Non-phosphorylated Peptides Research Articles

Development of a high-throughput kinase activity platform using nanoLC-MS/MS with DIA approach for studying the anti-cancer mechanism of Taxol in ovarian cancer

BackgroundProtein phosphorylation by protein kinases plays a pivotal role in increasing protein diversity, thereby influencing various cellular functions. However, due to the relatively low abundance of phosphopeptides in a mixture of peptides and the ion-suppression effect of non-phosphorylated peptides, the detection of phosphopeptides is not straightforward. ResultsHerein, a quantitative high-throughput platform was developed for assessing multikinase activity using nano-LC-MS/MS with a data-independent acquisition (DIA) approach. This platform was evaluated by studying the kinase activity in Taxol-treated SKOV3 cells. A library containing 38 peptide substrates was designed and analyzed to determine the activities of major kinases involved in cancer development. Twenty-three synthetic peptide substrates showed significant phosphorylation changes in triplicate biological experiments, as further verified by western blotting. Our findings reveal that Taxol suppressed SKOV3 cell survival by activating AMPK and suppressing the PI3K-Akt-dependent pathway, ultimately leading to mTOR inhibition. Furthermore, in combination with ERK, Akt, SGK, CK1, and ErbB2 inhibitors, Taxol enhanced the inhibitory effect on ovarian cancer. SignificanceThis platform can be an attractive approach for large-scale kinase activity studies to comprehensively uncover the mechanisms of drug-disease treatment and to investigate a more effective therapy strategy.

Pathogenic mutations of human phosphorylation sites affect protein–protein interactions

Despite their lack of a defined 3D structure, intrinsically disordered regions (IDRs) of proteins play important biological roles. Many IDRs contain short linear motifs (SLiMs) that mediate protein-protein interactions (PPIs), which can be regulated by post-translational modifications like phosphorylation. 20% of pathogenic missense mutations are found in IDRs, and understanding how such mutations affect PPIs is essential for unraveling disease mechanisms. Here, we employ peptide-based interaction proteomics to investigate 36 disease-associated mutations affecting phosphorylation sites. Our results unveil significant differences in interactomes between phosphorylated and non-phosphorylated peptides, often due to disrupted phosphorylation-dependent SLiMs. We focused on a mutation of a serine phosphorylation site in the transcription factor GATAD1, which causes dilated cardiomyopathy. We find that this phosphorylation site mediates interaction with 14-3-3 family proteins. Follow-up experiments reveal the structural basis of this interaction and suggest that 14-3-3 binding affects GATAD1 nucleocytoplasmic transport by masking a nuclear localisation signal. Our results demonstrate that pathogenic mutations of human phosphorylation sites can significantly impact protein-protein interactions, offering insights into potential molecular mechanisms underlying pathogenesis.

Modulation of biomineralization morphology by phosphorylated collagen peptides: insights into osteogenesis imperfecta pathophysiology.

Osteogenesis imperfecta (OI) is a hereditary skeletal disorder characterized by bone fragility and deformities, primarily attributed to defects in type I collagen, the most abundant structural protein in humans. Multiple phosphorylation sites have been detected within collagen, suggesting that phosphorylation may influence mineralization processes, thereby impacting the development of OI. In this study, we investigated the modulation of biomineralization morphology by phosphorylated collagen peptides mimicking Gly-Ser mutations in osteogenesis imperfecta. A series of collagen peptide sequences, including GPO13S, GPO13pS, GPO12S, GPO12pS, GPO11S, and GPO11pS, were synthesized to explore the role of phosphorylation in peptide stability and its templating effect on biomineralization. The CD results indicated that the phosphorylation of Gly-pSer mutants reduces the stability of collagen peptides. SEM images revealed that phosphorylated peptides acted as templates, guiding the morphology of calcium carbonate into either olive-like or spherical structures, depending on their conformational state of the peptides. Non-phosphorylated peptides maintained a calcite crystal structure. The XRD patterns predominantly exhibited peaks associated with calcite and vaterite for GPO13pS-CaCO3, GPO12pS-CaCO3, and GPO11pS-CaCO3, and peaks associated with calcite for GPO13S-CaCO3, GPO12S-CaCO3, and GPO11S-CaCO3, indicating a transformation of mesocrystals influenced by peptide phosphorylation. Our findings elucidate the crucial role of phosphorylated collagen peptides in mediating biomineralization morphology and polymorph selection, offering insights into the complex pathophysiology of OI.

