Fractionation Of Phosphopeptides Research Articles

Simultaneous enrichment and sequential separation of O-linked glycopeptides and phosphopeptides with immobilized titanium (IV) ion affinity chromatography materials

Protein phosphorylation and O-linked glycosylation are two critical posttranslational modifications (PTMs) and they both target identical amino acid residues, generating “crosstalk”. Analysis of the crosstalk between these PTMs is crucial for deciphering their biological functions. Although several strategies have been established to enrich N-linked glycopeptides and phosphopeptides simultaneously, they are not appropriate for O-linked glycopeptides and phosphopeptides. In this study, we established a method for the simultaneous enrichment and sequential elution of O-linked glycopeptides and phosphopeptides into two fractions using commercially available immobilized titanium (IV) ion affinity chromatography (Ti4+-IMAC) materials. The established method exhibited high selectivity, repeatability, and recovery of the targeted peptides. Particularly, the overlap between the O-linked glycopeptide and phosphopeptide fractions was very low (∼3%). The application of this method to cell lysates of the colon cancer HT29 cell line resulted in a comparable number of enriched phosphopeptides as the coeluted method. However, the number of identified O-linked glycopeptides with our established method was 9.7-fold higher than that with the coelution method. Our study demonstrated that the established strategy was beneficial for the simultaneous analysis of O-linked glycopeptides and phosphopeptides, which might facilitate the study of the biological function of PTM crosstalk.

Comprehensive Evaluation of Different TiO2-Based Phosphopeptide Enrichment and Fractionation Methods for Phosphoproteomics.

Protein phosphorylation is an essential post-translational modification that regulates multiple cellular processes. Due to their low stoichiometry and ionization efficiency, it is critical to efficiently enrich phosphopeptides for phosphoproteomics. Several phosphopeptide enrichment methods have been reported; however, few studies have comprehensively compared different TiO2-based phosphopeptide enrichment methods using complex proteomic samples. Here, we compared four TiO2-based phosphopeptide enrichment methods that used four non-phosphopeptide excluders (glutamic acid, lactic acid, glycolic acid, and DHB). We found that these four TiO2-based phosphopeptide enrichment methods had different enrichment specificities and that phosphopeptides enriched by the four methods had different physicochemical characteristics. More importantly, we discovered that phosphopeptides had a higher deamidation ratio than peptides from cell lysate and that phosphopeptides enriched using the glutamic acid method had a higher deamidation ratio than the other three methods. We then compared two phosphopeptide fractionation methods: ammonia- or TEA-based high pH reversed-phase (HpH-RP). We found that fewer phosphopeptides, especially multi-phosphorylated peptides, were identified using the ammonia-based method than using the TEA-based method. Therefore, the TEA-based HpH-RP fractionation method performed better than the ammonia method. In conclusion, we comprehensively evaluated different TiO2-based phosphopeptide enrichment and fractionation methods, providing a basis for selecting the proper protocols for comprehensive phosphoproteomics.

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.

Phosphoproteomic Analysis of Signaling Pathways in Lymphomas.

Cell fate decisions are controlled by complex signal transduction processes that transmit information via posttranslational protein modifications such as phosphorylation. In lymphoma, as in other cancer types, these signaling networks are often dysregulated and thus contribute to malignant transformation and tumor maintenance. For example, B-cell antigen receptor signals are rewired in certain lymphoma types, such as diffuse large B-cell lymphomas, to promote cell growth and survival of the malignant cell clones. Hence, global elucidation of such intricate signaling networks is important for an improved understanding of the biology of these tumors and the identification of target proteins for therapeutic purposes.We describe here a mass spectrometry-based phosphoproteomic approach for characterization of intracellular signaling events and their dynamics. This integrated phosphoproteomic technology combines phosphopeptide enrichment and fractionation with liquid-chromatography-coupled mass spectrometry for the site-specific mapping and quantification of thousands of phosphorylation events in a given cell type. Such global signaling analyses provide valuable insights into oncogenic signaling networks and can inform drug development efforts.

