Titanium Dioxide Chromatography Research Articles

TiCPG - a strategy for the simultaneous enrichment of reversibly modified cysteine peptides, phosphopeptides, and sialylated N-Glycopeptides to study cytokines stimulated beta-cells

Diverse post-translational modifications (PTMs) regulate protein function and interaction to fine-tune biological processes. Reversible phosphorylation, cysteines (Cys) modifications, and N-linked glycosylation are all essentially involved in cellular signaling pathways, such as those initiated by the action of pro-inflammatory cytokines, which can induce pancreatic β-cell death and diabetes. Here we have developed a novel strategy for the simultaneous and comprehensive characterization of the proteome and three PTMs including reversibly modified Cysteines (rmCys), phosphorylation, and sialylated N-linked glycosylation from low amount of sample material. This strategy, termed TiCPG, is based on a combination of chemical labeling and titanium dioxide (TiO2) chromatography. We applied the TiCPG strategy to study the proteome and the three PTMs changes in β-cells subject to pro-inflammatory cytokines stimulation. It enabled quantitative analysis of 8346 rmCys sites, 10,321 phosphosites and 962 sialylated N-glycosites from 5496 proteins. Significant regulation was found on 100 proteins at the expression level, while 3020 PTM peptide isoforms from 1468 proteins were significantly regulated. The three PTMs were involved in cytokine mediated β-cell apoptosis, such as the NFκB and the inducible NO synthase signaling pathways. Overall, the TiCPG strategy is a cheap, straightforward, and powerful tool for studies targeting the three PTMs described above. SignificanceThe present study presents a fast and easy method for quantitative assessment of the proteome and three PTMs from minimal amount of sample material. This simple method provides comprehensive and significant knowledge on biological systems and cellular signaling with relatively low analysis time, suitable for younger researchers and researchers that do not have direct access to LC-MSMS in their laboratories. From sub-milligram amount of material, we were able to map known cellular signaling events of proinflammatory cytokine effect on beta-cells and to discover novel PTMs involved in several known signaling pathways.

Phosphoproteomics reveals conserved exercise-stimulated signaling and AMPK regulation of store-operated calcium entry.

Exercise stimulates cellular and physiological adaptations that are associated with widespread health benefits. To uncover conserved protein phosphorylation events underlying this adaptive response, we performed mass spectrometry-based phosphoproteomic analyses of skeletal muscle from two widely used rodent models: treadmill running in mice and insitu muscle contraction in rats. We overlaid these phosphoproteomic signatures with cycling in humans to identify common cross-species phosphosite responses, as well as unique model-specific regulation. We identified >22,000 phosphosites, revealing orthologous protein phosphorylation and overlapping signaling pathways regulated by exercise. This included two conserved phosphosites on stromal interaction molecule 1 (STIM1), which we validate as AMPK substrates. Furthermore, we demonstrate that AMPK-mediated phosphorylation of STIM1 negatively regulates store-operated calcium entry, and this is beneficial for exercise in Drosophila. This integrated cross-species resource of exercise-regulated signaling in human, mouse, and rat skeletal muscle has uncovered conserved networks and unraveled crosstalk between AMPK and intracellular calcium flux.

Characterization of Macrophage Endogenous S-Nitrosoproteome Using a Cysteine-Specific Phosphonate Adaptable Tag in Combination with TiO2 Chromatography

Protein S-nitrosylation is a cysteine post-translational modification mediated by nitric oxide. An increasing number of studies highlight S-nitrosylation as an important regulator of signaling involved in numerous cellular processes. Despite the significant progress in the development of redox proteomic methods, identification and quantification of endogeneous S-nitrosylation using high-throughput mass-spectrometry-based methods is a technical challenge because this modification is highly labile. To overcome this drawback, most methods induce S-nitrosylation chemically in proteins using nitrosylating compounds before analysis, with the risk of introducing nonphysiological S-nitrosylation. Here we present a novel method to efficiently identify endogenous S-nitrosopeptides in the macrophage total proteome. Our approach is based on the labeling of S-nitrosopeptides reduced by ascorbate with a cysteine specific phosphonate adaptable tag (CysPAT), followed by titanium dioxide (TiO2) chromatography enrichment prior to nLC-MS/MS analysis. To test our procedure, we performed a large-scale analysis of this low-abundant modification in a murine macrophage cell line. We identified 569 endogeneous S-nitrosylated proteins compared with 795 following exogenous chemically induced S-nitrosylation. Importantly, we discovered 579 novel S-nitrosylation sites. The large number of identified endogenous S-nitrosylated peptides allowed the definition of two S-nitrosylation consensus sites, highlighting protein translation and redox processes as key S-nitrosylation targets in macrophages.