Packed in-tube solid-phase microextraction with mesoporous zirconium titanium oxides for highly efficient phosphopeptide enrichment and analysis

Phosphorylated peptides are crucial for understanding biological regulation processes. However, the detection of phosphorylated peptides remains challenging in mass spectrometry (MS) due to their high hydration, low abundance, and the significant signal suppression caused by non-phosphorylated peptides. It is essential to enhance the efficiency of phosphopeptide enrichment from complex biological samples to achieve comprehensive analysis and identification of phosphopeptides. Herein, a packed in-tube solid-phase microextraction (IT-SPME) method was developed based on mesoporous zirconium titanium oxides for the selective enrichment and analysis of phosphopeptides. The IT-SPME system achieved the efficient capture of phosphopeptides from complex biological samples followed by high-sensitivity detection (5 fmol). This exceptional performance can be attributed to the high surface area (289.77 m2/g) of mesoporous zirconium titanium oxides, which provided abundant active sites for a high loading capacity. The zirconium titanium oxides with a mesoporous structure (∼4.5 nm) was prepared through a sol–gel and solvothermal process. Importantly, this method proved effective in enriching phosphopeptides from practical samples such as non-fat milk and egg white. It is the first attempt to employ a mesoporous packed IT-SPME approach for phosphopeptide enrichment. These results demonstrate the significant application potential of mesoporous zirconium titanium oxides in phosphoproteomics.

RB798, RB799, RB800, RB801, RB802, RB803, RB804, RB805, RB806 and RB807 antibodies recognize a human Aurora kinase B (AURKB) phosphorylated peptide by ELISA

The recombinant antibodies RB798, RB799, RB800, RB801, RB802, RB803, RB804, RB805, RB806, and RB807 were tested by ELISA for their binding to a synthetic phosphorylated peptide from the human Aurora kinase B (AurKB) protein. Antibodies RB805 and RB806 detected both the phosphorylated and non-phosphorylated peptides, whereas the remaining antibodies selectively recognized the phosphorylated peptide.

Mass spectrometric simultaneous quantification of tau species in plasma shows differential associations with amyloid and tau pathologies

Blood phosphorylated tau (p-tau) biomarkers, at differing sites, demonstrate high accuracy to detect Alzheimerʼs disease (AD). However, knowledge on the optimal marker for disease identification across the AD continuum and the link to pathology is limited. This is partly due to heterogeneity in analytical methods. In this study, we employed an immunoprecipitation mass spectrometry method to simultaneously quantify six phosphorylated (p-tau181, p-tau199, p-tau202, p-tau205, p-tau217 and p-tau231) and two non-phosphorylated plasma tau peptides in a total of 214 participants from the Paris Lariboisière and Translational Biomarkers of Aging and Dementia cohorts. Our results indicate that p-tau217, p-tau231 and p-tau205 are the plasma tau forms that best reflect AD-related brain changes, although with distinct emergences along the disease course and correlations with AD features—amyloid and tau. These findings support the differential association of blood p-tau variants with AD pathology, and our method offers a potential tool for disease staging in clinical trials.

Abstract P2-24-03: Z-Endoxifen Allosterically Inhibits PKCβI and its Paradoxical Membrane Translocation

