrypsin to improve sequence coverage of membrane protein by mass spectrometry].

In recent years, mass spectrometry has been widely used to study membrane protein structure and function. However, the application of mass spectrometry to study integral membrane protein is limited because there are many hydrophobic amino acids in the trans-membrane domain of integral membrane protein to cause low sequence coverage detected by LC-MS/MS. Therefore, we used vitamin K epoxide reductase (VKORC1), a human integral membrane protein, as a model to optimize the digestion conditions of chymotrypsin, and developed an in-gel digestion method of chymotrypsin to improve sequence coverage of membrane protein by mass spectrometry. By exploring the effects of calcium concentration, pH value and buffer system on the percentage of sequence coverage, number of total detected and types of unique peptide, and the size of unique peptide, sequence coverage and peptide diversity could be considered under condition of Tris-HCl buffer with 5-10 mmol/L calcium ion concentration and pH value 8.0-8.5. This method could make the sequence coverage of membrane protein to reach more than 80%. It could be widely used in the study of membrane protein structure and function, identification of interaction site between membrane proteins, and identification of binding site between membrane protein and small molecular drug.

Proteomic profiles of unilateral cryptorchidism in pigs at different ages using MALDI-TOF mass spectrometry and in-gel digestion coupled with mass spectrometry (GeLC-MS/MS) approaches.

BACKGROUND: Cryptorchidism is a condition that occurs when one or both testes fail to descend into the scrotum. It is a common congenital disorder, causing economic loss in pig production. However, there have been only limited studies of differential protein expression profiles in undescended testes (UDTs) in the abdomen and descended testes (DTs) in cryptorchid pigs, especially at the peptidome and proteome levels. The present study aimed to analyze the peptidome of UDTs and DTs in unilateral cryptorchid pigs aged 1-2, 6, 15 and 20 weeks and in normal testes of healthy pigs aged 1-2 and 12 weeks, using peptide mass fingerprinting and three-dimensional principal component analysis (3D-PCA) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and to identify potential protein candidates, using in-gel digestion coupled with mass spectrometry (GeLC-MS/MS). Western blot analysis was used to verify protein expression. Protein sequence was affirmed by liquid chromatography-tandem mass spectrometry. RESULTS: A PCA plot showed a discrete cluster for each sample group. Peptide mass fingerprints (PMFs) demonstrated unique peptide fragments in UDTs at different ages. A number of markedly expressed proteins from GeLC-MS/MS were identified, including the multifunctional tumor necrosis factor receptor superfamily member 18 (TNFRSF18), in DTs at 1-2 and 6 weeks and in UDTs at 15 and 20 weeks of age. Using western blot analysis, high expression of TNFRSF18 was observed in the UDTs at 15 weeks. Using the STITCH database, this protein was found to be related to apoptosis, corresponding to the previous report in the UDTs at the same age. CONCLUSIONS: The present study revealed the specific PMFs and clusters for porcine cryptorchidism, and a novel protein, TNFRSF18, associated with the disease mechanism. These results could provide further insights into the pathogenesis of the disease.

In-gel digestion coupled with mass spectrometry (GeLC-MS/MS)-based salivary proteomic profiling of canine oral tumors.

BACKGROUND: Various types of oral tumors, either benign or malignant, are commonly found in dogs. Since saliva directly contacts the tumors and saliva collection is non-invasive, easily accessible and cost effective, salivary biomarkers are practical to be used for the diagnosis and/or prognosis of these diseases. However, there is limited knowledge of protein expression in saliva for canine oral tumors. The present study aimed to investigate novel biomarkers from the salivary proteome of dogs with early- and late-stage oral melanoma (EOM and LOM, respectively), oral squamous cell carcinoma (OSCC), benign oral tumors (BN), and periodontitis and healthy controls (CP), using an in-gel digestion coupled with mass spectrometry (GeLC-MS/MS). The relationships between protein candidates and chemotherapy drugs were explored and the expression of potential biomarkers in saliva and tissues was verified by western blot analysis. RESULTS: For saliva samples, increased expression of protein tyrosine phosphatase non-receptor type 5 (PTPN5) was shown in all tumor groups compared with the CP group. Marked expression of PTPN5 was also observed in LOM and OSCC compared with that in BN and EOM. In addition, tumor protein p53 (p53), which appeared in the PTPN5-drug interactions, was exhibited to be expressed in all tumor groups compared with that in the CP group. For tissue samples, increased expression of p53 was shown in LOM compared with the control group. CONCLUSION: PTPN5 and p53 were proposed to be potential salivary biomarkers of canine oral tumors.