Mercury alters endogenous phosphorylation profiles of SYK in murine B cells

BackgroundEpidemiological evidence and animal models suggest that exposure to low and non-neurotoxic concentrations of mercury may contribute to idiosyncratic autoimmune disease. Since defects in function and signaling in B cells are often associated with autoimmunity, we investigated whether mercury exposure might alter B cell responsiveness to self-antigens by interfering with B cell receptor (BCR) signal transduction. In this study we determined the effects of mercury on the protein tyrosine kinase SYK, a critical protein involved in regulation of the BCR signaling pathway.MethodsPhosphorylation sites of murine SYK were mapped before and after treatment of WEHI cell cultures with mercury, or with anti-IgM antibody (positive control) or pervanadate (a potent phosphatase inhibitor). Phosphopeptides were enriched by either titanium dioxide chromatography or anti-phosphotyrosine immunoaffinity, and analyzed by liquid chromatography-mass spectrometry. Select SYK phosphosite cluster regions were profiled for responsiveness to treatments using multiple reaction monitoring (MRM) methodology.ResultsA total of 23 phosphosites were identified with high probability in endogenous SYK, including 19 tyrosine and 4 serine residues. For 10 of these sites phosphorylation levels were increased following BCR activation. Using MRM to profile changes in phosphorylation status we found that 4 cluster regions, encompassing 8 phosphosites, were activated by mercury and differentially responsive to all 3 treatments. Phosphorylation of tyrosine-342 and -346 residues were most sensitive to mercury exposure. This cluster is known to propagate normal BCR signal transduction by recruiting adaptor proteins such as PLC-γ and Vav-1 to SYK during formation of the BCR signalosome.ConclusionsOur data shows that mercury alters the phosphorylation status of SYK on tyrosine sites known to have a role in promoting BCR signals. Considering the importance of SYK in the BCR signaling pathway, these data suggest that mercury can alter BCR signaling in B cells, which might affect B cell responsiveness to self-antigen and have implications with respect to autoimmunity and autoimmune disease.

Simultaneous Enrichment of Cysteine-containing Peptides and Phosphopeptides Using a Cysteine-specific Phosphonate Adaptable Tag (CysPAT) in Combination with titanium dioxide (TiO2) Chromatography

Cysteine is a rare and conserved amino acid involved in most cellular functions. The thiol group of cysteine can be subjected to diverse oxidative modifications that regulate many physio-pathological states. In the present work, a Cysteine-specific Phosphonate Adaptable Tag (CysPAT) was synthesized to selectively label cysteine-containing peptides (Cys peptides) followed by their enrichment with titanium dioxide (TiO2) and subsequent mass spectrometric analysis. The CysPAT strategy was developed using a synthetic peptide, a standard protein and subsequently the strategy was applied to protein lysates from Hela cells, achieving high specificity and enrichment efficiency. In particular, for Cys proteome analysis, the method led to the identification of 7509 unique Cys peptides from 500 μg of HeLa cell lysate starting material. Furthermore, the method was developed to simultaneously enrich Cys peptides and phosphorylated peptides. This strategy was applied to SILAC labeled Hela cells subjected to 5 min epidermal growth factor (EGF) stimulation. In total, 10440 unique reversibly modified Cys peptides (3855 proteins) and 7339 unique phosphopeptides (2234 proteins) were simultaneously identified from 250 μg starting material. Significant regulation was observed in both phosphorylation and reversible Cys modification of proteins involved in EGFR signaling. Our data indicates that EGF stimulation can activate the well-known phosphorylation of EGFR and downstream signaling molecules, such as mitogen-activated protein kinases (MAPK1 and MAPK3), however, it also leads to substantial modulation of reversible cysteine modifications in numerous proteins. Several protein tyrosine phosphatases (PTPs) showed a reduction of the catalytic Cys site in the conserved putative phosphatase HC(X)5R motif indicating an activation and subsequent de-phosphorylation of proteins involved in the EGF signaling pathway. Overall, the CysPAT strategy is a straight forward, easy and promising method for studying redox proteomics and the simultaneous enrichment strategy offers an excellent solution for characterization of cross-talk between phosphorylation and redox induced reversible cysteine modifications.