Abstract Z-Endoxifen Allosterically Inhibits PKCβI and its Paradoxical Membrane Translocation Sayantani Sarkar Bhattacharya1, Taylor L. Witter1, Anh Q. T. Cong1, Elizabeth Bruinsma2, Swaathi Jayaraman2, Matthew P. Goetz2, John R. Hawse1, and Matthew J. Schellenberg1 1Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN USA 55905 2Department of Oncology, Mayo Clinic, Rochester, MN USA 55905 Background: Z-Endoxifen (ENDX), the active metabolite of tamoxifen (TAM) and a selective estrogen receptor modulator (SERM), exhibited high antitumor activity in endocrine-resistant hormone receptor-positive breast and other gynecologic cancer. ENDX has also been shown to be a protein kinase C (PKC) inhibitor. PKCs participate in diverse cellular functions and their activity is often elevated in breast tumors. Guided by mechanistic insights from our recently determined crystal structure of PKCβI, we sought to determine the effects of ENDX on PKCβI in a breast cancer cell line model. Methods: To determine how ENDX regulates PKC activity, we used a Z’LYTE kinase activity assay. This Fluorescence Resonance Energy Transfer (FRET) based biochemical method can detect differential sensitivity of phosphorylated and non-phosphorylated peptides to proteolytic cleavage. We probed changes in activity for conventional and novel PKCs, as well as the purified catalytic domain of conventional PKCs in vitro. Alongside, as kinase activity of PKC relies on its spatial assembly, therefore we studied its intracellular localization using live cell confocal imaging. MCF7 cells expressing YFP-tagged PKCβI were grown in a glass bottom chamber and treated with ENDX and other modulators for relevant time and doses for this study. Images were taken using Zeiss LSM 780 confocal laser scanning microscope and analyzed in Zeiss-ZEN microscope software and GraphPad Prism 9. Results: Our data from an in vitro kinase assay indicates that ENDX inhibits the kinase activity of conventional (PKCβI) and novel (PKC𝛿) PKC isoforms with a similar IC50, however PKCβI catalytic domain is less sensitive to ENDX. We also identified a multi-domain mechanism of PKC inhibition through an allosteric inhibitory mechanism. Our live cell imaging study demonstrated that ENDX promotes the recruitment of PKCβI to the cell membrane in both a dose and time-dependent manner. Moreover, this translocation can also be mitigated by co-treatment with inhibitors of the PKC-regulator PHLPP phosphatases. Conclusion: Taken together, these results suggest the allosteric effects of ENDX trigger PKCβI recruitment to the cell membrane, yet since ENDX also inhibits kinase activity it suggests that ENDX triggers a non-productive interaction with the enzyme and "breaks" the well-known mechanism of PKC activation upon binding the cell membrane. Furthermore, ENDX is likely to trigger dephosphorylation and ultimately degradation, suggesting that ENDX represents a new mechanistic basis for targeting and downregulating PKC in cancer cells. Hence, the current study provides an integrated pattern of highly specific treatments regimen that can exploit PKCβI as a repurposing clinical target in breast cancer. Citation Format: Sayantani Sarkar Bhattacharya, Taylor L. Witter, Anh T. Cong, Elizabeth S. Bruinsma, Swaathi Jayaraman, Matthew P. Goetz, John Hawse, Matthew Schellenberg. Z-Endoxifen Allosterically Inhibits PKCβI and its Paradoxical Membrane Translocation [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-24-03.

Characterization of Phosphorylation-Dependent Antibody Binding to Cancer-Mutated Linkers of C2H2 Zinc Finger Proteins by Intact Transition Epitope Mapping-Thermodynamic Weak-Force Order Analysis.

With Intact Transition Epitope Mapping-Thermodynamic Weak-force Order (ITEM-TWO) analysis in combination with molecular modeling, the phosphorylation-dependent molecular recognition motif of the anti-HpTGEKP antibody has been investigated with binary and ternary component mixtures consisting of antibody and (phospho-) peptides. Amino acid sequences have been selected to match either the antibody's recognition motif or the cancer-related zinc finger protein mutations and phosphorylations of the respective amino acid residues. Upon electrospraying of all the components of the mixtures, that is, hexapeptides, antibody, and intact immune complexes, the produced ions were subjected to mass spectrometric mass filtering. The antibody ions as well as the immune complex ions traversed into the mass spectrometer's collision chamber, whereas paths of unbound peptide ions were blocked prior to entering the collision cell. After dissociation of the multiply charged immune complexes in the gas phase, the complex-released peptide ions were recorded after having traversed the second mass filter. Complex-released peptides were unambiguously identified by their masses using mass analysis with isotope resolution. From the results of our studies with seven (phospho-) peptides with distinct amino acid sequences, which resembled either the antibody's binding motif or mutations, we conclude the following: (i) A negatively charged phospho group, located near the peptide's N-terminus is mandatory for antibody binding when placed on the peptide surface at a precise distance to the C-terminally located positively charged ε-amino group of a lysinyl residue. (ii) A bulky amino acid residue, such as the tyrosinyl residue at the N-terminal position of the (phospho-) threoninyl residue, abolishes antibody binding. (iii) Two closely spaced phospho groups negatively interfere with the surface polarity pattern and abolish antibody binding as well. (iv) Non-phosphorylated peptides are not binding partners of the anti-HpTGEKP antibody.

