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FAIMS interface is gaining popularity because of the impressive 100-fold signal to noise enhancement in addition to the recent coupling to the Orbitrap technology, the most important analyzer developed in the last 20 years. The selection of group of ions and effective removal of single-charged ones at particular compensation voltages increases around 50% the proteome coverage at expenses of lower peptides coverage. However, specific setting for phosphoproteome analysis is yet poorly described. Here we have found the maximum transmission for several tryptic phosphopeptides isolated from a single complex mixture and we have set an experimental method based on five compensation voltages partially different to the ones described previously, demonstrating the relevance of voltages higher than 47 V, with an increase of around 20% of unique phosphopeptides. Using this experimental setup two complex phosphoproteomes isolates (SH-SY5Y cell line and plasma) were analyzed and found increments of 50% on phosphopeptides identification with the proposed method with respect to a previous one, for the cell line extract. Meanwhile for plasma 109 of the detected phosphopeptides are found for first time in this body fluid, presumably due to the release of intracellular proteins. With this FAIMS setup, 60% of the proteins identified are classified as very low abundant proteins.
Collisionally activated dissociation (CAD), infrared multiphoton dissociation (IRMPD), ultraviolet photodissociation (UVPD), electron capture dissociation and electron detachment dissociation (EDD) experiments were conducted on a set of phosphopeptides, in a Fourier transform ion cyclotron resonance mass spectrometer. The fragmentation patterns were compared and varied according to the fragmentation mechanisms and the composition of the peptides. CAD and IRMPD produced similar fragmentation profiles of the phosphopeptides, while UVPD produced a large number of complementary fragments. Electron-based dissociation techniques displayed lower fragmentation efficiencies, despite retaining the labile phosphate group, and drastically different fragmentation profiles. EDD produced complex spectra whose interpretation proved challenging.
The uranyl ion (UO22+ ) binds phosphopeptides with high affinity, and when irradiated with UV-light, it can cleave the peptide backbone. In this study, high-accuracy tandem mass spectrometry and enzymatic assays were used to characterise the photocleavage products resulting from the uranyl photocleavage reaction of a tetraphosphorylated β-casein model peptide. We show that the primary photocleavage products of the uranyl-catalysed reaction are C-terminally amidated. This could be of great interest to the pharmaceutical industry, as efficient peptide amidation reactions are one of the top challenges in green pharmaceutical chemistry.
Mass spectrometry (MS)-based large scale phosphoproteomics has facilitated the investigation of plant phosphorylation dynamics on a system-wide scale. However, generating large scale data sets for membrane phosphoproteins usually requires fractionation of samples and extended hands-on laboratory time. To overcome these limitations, we developed "ShortPhos," an efficient and simple phosphoproteomics protocol optimized for research on plant membrane proteins. The optimized workflow allows fast and efficient identification and quantification of phosphopeptides, even from small amounts of starting plant materials. "ShortPhos" can produce label-free datasets with a high quantitative reproducibility. In addition, the "ShortPhos" protocol recovered more phosphorylation sites from membrane proteins, especially plasma membrane and vacuolar proteins, when compared to our previous workflow and other membrane-based data in the PhosPhAt 4.0 database. We applied "ShortPhos" to study kinase-substrate relationships within a nitrate-induction experiment on Arabidopsis roots. The "ShortPhos" identified significantly more known kinase-substrate relationships compared to previous phosphoproteomics workflows, producing new insights into nitrate-induced signaling pathways.