Sequential Elution from IMAC (SIMAC): An Efficient Method for Enrichment and Separation of Mono- and Multi-phosphorylated Peptides.

Phosphoproteomics relies on methods for efficient purification and sequencing of phosphopeptides from highly complex biological systems, especially when using low amounts of starting material. Current methods for phosphopeptide enrichment, e.g., Immobilized Metal ion Affinity Chromatography and titanium dioxide chromatography provide varying degrees of selectivity and specificity for phosphopeptide enrichment. The number of multi-phosphorylated peptides identified in most published studies is rather low. Here we describe a protocol for a strategy that separates mono-phosphorylated peptides from multiply phosphorylated peptides using Sequential elution from Immobilized Metal ion Affinity Chromatography. The method relies on the initial enrichment and separation of mono- and multi-phosphorylated peptides using Immobilized Metal ion Affinity Chromatography and a subsequent enrichment of the mono-phosphorylated peptides using titanium dioxide chromatography. The two separate phosphopeptide fractions are then subsequently analyzed by mass spectrometric methods optimized for mono-phosphorylated and multi-phosphorylated peptides, respectively, resulting in improved identification of especially multi-phosphorylated peptides from a minimum amount of starting material.

Mass spectrometric phosphoproteome analysis of HIV-infected brain reveals novel phosphorylation sites and differential phosphorylation patterns.

To map the phosphoproteome and identify changes in the phosphorylation patterns in the HIV-infected and uninfected brain. Parietal cortex from individuals with and without HIV infection were lysed and trypsinized. The peptides were labeled with iTRAQ reagents, combined, phospho-enriched by titanium dioxide chromatography, and analyzed by LC-MS/MS with high resolution. Our phosphoproteomic workflow resulted in the identification of 112 phosphorylated proteins and 17 novel phosphorylation sites in all the samples that were analyzed. The phosphopeptide sequences were searched for kinase substrate motifs, which revealed potential kinases involved in important signaling pathways. The site-specific phosphopeptide quantification showed that peptides from neurofilament medium polypeptide, myelin basic protein, and 2'-3'-cyclic nucleotide-3' phosphodiesterase have relatively higher phosphorylation levels during HIV infection. This study has enriched the global phosphoproteome knowledge of the human brain by detecting novel phosphorylation sites on neuronal proteins and identifying differentially phosphorylated brain proteins during HIV infection. Kinases that lead to unusual phosphorylations could be therapeutic targets for the treatment of HIV-associated neurocognitive disorders.

Phosphoproteomics analysis of a clinical Mycobacterium tuberculosis Beijing isolate: expanding the mycobacterial phosphoproteome catalog.

Reversible protein phosphorylation, regulated by protein kinases and phosphatases, mediates a switch between protein activity and cellular pathways that contribute to a large number of cellular processes. The Mycobacterium tuberculosis genome encodes 11 Serine/Threonine kinases (STPKs) which show close homology to eukaryotic kinases. This study aimed to elucidate the phosphoproteomic landscape of a clinical isolate of M. tuberculosis. We performed a high throughput mass spectrometric analysis of proteins extracted from an early-logarithmic phase culture. Whole cell lysate proteins were processed using the filter-aided sample preparation method, followed by phosphopeptide enrichment of tryptic peptides by strong cation exchange (SCX) and Titanium dioxide (TiO2) chromatography. The MaxQuant quantitative proteomics software package was used for protein identification. Our analysis identified 414 serine/threonine/tyrosine phosphorylated sites, with a distribution of S/T/Y sites; 38% on serine, 59% on threonine and 3% on tyrosine; present on 303 unique peptides mapping to 214 M. tuberculosis proteins. Only 45 of the S/T/Y phosphorylated proteins identified in our study had been previously described in the laboratory strain H37Rv, confirming previous reports. The remaining 169 phosphorylated proteins were newly identified in this clinical M. tuberculosis Beijing strain. We identified 5 novel tyrosine phosphorylated proteins. These findings not only expand upon our current understanding of the protein phosphorylation network in clinical M. tuberculosis but the data set also further extends and complements previous knowledge regarding phosphorylated peptides and phosphorylation sites in M. tuberculosis.