Phosphorylated Serine Marks Proteins for Degradation By the Mitochondrial Matrix Protease, Clpxp

Mitochondrial ClpXP is an ATP-dependent protease complex in which the ClpX AAA+ chaperone binds, unfolds, and translocates specific protein substrates into the degradation chamber of ClpP. The serine protease ClpXP is located in the mitochondrial matrix and maintains the integrity of the respiratory chain by degrading damaged and/or misfolded proteins. This protease is overexpressed in 45% of AML patients. Genetic or chemical inhibition or hyperactivation of ClpXP kills leukemic cells and stem cells in vitro and in vivo. Yet, the degron sequences or post-translational modifications that are targeted by mitochondrial ClpXP remain to be elucidated. In Bacillus subtilis, the bacterial ClpXP homologue degrades proteins containing phospho-arginine. To determine if phosphorylated amino acids also mark proteins for mitochondrial ClpXP degradation, we evaluated human ClpXP protease activity in the presence of phosphorylated amino acids. Recombinant human ClpXP was incubated with FITC-tagged casein substrates and increasing concentrations of phospho-serine (pSer), phospho-threonine (pThr), phospho-arginine (pArg), or phospho-tyrosine (pTyr). The release of fluorogenic FITC-tag was measured to assess ClpXP protease activity. In a dose-dependent manner, pSer and pThr free amino acids inhibited casein cleavage by ClpXP. Similar inhibition was seen with short peptides containing pSer and pThr. In contrast, pTyr, pArg, free phosphates, and the dephosphorylated amino acids or peptides did not affect ClpXP protease activity in this assay. Although pSer and pThr inhibited the protease activity of ClpXP, they had no impact on ClpX ATPase activity nor the peptidase activity of isolated ClpP. We characterized ClpP peptidase activity and ClpX ATPase activity using the Ac-Trp-Leu-Ala-AMC (Ac-WLA-AMC) peptidase activity assay and the Malachite Green Phosphate assay, respectively. pSer and pThr had no effect on ClpP peptidase activity nor ClpX ATPase activity. Therefore, pSer and pThr specifically inhibited ClpXP's ability to degrade full-length proteins. Next, we investigated if pSer and pThr could bind to ClpX or ClpP. Thermal shift binding assays indicated that pSer and pThr but not pTyr and pArg bound to ClpX and none of the phosphorylated amino acids bound to ClpP. Similarly, Ala-pSer-Ala (ApSA), Arg-Arg-Ala-pSer-Val-Ala (RRApSVA) and Ala-pThr-Ala (ApTA) peptides bound to ClpX, while the non-phosphorylated peptides did not. Thus, taken together, pSer and pThr interact with ClpX at a site outside its ATP binding pocket. Previously we showed that ClpX and ClpP interact with respiratory chain complex II subunit SDHA. Moreover, inhibition of ClpP in AML cells increased misfolded or damaged SDHA protein and impaired respiratory chain complex II activity. Therefore, we asked if inhibition of ClpP and ClpX impacted levels of SDHA serine phosphorylation. We knocked down ClpX and ClpP individually in OCI-AML2 cells using shRNA. Then, mitochondrial lysates were prepared, serine phosphorylated proteins were immunoprecipitated and probed for SDHA. Increased abundance of serine phosphorylated SDHA was observed in AML cells after ClpP and ClpX knockdown. We also demonstrated that addition of recombinant ClpXP to the immunoprecipitates selectively degraded the fraction of serine phosphorylated SDHA. We then showed that serine phosphorylated SDHA was present only in the digitonin insoluble fraction of mitochondria proteins, indicating it is only found on damaged protein aggregates. In summary, we discovered that pSer and pThr promote the degradation of damaged respiratory chain proteins by mitochondrial ClpXP. Thus, this work highlights new mitochondrial biology and identifies a potential new strategy to develop ClpXP inhibitors for the treatment of AML.

