Mass spectrometry (MS)-based phosphoproteomics has achieved extraordinary success in qualitative and quantitative analysis of cellular protein phosphorylation. Considering that an estimated level phosphorylation a cell is placed at well above 100 000 sites, there still much room for improvement. Here, we attempt to extend the depth phosphoproteome coverage while maintaining realistic aspirations terms available material, robustness, instrument running time. We developed three strategies,...

The elucidation of protein post-translational modifications, such as phosphorylation, remains a challenging analytical task for proteomic studies. Since many the proteins targeted phosphorylation are low in abundance and is typically substoichiometric, prerequisite their identification specific enrichment phosphopeptide prior to mass spectrometric analysis. Here, we presented new method termed immobilized titanium ion affinity chromatography (Ti4+-IMAC) enriching phosphopeptides. A phosphate...

The mixture of phosphopeptides enriched from proteome samples are very complex. To reduce the complexity it is necessary to fractionate phosphopeptides. However, conventional enrichment methods typically only enrich but not In this study, application strong anion exchange (SAX) chromatography for and fractionation was presented. It found that were highly by SAX majority unmodified peptides did bind onto SAX. Compared with Fe(3+) immobilized metal affinity (Fe(3+)-IMAC), almost double...

We recently introduced a novel scheme combining electron-transfer and higher-energy collision dissociation (termed EThcD), for improved peptide ion fragmentation identification. reasoned that phosphosite localization, one of the major hurdles in high-throughput phosphoproteomics, could also highly benefit from generation such EThcD spectra. Here, we systematically assessed impact on localization utilizing comparison to methods employing either ETD or HCD, respectively, using defined...

Phosphorylation is one of the most important post-translational modifications proteins, which modulates a wide range biological functions and activity proteins. The analysis phosphopeptides still challenging tasks in proteomics research by mass spectrometry. In this study, novel phosphopeptide enrichment approach based on strong interaction zirconium phosphonate (ZrP) modified surface with has been developed. ZrP porous silicon (ZrP-pSi) wafer was prepared to specifically capture from...

Phosphorylation is one of the most important post-translational modifications proteins, which modulates a wide range biological functions and activities proteins. The phosphorylation proteins also associated with pathway cancer cells. We have previously enriched low molecular weight proteome from human plasma based on combination size exclusion adsorption mechanism by using highly ordered mesoporous silica particles. Herein, particles were modified titanium phosphonate to selectively capture...

Inducing post-translational protein knockdown is an important approach to probe biology and validate drug targets. An efficient strategy achieve this involves expression of a interest fused exogenous tag, allowing tag-directed chemical degraders mediate ubiquitylation proteasomal degradation. Here, we combine improved HaloPROTAC degrader probes with CRISPR/Cas9 genome editing technology trigger rapid degradation endogenous target proteins. Our optimized probe, HaloPROTAC-E, chloroalkane...

SGK3 is a PX domain containing protein kinase activated at endosomes downstream of class 1 and 3 PI3K family members by growth factors oncogenic mutations. plays key role in mediating resistance breast cancer cells to or Akt inhibitors, substituting for the loss activity restoring proliferative pathways such as mTORC1 signaling. It therefore critical develop tools potently target obstruct its inhibitor resistance. Here, we describe development SGK3-PROTAC1, PROTAC conjugate 308-R SGK with...

Large-scale characterization of phosphoproteins requires highly specific methods for the purification phosphopeptides because low abundance and substoichiometry phosphorylation. A phosphopeptide enrichment method using ZrO2 nanoparticles is presented. The high specificity this approach was demonstrated by isolation from digests model phosphoproteins. strong affinity to enables a complex peptide mixture in which two orders magnitude lower than that nonphosphopeptides. Superior selectivity...

Protein phosphorylation is instrumental to early signaling events. Studying system-wide in relation processes under investigation requires a quantitative proteomics approach. In Arabidopsis, auxin application can induce pericycle cell divisions and lateral root formation. Initiation of formation transcriptional reprogramming following auxin-mediated degradation repressors. The immediate events prior this derepression are virtually uncharacterized. To identify the signal molecules responding...