A casein phosphopeptide (CPP) fraction derived from tryptic hydrolysis of bovine casein was evaluated for antioxidant activity. Conjugations or mixtures of CPP with polysaccharide, galactomannan (Gal), or xyloglucan (Xyl) were prepared to evaluate potential enhancement of CPP antioxidant activity. The effect of calcium was also investigated. The CPP preparation alone was effective at scavenging hydroxyl radicals and sequestering Fe2+ to protect against Fenton reaction-induced deoxyribose oxidation in non-site-specific (up 63.3% inhibition) and site-specific (up 32.1% inhibition) binding assays, respectively. CPP also effectively quenched 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid radicals (ABTS•+) to an extent of 67.6% scavenging in an aqueous system. In a soybean lecithin liposome system, CPP exhibited effective protection against peroxyl radical-induced liposomal peroxidation (38.3% of control in terms of rate of propagation). Conjugating CPP with Gal or Xyl polysaccharides using Maillard reaction conditions significantly reduced activity in the Fenton reaction-deoxyribose assays, while exhibiting no effect on the antioxidant activity of native CPP in both the ABTS and liposome assays, respectively. These results represent comparative antioxidant capacity of the native CPP and associated conjugates in phases that varied in relative hydrophilic and hydrophobic character. We conclude that CPP has the potential to act as both a primary and secondary antioxidant by displaying transition metal ion sequestering activity and free radical quenching activity. Improvements in antioxidant activity of CPP by Maillard-type conjugation with Xyl or Gal were relatively small and model-specific.
Protein phosphorylation and glycosylation coordinately regulate numerous complex biological processes. However, the main methods to simultaneously enrich them are based on the coordination interactions or Lewis acid-base interactions, which suffer from low coverage of target molecules due to strong intermolecular interactions. Here, we constructed a poly-histidine modified silica (SiO2@Poly-His) microspheres-based method for the simultaneous enrichment, sequential elution and analysis of phosphopeptides and glycopeptides. The SiO2@Poly-His microspheres driven by hydrophilic interactions and multiple hydrogen bonding interactions exhibited high selectivity and coverage for simultaneous enrichment of phosphopeptides and glycopeptides from 1,000 molar folds of bovine serum albumin interference. Furthermore, "on-line deglycosylation" strategy allows sequential elution of phosphopeptides and glycopeptides, protecting phosphopeptides from hydrolysis during deglycosylation and improving the coverage of phosphopeptides. The application of our established method to HT29 cell lysates resulted in a total of 1,601 identified glycopeptides and 694 identified phosphopeptides, which were 1.2-fold and 1.5-fold higher than those obtained from the co-elution strategy, respectively. The SiO2@Poly-His based simultaneous enrichment and sequential separation strategy might have great potential in co-analysis of PTMs-proteomics of biological and clinic samples.
Reversible protein glycosylation and phosphorylation tightly modulate important cellular processes and are closely involved in pathological processes in a crosstalk dependent manner. Because of their significance and low abundances of glyco- and phosphopeptides, several strategies have been developed to simultaneously enrich and co-elute glyco- and phosphopeptides. However, the co-existence of deglycosylated peptides and phosphopeptides aggravates the mass spectrometry analysis. Herein we developed a novel strategy to analyze glyco- and phosphopeptides based on simultaneous enrichment with TiO2, on-line deglycosylation and collection of deglycosylated peptides, and subsequent elution of phosphopeptides. To optimize on-line deglycosylation conditions, the solution pH, buffer types and concentrations, and deglycosylation time were investigated. The application of this novel strategy to 100 μg mouse brain resulted in 355 glycopeptides and 1,975 phosphopeptides, which were 2.5 and 1.4 folds of those enriched with the reported method. This study will expand the application of TiO2 and may shed light on simultaneously monitoring protein multiple post-translational modifications.
Casein phosphopeptides have been studied widely for their ability to chelate calcium. However, systematic studies on the effects of casein phosphopeptides (CPP) on calcium absorption in vitro and in vivo are scarce. The purities of two commercially available products, CPP1 and CPP2, are 18.37 and 25.12%, respectively. Here, the in vitro calcium binding capacity of CPP2 was 142.56 ± 7.39 mg/g, which was higher than that of CPP1 (107.15 ± 6.27 mg/g). The calcium transport results in a Caco-2 monolayer model indicated that, relative to controls, CPP1 and CPP2 increased calcium transport by 21.78 and 53.68%, respectively. Subsequent animal experiments showed that the CPP2-Ca-H group (1% Ca, 0.4% CPP2) had significant increases in the femur index, serum Ca2+ and serum osteocalcin levels, and femoral Ca content. The CPP2-Ca-H animal also had decreased serum alkaline phosphatase levels, parathyroid hormone content, and urinary pyridinoline content. Overall, our results demonstrated that CPP2 had stronger effects on promoting calcium uptake than CPP1.
