• Molecules and Cells
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• v.24 no.2
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• pp.268-275
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• 2007

Protein phosphorylation plays a key role in signal transduction in cells. Since phosphoproteins are present in low abundance, enrichment methods are required for their purification and analysis. Chemical derivatization strategies have been devised for enriching phosphoproteins and phosphopeptides. In this report, we employed a strategy that replaces the phosphate moieties on serine and threonine residues with a biotin-containing tag via a series of chemical reactions. Ribulose 1,5-bisphosphate carboxylase/oxygenase (RUBISCO)-depleted protein extracts prepared from Arabidopsis seedlings were chemically modified for 'biotin-tagging'. The biotinylated (previously phosphorylated) proteins were then selectively isolated by avidin-biotin affinity chromatography, followed by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). This led to the identification of 31 protein spots, representing 18 different proteins, which are implicated in a variety of cellular processes. Despite its current technical limitations, with further improvements in tools and techniques this strategy may be developed into a useful approach.

• Mass Spectrometry Letters
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• v.12 no.4
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• pp.163-171
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• 2021

Emiliania huxleyi is a marine phytoplankton that plays a critical role in global carbon and sulfur cycling. The genome of E. huxleyi has been sequenced, and an in-depth proteomic profile of this organism has been reported. This study analyzed the phosphoproteome of E. huxleyi and identified its changes under calcium-limited conditions. A TiO₂ microcolumn was used for phosphopeptide enrichment, followed by liquid chromatography-tandem mass spectrometry analysis. Overall, we identified 7,010 phosphorylated sites on 3,355 phosphopeptides associated with 2,929 phosphoproteins in E. huxleyi. Quantitative analysis revealed changes in the phosphoproteome in E. huxleyi when ambient conditions changed to calcium-limited conditions, notably the phosphorylation of some transporters was altered. This study provides an overview of protein phosphorylation in E. huxleyi and paves the way for further investigations of its biological functions.

• BMB Reports
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• v.40 no.5
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• pp.765-772
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• 2007

Eosinophils act as effectors in the inflammatory reactions of allergic diseases including atopic dermatitis. Atopic dermatitis patients and others with allergic disorders suffer from eosinophilia, an accumulation of eosinophils due to increased survival or decreased apoptosis of eosinophils. In this study, a differential phosphoproteome analysis of AML14.3D10 eosinophil cell line after treatment with IL-5 or dexamethasone was conducted in an effort to identify the phosphoproteins involved in the proliferation or apoptosis of eosinophils. Proteins were separated by 2-DE and alterations in phosphoproteins were then detected by Pro-Q Diamond staining. The significant quantitative changes were shown in nineteen phosphoproteins including retinoblastoma binding protein 7, MTHSP75, and lymphocyte cytosolic protein 1. In addition, seven phosphoproteins including galactokinase I, and proapolipoprotein, were appeared after treatment with IL-5 or dexamethasone. Especially, the phospho-APOE protein was down-regulated in IL-5 treated AML14.3D10, while the more heavily phosphorylated APOE form was induced after dexamethasone treatment. These phosphoproteome data for the AML14.3D10 cell line may provide clues to understand the mechanism of eosinophilia as well as allergic disorders including atopic dermatitis.

• Mass Spectrometry Letters
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• v.3 no.2
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• pp.39-42
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• 2012

Phosphorylation and glycosylation are two of the most important and widespread post-translational modifications (PTMs) in an organism. Proteomics analysis of the PTMs has been challenged by low stoichiometry of the modified proteins and suppression effects by high abundance proteins, typically no-functional house-keeping proteins. In this study, a novel method was applied for not only isolating PTM peptides from intact peptides but also concurrently characterizing of glyco- and phosphoproteome using electrostatic repulsion hydrophilic interaction chromatography (ERLIC) packed with silica coated by crosslinked polyethyleneimine. For 2 mg tryptic digest of mouse proteome of epicardial adipose tissue with fat diet, 802 N-glycosylated peptides of 316 glycoproteins and 159 phosphorylated peptides of 75 phosphoproteins were identified using HPLC chip/quadrupole time-of-flight (Q-OF) tandem mass spectrometer.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.3
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• pp.1011-1018
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• 2015

This study was conducted to investigate the plasma phosphoproteome and differential plasma phosphoproteins in cases of of Opisthorchis viverrini (OV)-related cholangiocarcinoma (CCA). Plasma phosphoproteomes from CCA patients (10) and non-CCA subjects (5 each for healthy subjects and OV infection) were investigated using gel-based and solution-based LC-MS/MS. Phosphoproteins in plasma samples were enriched and analyzed by LC-MS/MS. STRAP, PANTHER, iPath, and MeV programs were applied for the identification of their functions, signaling and metabolic pathways; and for the discrimination of potential biomarkers in CCA patients and non-CCA subjects, respectively. A total of 90 and 60 plasma phosphoproteins were identified by gel-based and solution-based LC-MS/MS, respectively. Most of the phosphoproteins were cytosol proteins which play roles in several cellular processes, signaling pathways, and metabolic pathways (STRAP, PANTHER, and iPath analysis). The absence of serine/arginine repetitive matrix protein 3 (A6NNA2), tubulin tyrosine ligase-like family, member 6, and biorientation of chromosomes in cell division protein 1-like (Q8NFC6) in plasma phosphoprotein were identified as potential biomarkers for the differentiation of healthy subjects from patients with CCA and OV infection. To differentiate CCA from OV infection, the absence of both serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit beta isoform and coiled-coil domain-containing protein 126 precursor (Q96EE4) were then applied. A combination of 5 phosphoproteins may new alternative choices for CCA diagnosis.