Tip-Based Fractionation of Batch-Enriched Phosphopeptides Facilitates Easy and Robust Phosphoproteome Analysis.

The identification of large numbers of phosphopeptides from complex samples largely relies on sample fractionation to reduce complexity and allow using large amounts of starting material. For such experiments, commonly fractionation of whole cell lysate digests followed by enrichment of phosphopeptides from the single fractions is performed. We evaluated the tip-based fractionation of batch-enriched phosphopeptides as an alternative method. We compared three tip-based fractionation methods employing strong cation exchange (SCX), strong anion exchange (SAX), and C18 material for basic reversed-phase (BRP) fractionation using HeLa whole cell lysate digests. We show that SCX tips are superior to BRP and SAX tips due to a more efficient retention and distribution of phosphopeptides as well as a better resolution. Furthermore, we show that tip-based fractionation results in a similar performance as fractionation followed by phosphopeptide enrichment of the single fractions and outperforms analysis of unfractionated phosphopeptide-enriched samples with long chromatography gradients. Our fractionation approach using SCX tips is straightforward, reproducible, and requires a fraction of time, effort, and instrumentation compared to those of the fractionation of whole cell lysate digests with subsequent enrichment of phosphopeptides from the single fractions.

Phosphotyrosine Biased Enrichment of Tryptic Peptides from Cancer Cells by Combining pY-MIP and TiO2 Affinity Resins.

Protein phosphorylation at distinct tyrosine residues (pY) is essential for fast, specific, and accurate signal transduction in cells. Enrichment of pY-containing peptides derived from phosphoproteins is commonly facilitated by use of immobilized anti-pY antibodies prior to phosphoproteomics analysis by mass spectrometry. We here report on an alternative approach for pY-peptide enrichment using inexpensive pY-imprinted polymer (pY-MIP). We assessed by mass spectrometry the performance of pY-MIP for enrichment and sequencing of phosphopeptides obtained by tryptic digestion of protein extracts from HeLa cells. The combination of pY-MIP- and TiO2-based phosphopeptide enrichment provided more than 90% selectivity for phosphopeptides. Mass spectrometry signal intensities were enhanced for most pY-phosphopeptides (approximately 70%) when using the pY-MIP-TiO2 combination as compared to TiO2 alone. pY constituted up to 8% of the pY-MIP-TiO2-enriched phosphopeptide fractions. The pY-MIP-TiO2 and the TiO2 protocols yielded comparable numbers of distinct phosphopeptides, 1693 and 1842, respectively, from microgram levels of peptide samples. Detailed analysis of physicochemical properties of pY-MIP-TiO2-enriched phosphopeptides demonstrated that this protocol retrieved phosphopeptides that tend to be smaller (<24 residues), less acidic, and almost exclusively monophosphorylated, as compared to TiO2 alone. These unique properties render the pY-MIP-based phosphopeptide enrichment technique an attractive alternative for applications in phosphoproteomics research.

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.

Fast and easy phosphopeptide fractionation by combinatorial ERLIC-SCX solid-phase extraction for in-depth phosphoproteome analysis.

Mass spectrometry-based phosphoproteomic analysis is a powerful method for gaining a global, unbiased understanding of cellular signaling. Its accuracy and comprehensiveness stands or falls with the quality and choice of the applied phosphopeptide prefractionation strategy. This protocol covers a powerful but simple and rapid strategy for phosphopeptide prefractionation. The combinatorial use of two distinct chromatographic techniques that address the inverse physicochemical properties of peptides allows for superior fractionation efficiency of multiple phosphorylated peptides. In the first step, multiphosphorylated peptides are separated according to the number of negatively charged phosphosites by electrostatic repulsion-hydrophilic interaction chromatography (ERLIC). A subsequent strong cation exchange (SCX) step separates mostly singly phosphorylated peptides in the ERLIC flow-through according to their positive charge. The presented strategy is inexpensive and adaptable to large and small amounts of starting material, and it allows highly multiplexed sample preparation. Because of its implementation as solid-phase extraction, the entire workflow takes only 2 h to complete.