Proteomic Approaches to Identify Cold-Regulated Plasma Membrane Proteins.

Plasma membrane is the primary determinant of freezing tolerance in plants because of its central role in freeze-thaw cycle. Changes in plasma membrane protein composition have been one of the major research areas in plant cold acclimation. To obtain comprehensive profiles of the plasma membrane proteomes and their changes during the cold acclimation process, a plasma membrane purification method using a dextran-polyethylene glycol two polymer system and a mass spectrometry-based shotgun proteomics method using nano-LC-MS/MS for the plasma membrane proteins are described. The proteomic results obtained are further applied to label-free protein semiquantification.

Shotgun Proteomics of Plant Plasma Membrane and Microdomain Proteins Using Nano-LC-MS/MS.

Shotgun proteomics allows for the comprehensive analysis of proteins extracted from plant cells, subcellular organelles, and membranes. Previously, two-dimensional gel electrophoresis-based proteomics was used for mass spectrometric analysis of plasma membrane proteins. However, this method is not fully applicable for highly hydrophobic proteins with multiple transmembrane domains. In order to solve this problem, we here describe a shotgun proteomics method using nano-LC-MS/MS for proteins in the plasma membrane and plasma membrane microdomain fractions. The results obtained are easily applicable to label-free protein semiquantification.

Recovery of Extra-Radical Fungal Peptides Amenable for Shotgun Protein Profiling in Arbuscular Mycorrhizae.

In arbuscular mycorrhizal symbiosis, the belowground mycelium that develops into the soil, not only provides extensive pathways for nutrient fluxes, the occupation of different niches, and dispersal of propagules, but also has strong influences upon biogeochemical cycling. By providing a valuable overview of expression changes of most proteins, shotgun proteomics can help decipher key metabolic pathways involved in the functioning of fungal mycelia. In this protocol, we describe the combination of extra-radical mycelium growth systems with gel-based extraction of fungal peptides amenable for shotgun protein profiling, which allows gaining information about the extra-radical proteome.

An in-gel digestion method of chymotrypsin to improve sequence coverage of membrane protein by mass spectrometry / 生物工程学报

In recent years, mass spectrometry has been widely used to study membrane protein structure and function. However, the application of mass spectrometry to study integral membrane protein is limited because there are many hydrophobic amino acids in the trans-membrane domain of integral membrane protein to cause low sequence coverage detected by LC-MS/MS. Therefore, we used vitamin K epoxide reductase (VKORC1), a human integral membrane protein, as a model to optimize the digestion conditions of chymotrypsin, and developed an in-gel digestion method of chymotrypsin to improve sequence coverage of membrane protein by mass spectrometry. By exploring the effects of calcium concentration, pH value and buffer system on the percentage of sequence coverage, number of total detected and types of unique peptide, and the size of unique peptide, sequence coverage and peptide diversity could be considered under condition of Tris-HCl buffer with 5-10 mmol/L calcium ion concentration and pH value 8.0-8.5. This method could make the sequence coverage of membrane protein to reach more than 80%. It could be widely used in the study of membrane protein structure and function, identification of interaction site between membrane proteins, and identification of binding site between membrane protein and small molecular drug.

An Effective Protocol for Proteome Analysis of Medaka (Oryzias latipes) after Acute Exposure to Ionizing Radiation.