• Mass Spectrometry Letters
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• v.6 no.2
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• pp.31-37
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• 2015

Phosphorylation upon protein is well known to a key regulator that implicates in modulating many cellular processes like growth, migration, and differentiation. Up to date, grafting of multidimensional separation techniques onto advanced mass spectrometry (MS) has emerged as a promising tool for figuring out the biological functions of phosphorylation in a cell. However, advanced MS-based phosphoproteomics is still challenging, due to its intrinsic issues, i.e., low stoichiometry, less susceptibility in positive ion mode, and low abundance in biological sample. To overcome these bottlenecks, diverse techniques (e.g., SCX, HILIC, ERLIC, IMAC, TiO₂, etc.) are continuously developed for on-/off-line enrichment of phosphorylated protein (or peptide) from biological samples, thereby helping qualitative/quantitative determination of phosphorylated protein and its phosphorylated sites. In this review, we introduce to the overall views of enrichment tools that are universally used to selectively isolate targeted phosphorylated protein (or peptide) from ordinary ones before MS-based phospoproteomic analysis.

• Mass Spectrometry Letters
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• v.5 no.3
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• pp.70-78
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• 2014

Microglia are the confined immune cells of the central nervous system (CNS). In response to injury or infection, microglia readily become activated and release proinflammatory mediators that are believed to contribute to microglia-mediated neurodegeneration. In the present study, inflammation was induced in the immortalized murine microglial cell line BV-2 by lipopolysaccharide (LPS) treatment. We firstly performed phosphoproteomics analysis and phosphoinositide lipidomics analysis with LPS activated microglia in order to compare phosphorylation patterns in active and inactive microglia and to detect the pattern of changes in phosphoinositide regulation upon activation of microglia. Mass spectrometry analysis of the phosphoproteome of the LPS treatment group compared to that of the untreated control group revealed a notable increase in the diversity of cellular phosphorylation upon LPS treatment. Additionally, a lipidomics analysis detected significant increases in the amounts of phosphoinositide species in the LPS treatment. This investigation could provide an insight for understanding molecular mechanisms underlying microglia-mediated neurodegenerative diseases.

• Journal of Applied Biological Chemistry
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• v.54 no.2
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• pp.101-107
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• 2011

Aging in females is associated with a reduced metabolic function, increased incidence of neurodegenerative diseases, and cognitive dysfunction, as a result of loss in gonadal function. The change can alter the states of phosphorylation on the proteins, which cause dramatic changes in the cellular location or activity of the proteins. In this study, the differential phosphorylation of the proteins responsible for the functions related to cognition was studied using the ovariectomized adult rats. Phosphoproteomic analysis using the cerebral and hippocampal tissues could identify 51 differentially phosphorylated proteins including 12 proteins for energy metabolism, 8 cytoskeletal proteins, 6 signaling proteins, and other functional proteins in the ovariectomized rats. Further, anti-oxidative enzymes, superoxide dismutase and peroxiredoxin-2, which are known to be inactivated by phosphorylation, were found to be differentially phosphorylated in the cerebellum and hippocampus of the ovariectomized rats, respectively. Many of the deactivated proteins by differential phosphorylation identified in this study were overlapped to those of Alzheimer's disease cases. These results will provide information for neurodegenerative learning and memory impairments in women as brought about by menopause.

• Mass Spectrometry Letters
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• v.3 no.4
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• pp.93-100
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• 2012

Cellular processes such as proliferation, differentiation, and adaptation to environmental changes are regulated by protein phosphorylation. In order to enhance the understanding of molecular dynamics for biological process in detail, it is necessary to develop sensitive and comprehensive analytical methods for the determination of protein phosphorylation. Neural stem cells hold great promise for neural repair following an injury or disease. In this study, we made differentiated oligodendrocytes from human neural stem cells using over-expression of olig2 gene. We confirmed using quantitative phosphoproteome analysis approach that combines stable isotope labeling by amino acids in cell culture (SILAC) and $TiO_2$ micro-column for phosphopeptide enrichment with $MS^2$ and $MS^3$ mass spectrometry. We detected 275 phosphopeptides which were modulated at least 2-fold between human neural stem cells and oligodendrocytes. Among them, 23 phosphoproteins were up-regulated in oligodendrocytes and 79 phosphoproteins were up-regulated in F3 cells.