Phosphoproteomic Analysis of Protein Phosphorylation Networks in Tetrahymena thermophila, a Model Single-celled Organism

Tetrahymena thermophila is a widely used unicellular eukaryotic model organism in biological research and contains more than 1000 protein kinases and phosphatases with specificity for Ser/Thr/Tyr residues. However, only a few dozen phosphorylation sites in T. thermophila are known, presenting a major obstacle to further understanding of the regulatory roles of reversible phosphorylation in this organism. In this study, we used high-accuracy mass-spectrometry-based proteomics to conduct global and site-specific phosphoproteome profiling of T. thermophila. In total, 1384 phosphopeptides and 2238 phosphorylation sites from 1008 T. thermophila proteins were identified through the combined use of peptide prefractionation, TiO2 enrichment, and two-dimensional LC-MS/MS analysis. The identified phosphoproteins are implicated in the regulation of various biological processes such as transport, gene expression, and mRNA metabolic process. Moreover, integrated analysis of the T. thermophila phosphoproteome and gene network revealed the potential biological functions of many previously unannotated proteins and predicted some putative kinase-substrate pairs. Our data provide the first global survey of phosphorylation in T. thermophila using a phosphoproteomic approach and suggest a wide-ranging regulatory scope of this modification. The provided dataset is a valuable resource for the future understanding of signaling pathways in this important model organism.

Global Analysis of Phosphoproteome Regulation by the Ser/Thr Phosphatase Ppt1 in Saccharomyces cerevisiae

Even though protein phosphatases are key regulators of signal transduction, their cellular mechanisms of action are poorly understood. Here, we undertook a large-scale proteomics survey to identify cellular protein targets of a serine/threonine phosphatase. We used SILAC-based quantitative MS to measure differences in protein expression and phosphorylation upon ablation of the serine/threonine phosphatase Ppt1 in Saccharomyces cerevisiae. Phosphopeptide fractionation by strong cation exchange chromatography combined with immobilized metal affinity chromatography (IMAC) enrichment enabled quantification of more than 8000 distinct phosphorylation sites in Ppt1 wild-type versus Ppt1-deficient yeast cells. We further quantified the relative expression of 1897 yeast proteins and detected no major protein changes accompanying Ppt1 deficiency. Notably, we found 33 phosphorylation sites to be significantly and reproducibly up-regulated while no phosphorylation events were repressed in cells lacking Ppt1. Ppt1 acted on its cellular target proteins in a sequence- and site-specific fashion. Several of the regulated phosphoproteins were involved in the response to heat stress in agreement with known Ppt1 functions. Additionally, biosynthetic enzymes were particularly prominent among Ppt1-regulated phosphoproteins, pointing to unappreciated roles of Ppt1 in the control of various metabolic functions. These results demonstrate the utility of large-scale and quantitative phosphoproteomics to identify cellular sites of serine/threonine phosphatase action in an unbiased manner.

Phosphopeptide enrichment and fractionation by using Click OEG-CD matrix

Reversible protein phosphorylation regulates many significant cellular processes. Identification of phosphorylation sites is vital to elucidate their biofunctions. To aid in phosphoproteome characterization, several selective enrichment methods have been developed, including immobilized metal-ion affinity chromatography (IMAC) and titanium dioxide (TiO2). However, the high pH elution step applied in these two methods, which tends to degrade phosphopeptides. In order to improve phosphopeptide enrichment efficiency, a hydrophilic interaction liquid chromatography (HILIC) based material, cyclodextrin (CD) bonded silica (Click OEG-CD), was synthesized in our group and applied to phosphopeptide enrichment and fractionation. Taking tryptic digest of standard protein α-casein as model sample, the performance of Click OEG-CD in phosphopeptide isolation was investigated. It was found that both the acetonitrile and salt concentrations in mobile phase influence the phosphopeptide enrichment selectivity of the matrix. Under optimized enrichment condition, Click OEG-CD has a similar phosphopeptide enrichment selectivity as commercial TiO2. Meanwhile, phosphopeptides with same charges could be fractionated according to hydrophilicity difference on Click OEG-CD under acetonitrile gradient. Different selectivity is proved with Click OEG-CD from conventional SAX. We demonstrate that Click OEG-CD is an efficient matrix in phosphopeptide enrichment and fractionation.