There is a pressing need for novel immunotherapeutic targets in colorectal cancer (CRC). Cytotoxic T cell infiltration is well established as a key prognostic indicator in CRC, and it is known that these tumor infiltrating lymphocytes (TILs) target and kill tumor cells. However, the specific antigens that drive these CD8+ T cell responses have not been well characterized. Recently, phosphopeptides have emerged as strong candidates for tumor-specific antigens, as dysregulated signaling in cancer leads to increased and aberrant protein phosphorylation. Here, we identify 120 HLA-I phosphopeptides from primary CRC tumors, CRC liver metastases and CRC cell lines using mass spectrometry and assess the tumor-resident immunity against these posttranslationally modified tumor antigens. Several CRC tumor-specific phosphopeptides were presented by multiple patients' tumors in our cohort (21% to 40%), and many have previously been identified on other malignancies (58% of HLA-A*02 CRC phosphopeptides). These shared antigens derived from mitogenic signaling pathways, including p53, Wnt and MAPK, and are therefore markers of malignancy. The identification of public tumor antigens will allow for the development of broadly applicable targeted therapeutics. Through analysis of TIL cytokine responses to these phosphopeptides, we have established that they are already playing a key role in tumor-resident immunity. Multifunctional CD8+ TILs from primary and metastatic tumors recognized the HLA-I phosphopeptides presented by their originating tumor. Furthermore, TILs taken from other CRC patients' tumors targeted two of these phosphopeptides. In another cohort of CRC patients, the same HLA-I phosphopeptides induced higher peripheral T cell responses than they did in healthy donors, suggesting that these immune responses are specifically activated in CRC patients. Collectively, these results establish HLA-I phosphopeptides as targets of the tumor-resident immunity in CRC, and highlight their potential as candidates for future immunotherapeutic strategies.
The 14-3-3 family of phosphoprotein-binding proteins regulates many cellular processes by docking onto pairs of phosphorylated Ser and Thr residues in a constellation of intracellular targets. Therefore, there is a pressing need to develop new prediction methods that use an updated set of 14-3-3-binding motifs for the identification of new 14-3-3 targets and to prioritize the downstream analysis of >2000 potential interactors identified in high-throughput experiments.
Certain phosphorylated peptides are differentially presented by MHC molecules on cancer cells characterized by aberrant phosphorylation. Phosphopeptides presented in complex with the human leukocyte antigen HLA-A*02:01 provide a stability advantage over their nonphosphorylated counterparts. This stability is thought to contribute to enhanced immunogenicity. Whether tumor-associated phosphopeptides presented by other common alleles exhibit immunogenicity and structural characteristics similar to those presented by A*02:01 is unclear. Therefore, we determined the identity, structural features, and immunogenicity of phosphopeptides presented by the prevalent alleles HLA-A*03:01, -A*11:01, -C*07:01, and - C*07:02.
Post-translational phosphorylation is essential to human cellular processes, but the transient, heterogeneous nature of this modification complicates its study in native systems. We developed an approach to interrogate phosphorylation and its role in protein-protein interactions on a proteome-wide scale. We genetically encoded phosphoserine in recoded E. coli and generated a peptide-based heterologous representation of the human serine phosphoproteome. We designed a single-plasmid library encoding >100,000 human phosphopeptides and confirmed the site-specific incorporation of phosphoserine in >36,000 of these peptides. We then integrated our phosphopeptide library into an approach known as Hi-P to enable proteome-level screens for serine-phosphorylation-dependent human protein interactions. Using Hi-P, we found hundreds of known and potentially new phosphoserine-dependent interactors with 14-3-3 proteins and WW domains. These phosphosites retained important binding characteristics of the native human phosphoproteome, as determined by motif analysis and pull-downs using full-length phosphoproteins. This technology can be used to interrogate user-defined phosphoproteomes in any organism, tissue, or disease of interest.