Metal and metal oxide chelating-based phosphopeptide enrichment technologies provide powerful tools for the in-depth profiling of phosphoproteomes. One weakness inherent to current strategies is poor binding phosphopeptides containing multiple basic residues. The problem exacerbated when strong cation exchange (SCX) used pre-fractionation, as under low pH SCX conditions phosphorylated peptides with residues elute bulk tryptic digest therefore require more stringent enrichment. Here, we...

K-RAS is known as the most frequently mutated oncogene. However, development of conventional inhibitors has been extremely challenging, with a mutation-specific inhibitor reaching clinical trials only recently. Targeted proteolysis emerged new modality in drug discovery to tackle undruggable targets. Our laboratory developed system for targeted using peptidic high-affinity binders, called "AdPROM." Here, we used CRISPR/Cas9 technology knock GFP tag on native gene A549 adenocarcinoma...

When cells are stressed, bulk translation is often downregulated to reduce energy demands while stress-response proteins simultaneously upregulated. To promote proteasome assembly and activity maintain cell viability upon TORC1 inhibition, 19S regulatory-particle chaperones (RPACs) selectively translated. However, the molecular mechanism for such selective translational upregulation unclear. Here, using yeast, we discover that remodelling of actin cytoskeleton important RPAC following...

CD44, a cell surface adhesion receptor and stem biomarker, is recently implicated in chronic metabolic diseases. Ablation of CD44 ameliorates adipose tissue inflammation insulin resistance obesity. Here, we investigated type-specific expression human mouse further studied how preadipocytes regulates adipocyte function. Using Crispr Cas9-mdediated gene deletion lentivirus-mediated re-expression, discovered that promotes differentiation adipogenesis, whereas re-expression abolishes this effect...

Abstract Immobilized metal affinity chromatography (IMAC) is a commonly used technique for phosphoproteome analysis due to its high adsorption of phosphopeptides. Miniaturization IMAC column essential the small amount sample. Nanoscale was prepared by chemical modification silica monolith with iminodiacetic acid (IDA) followed immobilization Fe 3+ ion inside capillary. It demonstrated that ‐IDA monolithic capillary could specifically capture phosphopeptides from tryptic digest α‐casein...

A molecularly imprinted polymer solid phase extraction (MISPE) method combined with flow-injection chemiluminescence (FI-CL) for the determination of residual tetracycline (TC) in fish samples is presented. The (MIP) TC was synthesized and particles this MIP were packed into a polytetrafluoroethylene (PTFE) tube, which connected sampling loop an eight-way injection valve served as MISPE column on-line selective adsorption TC. eluent (CH3CN : HNO3 (0.01 mol L−1) = 4 ∶ 1, v v) used extracting...

The highly selective capture of phosphopeptides from proteolytic digests is a great challenge for the identification phosphoproteins by mass spectrometry. In this work, zirconium phosphonate-modified magnetic Fe(3)O(4)/SiO(2) core/shell nanoparticles have been synthesized and successfully applied complex tryptic proteins before analysis MALDI-TOF spectrometry with desired convenience sample handling. ratio nanoparticle to protein incubation time capturing were investigated, optimized...

Mutations in the gene encoding CDKL5 kinase are among most common genetic causes of childhood epilepsy and can also give rise to severe neurodevelopmental condition CDD (CDKL5 deficiency disorder). Despite its importance for human health, phosphorylation targets cellular roles poorly understood, especially cell nucleus. Here, we report that is recruited sites DNA damage actively transcribed regions A quantitative phosphoproteomic screen nuclear substrates reveals a network transcriptional...

Alpha-zirconium phosphate nanoplatelets (alpha-ZrPN) were studied as a binding agent for phosphopeptides. Nanoplatelets of alpha-zirconium incubated overnight with zirconium oxychloride, followed by centrifugation, and washed twice water an aqueous solution 80% acetonitrile to form the agent. Alpha-ZrPN able specifically capture phosphoserine-containing peptides from tryptic digest complex peptide mixture in which its abundance was only 0.05%. also bound containing phosphothreonine...

Peptidome analysis has received increasing attention in recent years. Cancer diagnosis by serum peptidome also been reported peptides' profiling for discovery of peptide biomarkers. Tissue, which may have a higher biomarker concentration than blood, not investigated extensively means analysis. Here, method the mouse liver was developed combination size exclusion chromatography (SEC) prefractionation with nano-liquid chromatography−tamdem mass spectrometry (nanoLC−MS/MS) The extracted...