N- and O-Glycosylation in the Murine Synaptosome

We present the first large scale study characterizing both N- and O-linked glycosylation in a site-specific manner on hundreds of proteins. We demonstrate that a lectin-affinity fractionation step using wheat germ agglutinin enriches not only peptides carrying intracellular O-GlcNAc, but also those bearing ER/Golgi-derived N- and O-linked carbohydrate structures. Liquid chromatography-MS (LC/MS) analysis with high accuracy precursor mass measurements and high sensitivity ion trap electron-transfer dissociation (ETD) were utilized for structural characterization of glycopeptides. Our results reveal both the identity of the precise sites of glycosylation and information on the oligosaccharide structures possible on these proteins. We report a novel iterative approach that allowed us to interpret the ETD data set directly without making prior assumptions about the nature and distribution of oligosaccharides present in our glycopeptide mixture. Over 2500 unique N- and O-linked glycopeptides were identified on 453 proteins. The extent of microheterogeneity varied extensively, and up to 19 different oligosaccharides were attached at a given site. We describe the presence of the well-known mucin-type structures for O-glycosylation, an EGF-domain-specific fucosylation and a rare O-mannosylation on the transmembrane phosphatase Ptprz1. Finally, we identified three examples of O-glycosylation on tyrosine residues.

Dynamic Changes in Ribosome-Associated Proteome and Phosphoproteome During Deoxynivalenol-Induced Translation Inhibition and Ribotoxic Stress

Deoxynivalenol (DON), a trichothecene mycotoxin produced by Fusarium that commonly contaminates cereal-based food, interacts with the ribosome to cause translation inhibition and activate stress kinases in mononuclear phagocytes via the ribotoxic stress response (RSR). The goal of this study was to test the hypothesis that the ribosome functions as a platform for spatiotemporal regulation of translation inhibition and RSR. Specifically, we employed stable isotope labeling of amino acids in cell culture (SILAC)-based proteomics to quantify the early (≤ 30 min) DON-induced changes in ribosome-associated proteins in RAW 264.7 murine macrophage. Changes in the proteome and phosphoproteome were determined using off-gel isoelectric focusing and titanium dioxide chromatography, respectively, in conjunction with LC-MS/MS. Following exposure of RAW 264.7 to a toxicologically relevant concentration of DON (250 ng/ml), we observed an overall decrease in translation-related proteins interacting with the ribosome, concurrently with a compensatory increase in proteins that mediate protein folding, biosynthesis, and cellular organization. Alterations in the ribosome-associated phosphoproteome reflected proteins that modulate translational and transcriptional regulation, and others that converged with signaling pathways known to overlap with phosphorylation changes characterized previously in intact RAW 264.7 cells. These results suggest that the ribosome plays a central role as a hub for association and phosphorylation of proteins involved in the coordination of early translation inhibition as well as recruitment and maintenance of stress-related proteins-both of which enable cells to adapt and respond to ribotoxin exposure. This study provides a template for elucidating the molecular mechanisms of DON and other ribosome-targeting agents.

Optimization of Enrichment Conditions on TiO2Chromatography Using Glycerol As an Additive Reagent for Effective Phosphoproteomic Analysis

Metal oxide affinity chromatography (MOAC) represented by titanium dioxide (TiO2) chromatography has been used for phosphopeptide enrichment from cell lysate digests prior to mass spectrometry. For in-depth phosphoproteomic analysis, it is important for MOAC to achieve high phosphopeptide enrichment efficiency by optimizing purification conditions. However, there are some differences in phosphopeptide selectivity and specificity enriched by various TiO2 materials and procedures. Here, we report that binding/wash buffers containing polyhydric alcohols, such as glycerol, markedly improve phosphopeptide selectivity from complex peptide mixtures. In addition, the elution conditions combined with secondary amines, such as bis-Tris propane, made it possible to recover phosphopeptides with highly hydrophobic properties and/or longer peptide lengths. To assess the practical applicability of our improved method, we confirmed using PC3 prostate cancer cells. By combining the hydrophilic interaction chromatography (HILIC) with the optimized TiO2 enrichment method prior to LC-MS/MS analysis, over 8300 phosphorylation sites and 2600 phosphoproteins were identified. Additionally, some dephosphorylations of those were identified by treatment with dasatinib for a kinase inhibitor. These results indicate that our method is applicable to understanding the profiling of kinase inhibitors such as anticancer compounds, which will be useful for drug discovery and development.