MzServer: Web-based Programmatic Access for Mass Spectrometry Data Analysis

Continued progress toward systematic generation of large-scale and comprehensive proteomics data in the context of biomedical research will create project-level data sets of unprecedented size and ultimately overwhelm current practices for results validation that are based on distribution of native or surrogate mass spectrometry files. Moreover, the majority of proteomics studies leverage discovery-mode MS/MS analyses, rendering associated data-reduction efforts incomplete at best, and essentially ensuring future demand for re-analysis of data as new biological and technical information become available. Based on these observations, we propose to move beyond the sharing of interpreted spectra, or even the distribution of data at the individual file or project level, to a system much like that used in high-energy physics and astronomy, whereby raw data are made programmatically accessible at the site of acquisition. Toward this end we have developed a web-based server (mzServer), which exposes our common API (mzAPI) through very intuitive (RESTful) uniform resource locators (URL) and provides remote data access and analysis capabilities to the research community. Our prototype mzServer provides a model for lab-based and community-wide data access and analysis.

Nanodiamond-based two-step sampling of multiply and singly phosphorylated peptides for MALDI-TOF mass spectrometry analysis

Simultaneous detection of multiply and singly phosphorylated peptides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is challenging because of suppression effects during ionization. In oder to overcome this problem, this study presents a new approach to improve the detection of phosphopeptides by stepwise enrichment using polyarginine-coated (PA-coated) and titanium dioxide-coated (TiO(2)-coated) nanodiamonds for fractionation of multiply and singly phosphorylated peptides prior to on-probe MALDI MS analysis. The feasibility of this approach was demonstrated using synthetic peptides containing different numbers of phosphate groups, tryptic digests of α-casein, β-casein, and complex protein mixtures. The high specificity of the approach is shown in its effective enrichment and fractionation of phosphopeptides from the digest of β-casein and bovine serum albumin at a molar ratio as low as 1 : 1000, which out-performs the commercial Fe(3+)-IMAC and TiO(2) isolation kits. It offers a simple and effective alternative for the fractionation and identification of multiply and singly phosphorylated peptides by MALDI MS and allows for deduction of more information from limited starting materials.

Fractionation of phosphopeptides on strong anion‐exchange capillary trap column for large‐scale phosphoproteome analysis of microgram samples

It is one of the key issues to develop powerful fractionating method to increase the identification of the low-abundance phosphopeptides. In this study, a semi-online 2-D LC separation strategy based on three-step fractionation of the enriched peptides on strong anion-exchange trap column was developed. It was demonstrated that the sensitivity and phosphoproteome coverage obtained by this fractionating method with strong anion-exchange trap column is much higher than those by the conventional methods based on C18 trap column. In addition, when the same amount of sample was loaded, the number of identified phosphopeptides had increased 108%. Combination of this three-step fractionation method with RPLC-MS/MS analysis by 300 min RP-gradient separation was applied to phosphoproteome analysis of human liver proteins, and 853 unique phosphopeptides was positively identified from 500 microg tryptic digest of human liver proteins. After three cycles' consecutive analyses, 1554 unique phosphopeptides and 1566 phosphorylated sites were totally identified from 735 phosphorylated proteins at a false discovery rate of <1% in about 54 h of analysis time.

