Cytoplasmic Shotgun Proteomic Points to Key Proteins and Pathways in Temozolomide-Resistant Glioblastoma Multiforme.

Temozolomide (TMZ) is the first line of chemotherapy to treat primary brain tumors of the type glioblastoma multiforme (GBM). TMZ resistance (TMZR) is one of the main barriers to successful treatment and is a principal factor in relapse, resulting in a poor median survival of 15 months. The present paper focuses on proteomic analyses of cytosolic fractions from TMZ-resistant (TMZR) LN-18 cells. The experimental workflow includes an easy, cost-effective, and reproducible method to isolate subcellular fraction of cytosolic (CYTO) proteins, mitochondria, and plasma membrane proteins for proteomic studies. For this study, enriched cytoplasmic fractions were analyzed in replicates by nanoflow liquid chromatography tandem high-resolution mass spectrometry (nLC-MS/MS), and proteins identified were quantified using a label-free approach (LFQ). Statistical analysis of control (CTRL) and temozolomide-resistant (TMZR) proteomes revealed proteins that appear to be differentially controlled in the cytoplasm. The functions of these proteins are discussed as well as their roles in other cancers and TMZ resistance in GBM. Key proteins are also described through biological processes related to gene ontology (GO), molecular functions, and cellular components. For protein-protein interactions (PPI), network and pathway involvement analyses have been performed, highlighting the roles of key proteins in the TMZ resistance phenotypes. This study provides a detailed insight into methods of subcellular fractionation for proteomic analysis of TMZ-resistant GBM cells and the potential to apply this approach to future large-scale studies. Several key proteins, protein-protein interactions (PPI), and pathways have been identified, underlying the TMZ resistance phenotype and highlighting the proteins' biological functions.

Biological Applications for LC-MS-Based Proteomics.

Since its inception, liquid chromatography-mass spectrometry (LC-MS) has been continuously improved upon in many aspects, including instrument capabilities, sensitivity, and resolution. Moreover, the costs to purchase and operate mass spectrometers and liquid chromatography systems have decreased, thus increasing affordability and availability in sectors outside of academic and industrial research. Processing power has also grown immensely, cutting the time required to analyze samples, allowing more data to be feasibly processed, and allowing for standardized processing pipelines. As a result, proteomics via LC-MS has become popular in many areas of biological sciences, forging an important seat for itself in targeted and untargeted assays, pure and applied science, the laboratory, and the clinic. In this chapter, many of these applications of LC-MS-based proteomics and an outline of how they can be executed will be covered. Since the field of personalized medicine has matured alongside proteomics, it has also come to rely on various mass spectrometry methods and will be elaborated upon as well. As time goes on and mass spectrometry evolves, there is no doubt that its presence in these areas, and others, will only continue to grow.

Proteome Map of Pea (Pisum sativum L.) Embryos Containing Different Amounts of Residual Chlorophylls.

Due to low culturing costs and high seed protein contents, legumes represent the main global source of food protein. Pea (Pisum sativum L.) is one of the major legume crops, impacting both animal feed and human nutrition. Therefore, the quality of pea seeds needs to be ensured in the context of sustainable crop production and nutritional efficiency. Apparently, changes in seed protein patterns might directly affect both of these aspects. Thus, here, we address the pea seed proteome in detail and provide, to the best of our knowledge, the most comprehensive annotation of the functions and intracellular localization of pea seed proteins. To address possible intercultivar differences, we compared seed proteomes of yellow- and green-seeded pea cultivars in a comprehensive case study. The analysis revealed totally 1938 and 1989 nonredundant proteins, respectively. Only 35 and 44 proteins, respectively, could be additionally identified after protamine sulfate precipitation (PSP), potentially indicating the high efficiency of our experimental workflow. Totally 981 protein groups were assigned to 34 functional classes, which were to a large extent differentially represented in yellow and green seeds. Closer analysis of these differences by processing of the data in KEGG and String databases revealed their possible relation to a higher metabolic status and reduced longevity of green seeds.