Early Phosphoproteomic Changes in the Mouse Spleen During Deoxynivalenol-Induced Ribotoxic Stress

The trichothecene mycotoxin deoxynivalenol (DON) targets the innate immune system and is of public health significance because of its frequent presence in human and animal food. DON-induced proinflammatory gene expression and apoptosis in the lymphoid tissue have been associated with a ribotoxic stress response (RSR) that involves rapid phosphorylation of mitogen-activated protein kinases (MAPKs). To better understand the relationship between protein phosphorylation and DON's immunotoxic effects, stable isotope dimethyl labeling-based proteomics in conjunction with titanium dioxide chromatography was employed to quantitatively profile the immediate (≤ 30min) phosphoproteome changes in the spleens of mice orally exposed to 5mg/kg body weight DON. A total of 90 phosphoproteins indicative of novel phosphorylation events were significantly modulated by DON. In addition to critical branches and scaffolds of MAPK signaling being affected, DON exposure also altered phosphorylation of proteins that mediate phosphatidylinositol 3-kinase/AKT pathways. Gene ontology analysis revealed that DON exposure affected biological processes such as cytoskeleton organization, regulation of apoptosis, and lymphocyte activation and development, which likely contribute to immune dysregulation associated with DON-induced RSR. Consistent with these findings, DON impacted phosphorylation of proteins within diverse immune cell populations, including monocytes, macrophages, T cells, B cells, dendritic cells, and mast cells. Fuzzy c-means clustering analysis further indicated that DON evoked several distinctive temporal profiles of regulated phosphopeptides. Overall, the findings from this investigation can serve as a template for future focused exploration and modeling of cellular responses associated with the immunotoxicity evoked by DON and other ribotoxins.

Large-scale analysis of protein phosphorylation in Populus leaves

Protein phosphorylation is a key regulatory factor in all aspects of plant biology; most regulatory pathways are governed by the reversible phosphorylation of proteins. To better understand the role that phosphorylated proteins play in a woody model plant, we performed a systemic analysis of the phosphoproteome from Populus leaves using high accuracy NanoLC–MS/MS in combination with biochemical enrichments using strong cation exchange chromatography and titanium dioxide chromatography. We identified 104 phosphopeptides from 94 phosphoproteins and determined 111 phosphorylation sites including 93 occurring on serine residues and 18 on threonine residues. The identified phosphoproteins are involved in a wide variety of metabolic processes. Among these identified phosphoproteins, 68 phosphorylation sites (72 %) were located outside of conserved domains. The identified phosphopeptides share a common phosphorylation motif of pS/pT-P/D-S/A. These data suggest that the Populus metabolism and gene regulation machinery are major targets of phosphorylation. To our knowledge, this is the first gel-free, large-scale phosphoproteomics analysis in woody plants. The identified phosphorylation sites will be a valuable resource for many fields of plant biology, and information gained from the study will provide a better understanding of protein phosphorylation.

Global protein phosphorylation dynamics during deoxynivalenol-induced ribotoxic stress response in the macrophage

Deoxynivalenol (DON), a trichothecene mycotoxin produced by Fusarium that commonly contaminates food, is capable of activating mononuclear phagocytes of the innate immune system via a process termed the ribotoxic stress response (RSR). To encapture global signaling events mediating RSR, we quantified the early temporal (≤30min) phosphoproteome changes that occurred in RAW 264.7 murine macrophage during exposure to a toxicologically relevant concentration of DON (250ng/mL). Large-scale phosphoproteomic analysis employing stable isotope labeling of amino acids in cell culture (SILAC) in conjunction with titanium dioxide chromatography revealed that DON significantly upregulated or downregulated phosphorylation of 188 proteins at both known and yet-to-be functionally characterized phosphosites. DON-induced RSR is extremely complex and goes far beyond its prior known capacity to inhibit translation and activate MAPKs. Transcriptional regulation was the main target during early DON-induced RSR, covering over 20% of the altered phosphoproteins as indicated by Gene Ontology annotation and including transcription factors/cofactors and epigenetic modulators. Other biological processes impacted included cell cycle, RNA processing, translation, ribosome biogenesis, monocyte differentiation and cytoskeleton organization. Some of these processes could be mediated by signaling networks involving MAPK-, NFκB-, AKT- and AMPK-linked pathways. Fuzzy c-means clustering revealed that DON-regulated phosphosites could be discretely classified with regard to the kinetics of phosphorylation/dephosphorylation. The cellular response networks identified provide a template for further exploration of the mechanisms of trichothecenemycotoxins and other ribotoxins, and ultimately, could contribute to improved mechanism-based human health risk assessment.