Dysregulated post-translational modification provides a source of altered self-antigens that can stimulate immune responses in autoimmunity, inflammation, and cancer. In recent years, phosphorylated peptides have emerged as a group of tumour-associated antigens presented by MHC molecules and recognised by T cells, and represent promising candidates for cancer immunotherapy. However, the impact of phosphorylation on the antigenic identity of phosphopeptide epitopes is unclear. Here we examined this by determining structures of MHC-bound phosphopeptides bearing canonical position 4-phosphorylations in the presence and absence of their phosphate moiety, and examining phosphopeptide recognition by the T cell receptor (TCR). Strikingly, two peptides exhibited major conformational changes upon phosphorylation, involving a similar molecular mechanism, which focussed changes on the central peptide region most critical for T cell recognition. In contrast, a third epitope displayed little conformational alteration upon phosphorylation. In addition, binding studies demonstrated TCR interaction with an MHC-bound phosphopeptide was both epitope-specific and absolutely dependent upon phosphorylation status. These results highlight the critical influence of phosphorylation on the antigenic identity of naturally processed class I MHC epitopes. In doing so they provide a molecular framework for understanding phosphopeptide-specific immune responses, and have implications for the development of phosphopeptide antigen-specific cancer immunotherapy approaches.
Recent studies challenge the dogma that a 21-mer phosphopeptide P140 protects against direct cell damage in the phase-III clinical trial (NCT02504645) for lupus, involving reactive oxygen species (ROS)-dependent release of citrullinated histone H3 (H3cit)-linked neutrophil extracellular traps. An open question is the cellular location of ROS production and H3cit formation in lupus. In this study, we examined the effects of P140 peptides on ROS production and H3cit location in lupus with in vivo and situ fluorescence imaging with subcellular resolution. We developed a mouse model of the B6 strain harbouring a bioluminescent reporter under the control of the Lysozyme M promoter. Based on the imiquimod-induced disease model of B6 mice, we used bioluminescent imaging, flow cytometry analysis, and immunohistology staining to study the effects of P140 peptides in lupus. We found a profound accumulation of CX3CR1-positive macrophages in the lungs of lupus mice after the application of P140, accompanied by lung fibrosis formation. The defined P140-mediated macrophage responses were associated with an increase of H3cit in the cytosol, interleukin-1 receptor type 1 on the extracellular membrane, and intracellular production of ROS. Of interest, the disease of imiquimod-induced lupus was prevented with an antioxidant drug apocynin. This study shows that P140 peptides play a role in aggravated murine lupus in a manner dependent on ROS production and H3cit upregulation through pulmonary macrophages.
Liquid chromatography-mass spectrometry (LC-MS) is a major tool for the large-scale qualitative and/or quantitative analysis of protein phosphorylation in cells or tissues. The performance of LC is pivotal for the success of phosphoproteomics in both sensitivity and reproducibility. Here, we report that the widely used Easy-nLC 1200 has poor performance in analyzing phosphopeptides, particularly in terms of sensitivity and reproducibility, whereas its predecessor, Easy-nLC 1000, has a much better performance. Therefore, we suggest that Easy-nLC 1200 is not appropriate for LC-MS-based proteomics analysis for samples with a limited amount, particularly phosphopeptides from plants.
Electron capture dissociation mass spectrometry offers several advantages for the analysis of peptides, most notably that backbone c and z fragments typically retain labile modifications such as phosphorylation. We have shown previously that, in some cases, the presence of phosphorylation has a deleterious effect on peptide sequence coverage, and hypothesized that intramolecular interactions involving the phosphate group were preventing separation of backbone fragments. In the present work, we seek to rationalize the observed ECD behavior through a combination of ECD of model peptides, traveling wave ion mobility mass spectrometry and molecular dynamics simulations. The results suggest that for doubly protonated ions of phosphopeptide APLpSFRGSLPKSYVK a salt-bridge structure is favored, whereas for the doubly-protonated ions of APLSFRGSLPKpSYVK ionic hydrogen bonds predominate.