Antimicrobial, Polarizing Light, and Paired Helical Filament Properties of Fragmented Tau Peptides of Selected Putative Gingipains.

Tau is an established substrate for gingipains secreted by Porphyromonas gingivalis. Hyperphosphorylation of tau and neurofibrillary tangle (NFT) formation is a defining lesion of Alzheimer's disease (AD) where NFT distribution is related to Braak stage and disease severity. To assess gingipains'-fragmented tau peptides for their antimicrobial properties and for the likelihood of paired helical/straight filament (PHF/SF) formation with implications for the NFT lesion. Seven non-phosphorylated (A-G) and three phosphorylated (A-C) tau peptides, were tested for antimicrobial properties against P. gingivalis. Polarizing light properties were determined using Congo Red staining. Secondary and tertiary structures of peptides B-F were determined using transmission electron microscopy (TEM) and circular dichroism (CD) was undertaken for the soluble peptides A in phosphorylated and non-phosphorylated states. Phosphorylated tau peptide A displayed a significant effect against planktonic P. gingivalis. The CD results demonstrated that both peptides A, in phosphorylated and non-phosphorylated states, in aqueous solution, adopted mainly β-type structures. Non-phosphorylated peptides B-F and phosphorylated peptides B-C were insoluble and fibrillar under the TEM. The secondary and tertiary structures of the non-phosphorylated peptide B demonstrated fewer helical twists, whereas peptide C displayed significantly more helical twists along the whole fiber(s) length following its phosphorylation. Phosphorylated peptide A reduced P. gingivalis viability. CD spectroscopy demonstrated the phosphorylated and the non-phosphorylated peptide A predominantly formed from β-sheet structures in aqueous solution with potential antimicrobial activity. Phosphorylation of tau peptides physically changed their tertiary structure into PHFs with potential for self-aggregation and binding to the NFT lesion.

Surface plasma resonance biosensing of phosphorylated proteins via pH-adjusted specific binding of phosphate residues with UiO-66

The abnormal protein phosphorylation is closely related to a series of diseases including cancers, neurodegeneration, and other immune/inflammatory disorders. However, a simple, accurate, and pragmatic methodology for the direct detection of phosphorylation levels of target proteins is largely unavailable. Herein, a surface plasma resonance (SPR) biosensor was developed to detect phosphorylated protein based on significant SPR enhancement using UiO-66 and its high affinity for phosphate residues. Furthermore, the pH of the incubation buffer and the NaOH concentration in the washing solution to eliminate nonspecific adsorption of nanoparticles on the chip surface were optimized to adjust the surface charges of UiO-66. This improved the specificity and sensitivity of the proposed method. The designed biosensor could distinguish 1% phosphorylated peptides from non-phosphorylated peptides background. Moreover, the method could directly analyze the phosphorylation level of target protein in complex samples such as cell or tissue extracts without requiring a purification step in a single workflow. This simple and robust biosensor is a sensitive, accurate, and pragmatic toolbox to detect phosphorylated proteins without destroying the protein structure and can significantly facilitate phosphoproteomics and clinical diagnosis research.

Phosphopeptide Enrichment Techniques: A Pivotal Step for Phosphoproteomic Studies.

Many pathological conditions are caused by dysregulation of cell signaling, which can generate a cascade of abnormal responses and completely change the functions of a cell or tissue. A large portion of the regulation of these signals is via protein phosphorylation, in which cell responses can be activated or inhibited. Proteins that are both downstream and upstream of a phosphorylated protein can be modified, altering metabolism and other biological processes. Recently, the number of phosphoproteomic studies based on mass spectrometry has increased, constantly aiming to obtain a higher coverage of proteins and increase the number and location of their phospho-sites, as well as better understand their respective phosphorylation states. In this way, it is possible to better understand biological processes as a whole and their roles in cellular dysfunctions and diseases. To study changes at the phosphoproteome level, the stochiometric imbalance between the non-phosphorylated and phosphorylated peptides must be overcome, since higher quantities and comparatively better ionization of non-phosphorylated peptides can suppress the ion signals of the phosphorylated peptides. It is for this reason that phosphophopeptides are rarely found in samples that did not pass through a phospho-enrichment step, highlighting the importance of performing enrichment steps in phosphoproteomic studies. The numbers of identified phosphopeptides and phosphorylation sites are extremely important to the quality of a phosphoproteomic analysis; therefore, the efficiency of the enrichment process is critical. Here, phospho-enrichment techniques are presented to offer insight into the applicability of these methods to different experiment types and consequently support the growth of phosphoproteomic studies overall.