Modulation of Protein Phosphorylation, N-Glycosylation and Lys-Acetylation in Grape (Vitis vinifera) Mesocarp and Exocarp Owing to Lobesia botrana Infection

Grapevine (Vitis vinifera) is an economically important fruit crop that is subject to many types of insect and pathogen attack. To better elucidate the plant response to Lobesia botrana pathogen infection, we initiated a global comparative proteomic study monitoring steady-state protein expression as well as changes in N-glycosylation, phosphorylation, and Lys-acetylation in control and infected mesocarp and exocarp from V. vinifera cv Italia. A multi-parallel, large-scale proteomic approach employing iTRAQ labeling prior to three peptide enrichment techniques followed by tandem mass spectrometry led to the identification of a total of 3059 proteins, 1135 phosphorylation sites, 323 N-linked glycosylation sites and 138 Lys-acetylation sites. Of these, we could identify changes in abundance of 899 proteins. The occupancy of 110 phosphorylation sites, 10 N-glycosylation sites and 20 Lys-acetylation sites differentially changed during L. botrana infection. Sequence consensus analysis for phosphorylation sites showed eight significant motifs, two of which containing up-regulated phosphopeptides (X-G-S-X and S-X-X-D) and two containing down-regulated phosphopeptides (R-X-X-S and S-D-X-E) in response to pathogen infection. Topographical distribution of phosphorylation sites within primary sequences reveal preferential phosphorylation at both the N- and C termini, and a clear preference for C-terminal phosphorylation in response to pathogen infection suggesting induction of region-specific kinase(s). Lys-acetylation analysis confirmed the consensus X-K-Y-X motif previously detected in mammals and revealed the importance of this modification in plant defense. The importance of N-linked protein glycosylation in plant response to biotic stimulus was evident by an up-regulated glycopeptide belonging to the disease resistance response protein 206. This study represents a substantial step toward the understanding of protein and PTMs-mediated plant-pathogen interaction shedding light on the mechanisms underlying the grape infection.

Selective Enrichment of Endogenous Serum Phosphopeptides with Ultrafiltration Membrane Intercept-Titanium Dioxide Chromatography Coupled with Nano Ultra-High-Pressure Liquid Chromatography-Quadrupole-Time of Flight Mass Spectrometry

Abstract A novel method coupling ultrafiltration membrane with titanium dioxide (TiO 2 ) chromatography was developed to remove large amounts of high-molecular-weight compounds and enrich endogenous serum phosphopeptides. The enriched endogenous serum phosphopeptides were characterized by nano ultra-high-pressure liquid chromatography-quadrupole-flight time mass spectrometry (nano UPLC-Q-TOF MS). Up to 18 endogenous phosphopeptides could be successfully identified from the serum of lung cancer. The established method improved the sensitivity and efficiency of detection of serum phosphopeptides, which might be considered a milestone in tumour biomarker discovery.

Multidimensional Strategy for Sensitive Phosphoproteomics Incorporating Protein Prefractionation Combined with SIMAC, HILIC, and TiO2 Chromatography Applied to Proximal EGF Signaling