A Microdissection Protocol for Proteogenomic Analysis of Histological Sections to Advance Drug Development.

Combining proteogenomics with laser capture microdissection (LCM) in cancer research offers a targeted way to explore the intricate interactions between tumor cells and the different microenvironment components. This is especially important for immuno-oncology (IO) research where improvements in the predictability of IO-based drugs are sorely needed, and depends on a better understanding of the spatial relationships involving the tumor, blood supply, and immune cell interactions, in the context of their associated microenvironments. LCM is used to isolate and obtain distinct histological cell types, which may be routinely performed on complex and heterogeneous solid tumor specimens. Once cells have been captured, nucleic acids and proteins may be extracted for in-depth multimodality molecular profiling assays. Optimizing the minute tissue quantities from LCM captured cells is challenging. Following the isolation of nucleic acids, RNA-seq may be performed for gene expression and DNA sequencing performed for the discovery and analysis of actionable mutations, copy number variation, methylation profiles, etc. However, there remains a need for highly sensitive proteomic methods targeting small-sized samples. A significant part of this protocol is an enhanced liquid chromatography mass spectrometry (LC-MS) analysis of micro-scale and/or nano-scale tissue sections. This is achieved with a silver-stained one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) approach developed for LC-MS analysis of fresh-frozen tissue specimens obtained via LCM. Included is a detailed in-gel digestion method adjusted and specifically designed to maximize the proteome coverage from amount-limited LCM samples to better facilitate in-depth molecular profiling. Described is a proteogenomic approach leveraged from microdissected fresh frozen tissue. The protocols may also be applicable to other types of specimens having limited nucleic acids, protein quantity, and/or sample volume.

Genomic characterization of extensively drug-resistant Acinetobacter baumannii strain, KAB03 belonging to ST451 from Korea.

Extensively drug-resistant (XDR) Acinetobacter baumannii strains have emerged rapidly worldwide. The antibiotic resistance characteristics of XDR A. baumannii strains show regional differences; therefore, it is necessary to analyze both genomic and proteomic characteristics of emerging XDR A. baumannii clinical strains isolated in Korea to elucidate their multidrug resistance. Here, we isolated new sequence type of XDR A. baumannii clinical strain (KAB03) from Korean hospitals and performed comprehensive genome analyses. The strain belongs to new sequence type, ST451. Single nucleotide polymorphism (SNP) analysis with other types of A. baumannii strains revealed that KAB03 has unique SNP pattern in the regions of gyrB and gpi of MLST profiles. A. baumannii KAB03 harbours three antibiotic resistance islands (AbGRI1, 2, and 3). AbGRI1 harbours two copies of Tn2006 containing blaOXA-23, which play an important role in antibiotic resistance. AbGRI2 possesses aminoglycoside resistant gene aph(3')-Ic and class A ß-lactamase blaTEM. AbGIR3 has macrolide resistant genes and aminoglycoside resistant gene armA. A. baumannii KAB03 harbours mutations in pmrB and pmrC, which are believed to confer colistin resistance. In addition, proteomic and transcriptional analysis of KAB03 confirmed that ß-lactamases (ADC-73 and OXA-23), Ade efflux pumps (AdeIJK), outer membrane proteins (OmpA and OmpW), and colistin resistance genes (PmrCAB) were major proteins responsible for antibiotic resistance. Our proteogenomic results provide valuable information for multi-drug resistance in emerging XDR A. baumannii strains belonging to ST451.

Protocol for the Analysis of Laser Capture Microdissected Fresh-Frozen Tissue Homogenates by Silver-Stained 1D SDS-PAGE.