Protein tyrosine sulfation (sY) is a post-translational modification (PTM) catalyzed by Golgi-resident tyrosyl protein sulfo transferases (TPSTs). Information on sY in humans is currently limited to ∼50 proteins, with only a handful having verified sites of sulfation. As such, the contribution of sulfation to the regulation of biological processes remains poorly defined. Mass spectrometry (MS)-based proteomics is the method of choice for PTM analysis but has yet to be applied for systematic investigation of the "sulfome", primarily due to issues associated with discrimination of sY-containing from phosphotyrosine (pY)-containing peptides. In this study, we developed an MS-based workflow for sY-peptide characterization, incorporating optimized Zr4+ immobilized metal-ion affinity chromatography (IMAC) and TiO2 enrichment strategies. Extensive characterization of a panel of sY- and pY-peptides using an array of fragmentation regimes (CID, HCD, EThcD, ETciD, UVPD) highlighted differences in the generation of site-determining product ions and allowed us to develop a strategy for differentiating sulfated peptides from nominally isobaric phosphopeptides based on low collision energy-induced neutral loss. Application of our "sulfomics" workflow to a HEK-293 cell extracellular secretome facilitated identification of 21 new sulfotyrosine-containing proteins, several of which we validate enzymatically, and reveals new interplay between enzymes relevant to both protein and glycan sulfation.
The polychaete Neanthes arenaceodentata- is cosmopolitan in distribution-, has been used as a laboratory test animal. Life history of this species has several unique features; the female dies after spawning and the male incubates the fertilized eggs through the 21-segmented stage. The larvae leave the tube and commence feeding. Changes in protein abundance and phosphorylation were examined during early development of N. arenaceodentata. A gel-based approach and gel-free enrichment of phosphopeptides coupled with mass spectrometry were used to identify proteins and phosphopeptides in fertilized ova and larval stages. Patterns of proteins and phosphoproteins changed from fertilized ova to larval stages. Twelve proteins occurred in phosphorylated form and nine as stage specific proteins. Cytoskeletal proteins have exhibited differential phosphorylation from ova to larval stages; whereas, other proteins exhibited stage-specific phosphorylation patterns. Ten phosphopeptides were identified that showed phosphorylation sites on serine or threonine residues. Sixty percent of the identified proteins were related to structural reorganization and others with protein synthesis, stress response and attachment. The abundance and distribution of two cytoskeleton proteins were examined further by 2-DE Western blot analysis. This is the first report on changes in protein expression and phosphorylation sites at Thr/Ser in early development of N. arenaceodentata. The 2-DE proteome maps and identified phosphoproteins contributes toward understanding the state of fertilized ova and early larval stages and serves as a basis for further studies on proteomics changes under different developmental conditions in this and other polychaete species.
Single-crystalline hyperbranched nanostructures of iron hydroxyl phosphate Fe5(PO4)4(OH)3·2H2O (giniite) with orthorhombic phase were synthesized through a simple route. They have a well-defined dendrite fractal structure with a pronounced trunk and highly ordered branches. The toxicity test shows that the hyperbranched nanostructures have good biocompatibility and low toxicity level, which makes them have application potentials in life science. The study herein demonstrated that the obtained hyperbranched giniite nanostructures show highly selective capture of phosphopeptides and could be used as a kind of promising nanomaterial for the specific capture of phosphopeptides from complex tryptic digests with the detection of MALDI-TOF mass spectrometry.
Certain phosphorylated peptides are differentially presented by major histocompatibility complex (MHC) molecules on cancer cells characterized by aberrant phosphorylation. Phosphopeptides presented in complex with the human leukocyte antigen HLA-A*02:01 provide a stability advantage over their non-phosphorylated counterparts. This stability is thought to contribute to enhanced immunogenicity. Whether tumor-associated phosphopeptides presented by other common alleles exhibit immunogenicity and structural characteristics similar to those presented by A*02:01 is unclear. Therefore, we determined the identity, structural features, and immunogenicity of phosphopeptides presented by the prevalent alleles HLA-A*03:01, HLA-A*11:01, HLA-C*07:01, and HLA-C*07:02.
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