Comprehensive enrichment and fractionation is essential to obtain a broad coverage of the phosphoproteome. This inevitably leads to sample loss, and thus, phosphoproteomics studies are usually only performed on highly abundant samples. Here, we present a comprehensive phosphoproteomics strategy applied to 400 μg of protein from EGF-stimulated HeLa cells. The proteins are separated into membrane and cytoplasmic fractions using sodium carbonate combined with ultracentrifugation. The phosphopeptides were separated into monophosphorylated and multiphosphorylated pools using sequential elution from IMAC (SIMAC) followed by hydrophilic interaction liquid chromatography of the mono- and nonphosphorylated peptides and subsequent titanium dioxide chromatography of the HILIC fractions. This strategy facilitated the identification of >4700 unique phosphopeptides, while 636 phosphosites were changing following short-term EGF stimulation, many of which were not previously known to be involved in EGFR signaling. We further compared three different data processing programs and found large differences in their peptide identification rates due to different implementations of recalibration and filtering. Manually validating a subset of low-scoring peptides exclusively identified using the MaxQuant software revealed a large percentage of false positive identifications. This indicates that, despite having highly accurate precursor mass determination, peptides with low fragment ion scores should not automatically be reported in phosphoproteomics studies.

Selective enrichment of glycopeptides for mass spectrometry analysis using C18 fractionation and titanium dioxide chromatography

Comprehensive glycoprotein characterization based on mass spectrometry (MS) is challenging because of low concentration of glycopeptides and suppression effect of abundant non-glycosylated peptides in MS. Therefore, it is vital to enrich glycopeptides before MS analysis. A new method was developed to selectively enrich glycopeptides from complex sample by coupling C18 fractionation with titanium dioxide (TiO(2)) enrichment. The new method allows to selectively enrich N-linked glycopeptides with various glycan forms and different sequence lengths. Compared with single TiO(2) method, the established method demonstrated higher glycopeptide selectivity and higher glycosylation heterogeneity coverage. Further application of this method to mixture of non-glycosylated protein and glycoprotein digests at different levels reveals the feasibility of enrichment of tryptic glycopeptides from simple proteomics samples.

Titanium dioxide as chemo-affinity chromatographic sorbent of biomolecular compounds — Applications in acidic modification-specific proteomics

Characterization of the biomolecules involved in molecular processes occurring in biological systems such as the human cell remains central to biology, biotechnology, and medicine. One of the preferred methods of selectively purifying specific classes of biomolecules from complex biological matrices for further characterization is affinity chromatography, which relies on the specific interaction between an analyte in solution and a solid adsorbent. Titanium dioxide-based affinity chromatography has proven to be a versatile tool in enrichment of various compounds such as phosphorylated biomolecules due to its unique ion and ligand exchange properties and high stability towards pH and temperature. Recently, titanium dioxide chromatography was introduced in proteomics as a highly specific method for enriching phosphorylated peptides - a method, which has been widely adapted by the field of phosphoproteomics. Additional studies have shown the potential of this sorbent in purification of other acidic post-translational modified peptides, such as sialylated glycopeptides, thereby targeting the sialiome, defined as the content of sialic acid containing glycoproteins of a given cell, body fluid or tissue. The development of TiO(2)-based chromatographic strategies for separation of various biomolecules from its introduction for small molecules more than 20 years ago until recent proteomics applications today will be reviewed here.

Protein Phosphorylation Is a Key Event of Flagellar Disassembly Revealed by Analysis of Flagellar Phosphoproteins during Flagellar Shortening in Chlamydomonas

Cilia are disassembled prior to cell division, which is proposed to regulate proper cell cycle progression. The signaling pathways that regulate cilia disassembly are not well-understood. Recent biochemical and genetic data demonstrate that protein phosphorylation plays important roles in cilia disassembly. Here, we analyzed the phosphoproteins in the membrane/matrix fraction of flagella undergoing shortening as well as flagella from steady state cells of Chlamydomonas. The phosphopeptides were enriched by a combination of IMAC and titanium dioxide chromatography with a strategy of sequential elution from IMAC (SIMAC) and analyzed by tandem mass spectrometry. A total of 224 phosphoproteins derived from 1296 spectral counts of phosphopeptides were identified. Among the identified phosphoproteins are flagellar motility proteins such as outer dynein arm, intraflagellar transport proteins as well as signaling molecules including protein kinases, phosphatases, G proteins, and ion channels. Eighty-nine of these phosphoproteins were only detected in shortening flagella, whereas 29 were solely in flagella of steady growing cells, indicating dramatic changes of protein phosphorylation during flagellar shortening. Our data indicates that protein phosphorylation is a key event in flagellar disassembly, and paves the way for further study of flagellar assembly and disassembly controlled by protein phosphorylation.