Multiple phosphorylated protein selective analysis via fluorous derivatization and liquid chromatography-electrospray ionization-mass spectrometry analysis

Post-translational modification of proteins is involved in protein function and higher-order structure. Among such modification, phosphorylation is an important intracellular signal transduction pathway. Many studies on phosphorylated protein analysis using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) have been developed. However, there are few reports on the analysis of highly phosphorylated proteins because of their handling difficulty. Hence, we developed an analytical method that converts multiple phosphate groups contained in the peptides into perfluoroalkyl groups for selective analysis using fluorous affinity. Here, tryptic digested β-casein fragment peptides [RELEELNVPGEIVE(pSer)L(pSer)(pSer)(pSer)EESITR and FQ(pSer)EEQQQTEDELQDK] were used as model phosphorylated peptides. 1H,1H,2H,2H-Perfluorooctanethiol (PFOT) and 2,2,2-trifluoroethanethiol (TFET) were used as derivatization reagents for mono-phosphorylated peptides and multi-phosphorylated peptides, respectively, to derivatize via β-elimination/Michael addition. The derivatives were analyzed by LC-ESI-MS. A fluorous LC column is typically used to selectively retain the fluorous-derivatized peptides, which are expected to be separated from contaminants and non-phosphorylated peptides. When this method was applied to β-casein, TFET- and PFOT-derivatized peptides were strongly retained in the fluorous LC column and clearly separated from non-phosphorylated peptides on the chromatogram. Therefore, the developed method enables quantification of mono- and multi-phosphorylated peptides and is suitable for application in proteomics.

CIC Is a Mediator of the ERK1/2-DUSP6 Negative Feedback Loop.

SummaryDUSP6 functions as an important negative feedback component of the MAPK/ERK signaling pathway. Although DUSP6 expression is tightly regulated by ERK1/2 signaling, the molecular mechanism of this regulation remains partially understood. In this work, we show that the transcriptional repressor CIC functions downstream of the ERK1/2 signaling to negatively regulate DUSP6 expression. CIC directly represses DUSP6 transcription by binding to three cis-regulatory elements (CREs) in DUSP6 promoter. p90RSK, a downstream target of ERK1/2, phosphorylates CIC at S173 and S301 sites, which creates a 14-3-3 recognition motif, resulting in 14-3-3-mediated nuclear export of CIC and derepression of DUSP6. Finally, we demonstrate that the oncogenic CIC-DUX4 fusion protein acts as a transcriptional activator of DUSP6 and its nuclear/cytoplasmic distribution remains regulated by ERK1/2 signaling. These results complete an ERK1/2/p90RSK/CIC/DUSP6 negative feedback circuit and elucidate the molecular mechanism of how RTK/MAPK signaling harnesses the transcriptional repressor activity of CIC in mammalian cells.

Electrochemical assay of protein kinase activity based on the Fe3O4@PNE-Ti4+ functionalized PDMS microchip

A new strategy for the sensitive assay of protein kinase activity has been constructed using Fe3O4@PNE-Ti4+ nanocomposite as stationary phase in microchip electrophoresis. Through the self-polymerization of norepinephrine in weak alkaline solution, a thin poly(norepinephrine) (PNE) film can be spontaneously formed on the surface of Fe3O4 nanoparticles (NPs) to produce Fe3O4@PNE NPs. Due to the good magnetic property, Fe3O4@PNE NPs can be easily fixed in poly(dimethylsiloxane) (PDMS) channel under external magnetic field. On the other hand, the catechol hydroxyl groups in PNE can chelate Ti4+ to form Fe3O4@PNE-Ti4+ in PDMS channel. When peptides are phosphorylated by protein kinase A (PKA) in the presence of adenosine 5′-triphosphate (ATP), the Fe3O4@PNE-Ti4+ packed in PDMS channels can specifically coordinate with the phosphorylated sites of phosphopeptides, but the non-phosphorylated peptides cannot bind with Ti4+. Thus, the retention time of the phosphorylated peptides in the Fe3O4@PNE-Ti4+ packed PDMS channel was increased, and phosphorylated peptides and non-phosphorylated peptides can be separated successfully. Due to the high loading of Ti4+ on the Fe3O4@PNE, this strategy can be used for the enrichment of phosphorylated peptides and electrochemical assay of PKA activity. This method can also be used for quantitative analysis of PKA activity in cell lysate, indicating its potential application in clinical diagnosis.