All terrestrial organisms are subject to evolutionary pressures associated with natural sources of ionizing radiation (IR). The legacy of human-induced IR associated with energy, weapons production, medicine, and research has changed the distribution and magnitude of these evolutionary pressures. To date, no study has systematically examined the effects of environmentally relevant doses of radiation exposure across an organismal proteome. This void in knowledge has been due, in part, to technological deficiencies that have hampered quantifiable environmentally relevant IR doses and sensitive detection of proteomic responses. Here, we describe a protocol that addresses both needs, combining quantifiable IR delivery with a reliable method to yield proteomic comparisons of control and irradiated Medaka fish. Exposures were conducted at the Savannah River Ecology Laboratory (SREL, in Aiken, SC), where fish were subsequently dissected into three tissue sets (carcasses, organs and intestines) and frozen until analysis. Tissue proteins were extracted, resolved by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), and each sample lane was divided into ten equal portions. Following in-gel tryptic digestion, peptides released from each gel portion were identified and quantified by Liquid Chromatography-Mass Spectrometry (LC-MS/MS) to obtain the most complete, comparative study to date of proteomic responses to environmentally relevant doses of IR. This method provides a simple approach for use in ongoing epidemiologic studies of chronic exposure to environmentally relevant levels of IR and should also serve well in physiological, developmental, and toxicological studies.

Detection of Posttranslational Modifications by Fluorescent Staining of Two-Dimensional Gels.

Posttranslational modifications (PTMs) are key to the regulation of functional activities of proteins. Quantitative and qualitative information about PTM stages of proteins is crucial for the discovery of disease biomarkers. Fluorescent dyes specifically staining protein PTMs such as phosphorylation and glycosylation enable the specific detection of protein regulations taking place with respect to these modifications. Activity and molecular interactions of many proteins are determined by their extent of phosphorylation. In our search for biomarkers of neurodegenerative diseases such as multiple sclerosis (MS), using an animal model, experimental autoimmune encephalomyelitis (EAE), we have applied the phosphorylation-specific fluorescent dye, ProQ Diamond, to study changes taking place in the phosphoproteome. Subsequent colloidal Coomassie staining of the same gels detects the changes at the whole proteome level. We have detected many changes taking place in the CNS tissue of the EAE animals at the whole proteome as well as at the phosphoproteome level resulting in valuable insights into the pathophysiological mechanism of EAE and MS.

Proteome Analysis Using Gel-LC-MS/MS.

This article describes processing of protein samples using 1D SDS gels prior to protease digestion for proteomics workflows that subsequently utilize reversed-phase nanocapillary ultra-high-pressure liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS). The resulting LC-MS/MS data are used to identify peptides and thereby infer proteins present in samples ranging from simple mixtures to very complex proteomes. Bottom-up proteome studies usually involve quantitative comparisons across several or many samples. For either situation, 1D SDS gels represent a simple, widely available technique that can be used to either fractionate complex proteomes or rapidly clean up low microgram samples with minimal losses. After gel separation and staining/destaining, appropriate gel slices are excised, and in-gel reduction, alkylation, and protease digestion are performed. Digests are then processed for LC-MS/MS analysis. Protocols are described for either sample fractionation with high-throughput processing of many samples or simple cleanup without fractionation. An optional strategy is to conduct in-solution reduction and alkylation prior to running gels, which is advantageous when a large number of samples will be separated into large numbers of fractions. Optimization of trypsin digestion parameters and comparison to in-solution protease digestion are also described. © 2019 by John Wiley & Sons, Inc.

Gel Absorption-Based Sample Preparation Method for Shotgun Analysis of Membrane Proteome.

Membrane proteins solubilized in a starting buffer containing high concentration of SDS are directly entrapped and immobilized into gel matrix when the membrane protein solution is absorbed by the vacuum-dried polyacrylamide gel. After the detergent and other salts are removed by washing, the proteins are subjected to in-gel digestion, and the tryptic peptides are extracted and analyzed by CapLC-MS/MS. The newly developed method not only avoids protein loss and the adverse protein modifications during gel-embedment but also improves the subsequent in-gel digestion and the recovery of tryptic peptides, particularly hydrophobic peptides. Thus, this method facilitates the identification of membrane proteins, especially integral membrane proteins.