The heterogeneity present in solid tumors adds significant difficulty to scientific analysis and improved understanding. Fundamentally, solid tumor formation consists of cancer cells proper along with stromal elements. The burgeoning malignant process is dependent upon modified stromal elements. Collectively, the stroma forms an essential microenvironment, which is indispensable for the survival and growth of the malignant neoplasm. This cellular heterogeneity makes molecular profiling of solid tumors via mass spectrometry (MS)-based proteomics a daunting task. Laser capture microdissection (LCM) is commonly used to obtain distinct histological cell types (e.g., tumor parenchymal cells, stromal cells) from tumor tissue and attempt to address the tumor heterogeneity interference with downstream liquid chromatography (LC) MS analysis. To provide optimal LC-MS analysis of micro-scale and/or nano-scale tissue sections, we modified and optimized a silver-stained one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) protocol for the LC-MS analysis of LCM-procured fresh-frozen tissue specimens. Presented is a detailed in-gel digestion protocol adjusted specifically to maximize the proteome coverage of amount-limited LCM samples, and facilitate in-depth molecular profiling. Following LCM, targeted tissue sections are further fractionated using silver-stained 1D-SDS-PAGE to resolve and visualize tissue proteins prior to in-gel digestion and subsequent LC-MS analysis.

Allium cepa L. Response to Sodium Selenite (Se(IV)) Studied in Plant Roots by a LC-MS-Based Proteomic Approach.

Liquid chromatography-high-resolution mass spectrometry was used for the first time to investigate the impact of Se(IV) (10 mgSe L-1 as sodium selenite) on Allium cepa L. root proteome. Using MaxQuant platform, more than 600 proteins were found; 42 were identified based on at least 2 razor + unique peptides, score > 25, and were found to be differentially expressed in the exposed versus control roots with t-test difference > ±0.70 (p < 0.05, Perseus). Se(IV) caused growth inhibition and the decrease of total RNA in roots. Different abundances of proteins involved in transcriptional regulation, protein folding/assembly, cell cycle, energy/carbohydrate metabolism, stress response, and antioxidant defense were found in the exposed vs nonexposed roots. New evidence was obtained on the alteration of sulfur metabolism due to S-Se competition in A. cepa L. which, together with the original analytical approach, is the main scientific contribution of this study. Specifically, proteins participating in assimilation and transformation of both elements were affected; formation of volatile Se compounds seemed to be favored. Changes observed in methionine cycle suggested that Se(IV) stress might repress methylation capability in A. cepa L., potentially limiting accumulation of Se in the form of nonprotein methylated species and affecting adversely transmethylation-dependent signaling pathways.

An 8-Month Systems Toxicology Inhalation/Cessation Study in Apoe-/- Mice to Investigate Cardiovascular and Respiratory Exposure Effects of a Candidate Modified Risk Tobacco Product, THS 2.2, Compared With Conventional Cigarettes.

Smoking cigarettes is a major risk factor in the development and progression of cardiovascular disease (CVD) and chronic obstructive pulmonary disease (COPD). Modified risk tobacco products (MRTPs) are being developed to reduce smoking-related health risks. The goal of this study was to investigate hallmarks of COPD and CVD over an 8-month period in apolipoprotein E-deficient mice exposed to conventional cigarette smoke (CS) or to the aerosol of a candidate MRTP, tobacco heating system (THS) 2.2. In addition to chronic exposure, cessation or switching to THS2.2 after 2 months of CS exposure was assessed. Engaging a systems toxicology approach, exposure effects were investigated using physiology and histology combined with transcriptomics, lipidomics, and proteomics. CS induced nasal epithelial hyperplasia and metaplasia, lung inflammation, and emphysematous changes (impaired pulmonary function and alveolar damage). Atherogenic effects of CS exposure included altered lipid profiles and aortic plaque formation. Exposure to THS2.2 aerosol (nicotine concentration matched to CS, 29.9 mg/m(3)) neither induced lung inflammation or emphysema nor did it consistently change the lipid profile or enhance the plaque area. Cessation or switching to THS2.2 reversed the inflammatory responses and halted progression of initial emphysematous changes and the aortic plaque area. Biological processes, including senescence, inflammation, and proliferation, were significantly impacted by CS but not by THS2.2 aerosol. Both, cessation and switching to THS2.2 reduced these perturbations to almost sham exposure levels. In conclusion, in this mouse model cessation or switching to THS2.2 retarded the progression of CS-induced atherosclerotic and emphysematous changes, while THS2.2 aerosol alone had minimal adverse effects.