Fractionation of Enriched Phosphopeptides Using pH/Acetonitrile-Gradient-Reversed-Phase Microcolumn Separation in Combination with LC-MS/MS Analysis.

Mass spectrometry (MS) is a powerful and sensitive method often used for the identification of phosphoproteins. However, in phosphoproteomics, there is an identified need to compensate for the low abundance, insufficient ionization, and suppression effects of non-phosphorylated peptides. These may hamper the subsequent liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS) analysis, resulting in incomplete phosphoproteome characterization, even when using high-resolution instruments. To overcome these drawbacks, we present here an effective microgradient chromatographic technique that yields specific fractions of enriched phosphopeptides compatible with LC–MS/MS analysis. The purpose of our study was to increase the number of identified phosphopeptides, and thus, the coverage of the sample phosphoproteome using the reproducible and straightforward fractionation method. This protocol includes a phosphopeptide enrichment step followed by the optimized microgradient fractionation of enriched phosphopeptides and final LC–MS/MS analysis of the obtained fractions. The simple fractionation system consists of a gas-tight microsyringe delivering the optimized gradient mobile phase to reversed-phase microcolumn. Our data indicate that combining the phosphopeptide enrichment with the microgradient separation is a promising technique for in-depth phosphoproteomic analysis due to moderate input material requirements and more than 3-fold enhanced protein identification.

Application of functionalized magnetic nanomaterials in phosphopeptide enrichment

Protein phosphorylation is one of the most important and prevalent post-translational modifications. Mass spectrometry (MS) has become a powerful tool for the analysis of phosphopeptides. However, the low dynamic stoichiometry, poor ionization efficiency, and signal suppression by high-abundance non-phosphorylated peptides have hindered the direct applications in the analysis of phosphopeptides. Therefore, the development of highly selective and sensitive enrichment strategies is necessary for the identification of phosphopeptides prior to MS analysis. Magnetic nanomaterials have good magnetic responsiveness and can be separated rapidly from the solution with the help of external magnets. Functionalized magnetic nanomaterials have been widely used as a new analytical tool in proteomics research. This review surveys recently reported functionalized magnetic nanoparticles for the enrichment of phosphopeptides reported in recent years. Further trends in the enrichment of phosphopeptides with functionalized magnetic materials are also prospected.

Effect of shell structure of Ti-immobilized metal ion affinity chromatography core-shell magnetic particles for phosphopeptide enrichment

Magnetic materials in sample preparation for shotgun phosphoproteomics offer several advantages over conventional systems, as the enrichment can be achieved directly in solution, but they still suffer from some drawbacks, due to limited stability and selectivity, which is supposed to be affected by the hydrophilicity of the polymeric supports used for cation immobilization. The paper describes the development of an improved magnetic material with increased stability, thanks to a two-step covering of the magnetic core, for the enrichment of phosphopeptides in biological samples. Four materials were prepared featuring a polymeric shell with tunable hydrophilicity, obtained by “grafting from” polymerization of glycidyl methacrylate with 0–8.3% of polyethylene glycol methacrylate (PEGMA), the latter used to modulate the hydrophilicity of the material surface. Finally, the materials were functionalized with iminodiacetic acid for Ti4+ ion immobilization. The materials were analyzed for their composition by a combination of CHN elemental analysis and thermogravimetric analysis, also hyphenated to gas chromatography and mass spectrometric detection. Surface characteristics were evaluated by water contact angle measurements, scanning electron microscopy and energy dispersive X-ray spectrometry. These materials were applied to the enrichment of phosphopeptides from yeast protein digests. Peptides were identified by proteomics techniques using nano-high performance liquid chromatography coupled to mass spectrometry and bioinformatics. Qualitatively the peptides identified by the four systems were comparable, with 1606–1693 phosphopeptide identifications and a selectivity of 47–54% for all materials. The physico-chemical features of the identified peptides were also the same for the four materials. In particular, the grand average of hydropathy index values indicated that the enriched phosphopeptides were hydrophilic (ca. 90%), and only some co-enriched non-phosphorylated peptides were hydrophobic (21–28%), regardless of the material used for enrichment. Peptides had a pI ≤ 7, which indicated a well-known bias for acidic peptides binding, attributed to the interaction with the metal center itself. The results indicated that the enrichment of phosphopeptides and the co-enrichment of non-phosphorylated peptides is mainly driven by interactions with Ti4+ and does not depend on the amount of PEGMA chains in the polymer shell.

Application of mobile phase additives to reduce metal-ion mediated adsorption of non-phosphorylated peptides in RPLC/MS-based assays

Metal-ion mediated adsorption in liquid chromatography has been identified as a contributing factor in poor peak shape, tailing, and diminished recovery of compounds prone to cation exchange-like interaction with metal-based activity sites. Peptides that exhibit negative charge-bearing amino acids such as aspartic acid and glutamic acid are particularly sensitive to metal-ion mediated adsorption in RPLC/MS-based separations when using weak acids (e.g. formic acid) as mobile phase additives. Citric acid and medronic acid as metal complexing mobile phase additives were evaluated for their ability to mitigate metal-ion mediated adsorption in RPLC/MS-based peptide mapping assays. In this study, chromatographic performance was stabilized with peak tailing for peptides of interest reduced by as much as 40% in the presence of a chelator at a mobile phase concentration of 1 ppm. Performance gains were observed to be stable over a 67-hour time study with an average USP tailing factor of 1.00, % RSD = 0.64. The stabilizing effect of the chelator improved peptide mapping assay robustness with relative peak areas for target impurities calculated at 2.28% (% R.S.D. = 2.36) and 2.40% (% R.S.D. = 2.37). Collectively this study demonstrates that chelators as mobile phase additives offers a means to improve chromatographic performance for biomolecules sensitive to metal-ion mediated adsorption under formic acid-based RPLC conditions.

Core-shell magnetic bimetallic MOF material for synergistic enrichment of phosphopeptides

In proteomics, phosphorylation is an important process for protein post-translational modification (PTM), which greatly improves the diversity of proteomes. The PTM regulates almost all physiological and pathological processes such as signal transduction, cell division, proliferation, differentiation and metabolism. The abnormal expression of protein phosphorylation is also associated with cellular metabolic disorders and a range of diseases. However, in mass spectrometry-based phosphorylated peptideomics studies, phosphorylated peptide signals were inhibited by a high abundance of non-phosphorylated peptides; thus, highly selective enrichment was required. In this study, a newly designed material named Fe3O4@MIL(Fe/Ti) was synthesized using a layer-by-layer self-assembly technique that coats the surface of magnetic oxide nanospheres with bimetallic MOF of iron and titanium. The synergistic synthetic coating of the bimetallic MOF gives the material a large surface area and excellent hydrophilicity, which endow the nanoparticles with excellent phosphopeptide enrichment ability, high selectivity (β-casein/BSA molar ratio 1:500), a low detection limit (3 fmol), high recovery rate (85%), strong binding capacity, size exclusion ability, and ideal batch-to-batch repeatability. For comparison, we used Fe3O4@MIL(Fe/Ti) and two single-metal MOF materials Fe3O4@MIL-100(Fe) and Fe3O4@MIL-125(Ti), to enrich α-casein in the middle. Thus, the iron-titanium bimetallic MOF can not only enrich all the phosphorylated peptides enriched by Fe3O4@MIL-100(Fe) and Fe3O4@MIL-125(Ti), but can also specifically enrich four phosphorylated peptides. Encouraged by the excellent results of characterization and standard protein enrichment, we used this material to analyze human serum and found that bimetallic materials can effectively enrich all four phosphorylated peptides and exclude high molecular proteins. These experimental results indicate that the novel bimetallic MOF is a good candidate to analyze protein phosphorylation in complex samples.