Metal ion-mobilizing additives for comprehensive detection of femtomole amounts of phosphopeptides by reversed phase LC-MS

It is hypothesized that metal ion-mediated adsorption of phosphorylated peptides on stationary phases of LC-columns is the major cause for their frequently observed poor detection efficiency in LC-MS. To study this phenomenon in more detail, sample solutions spiked with metal ion-mobilizing additives were analyzed by reversed phase μLC-ICP-MS or nanoLC-ESI-MS. Using μLC-ICP-MS, metal ions were analyzed directly as atomic ions. Using electrospray ionization, either metal ion chelates or phosphopeptide standard mixtures injected in subpicomole amounts were analyzed. Deferoxamine, imidazole, ascorbate, citrate, EDTA, and the tetrapeptide pSpSpSpS were tested as sample additives for the interlinked purposes of metal ion-mobilization and improvement of phosphopeptide recovery. Iron probably represents the major metal ion contamination of reversed phase columns. Based on the certified iron level in LC-grade solvents, a daily metal ion load of >10pmol was estimated for typical nanoLC flow rates. In addition, phosphopeptide fractions from IMAC columns were identified as source for metal ion contamination of the LC column, as demonstrated for Ga(3+)-IMAC. The three metal ion-chelating additives, EDTA, citrate and pSpSpSpS, were found to perform best for improving the LC recovery of multiply phosphorylated peptides injected at subpicomole amounts. The benefits of metal ion-mobilizing LC (mimLC) characterized by metal ion complexing sample additives is demonstrated for three different instrumental setups comprising (a) a nanoUPLC-system with direct injection on the analytical column, (b) a nanoLC system with inclusion of a trapping column, and (c) the use of a HPLC-Chip system with integrated trapping and analytical column.

A Proteome-wide Analysis of Kinase-Substrate Network in the DNA Damage Response

The DNA damage checkpoint, consisting of an evolutionarily conserved protein kinase cascade, controls the DNA damage response in eukaryotes. Knowledge of the in vivo substrates of the checkpoint kinases is essential toward understanding their functions. Here we used quantitative mass spectrometry to identify 53 new and 34 previously known targets of Mec1/Tel1, Rad53, and Dun1 in Saccharomyces cerevisiae. Analysis of replication protein A (RPA)-associated proteins reveals extensive physical interactions between RPA-associated proteins and Mec1/Tel1-specific substrates. Among them, multiple subunits of the chromatin remodeling complexes including ISW1, ISW2, INO80, SWR1, RSC, and SWI/SNF are identified and they undergo DNA damage-induced phosphorylation by Mec1 and Tel1. Taken together, this study greatly expands the existing knowledge of the targets of DNA damage checkpoint kinases and provides insights into the role of RPA-associated chromatins in mediating Mec1 and Tel1 substrate phosphorylation in vivo.

An integrated global strategy for cell lysis, fractionation, enrichment and mass spectrometric analysis of phosphorylated peptides

Recently, the field of phosphoproteomics has progressed to the point where thousands of protein phosphorylations can be analyzed simultaneously and used to address significant biological questions. However, several challenges still exist in current LC-MS/MS-based phosphoproteomics methods. Among these are the increased dynamic range of phosphoproteomics samples (due to low stoichiometry of most protein phosphorylations), insufficient inhibition of phosphatase activity, and neutral losses which occur during phosphopeptide fragmentation by MS(n). Here we present an improved method, free of conventional phosphatase inhibitors, for sample treatment to minimize phosphatase activity and improve the efficiency of phosphopeptide enrichment. We also present a solution-based IEF method for phosphopeptide fractionation and explore the utility of various fragmentation methods for identifying phosphopeptides and localizing phosphorylation sites.

Effect of Peptide-to-TiO2 Beads Ratio on Phosphopeptide Enrichment Selectivity

Titanium dioxide (TiO(2)) has been proven to be a highly efficient strategy and widely used for phosphopeptide enrichment. Many advances have been made recently, including online/offline mode and optimization of sample loading/elution buffer; however, beads usage has rarely been explored. In the current study, we found that the peptide-to-TiO(2) beads ratio was a significant factor for phosphopeptide enrichment, and insufficient or excessive beads could decrease the selectivity. Specifically, for HeLa total cell lysate, the optimum peptide-to-beads ratio is about 1:2-1:8 (mass/mass) to obtain the highest enrichment selectivity and the maximum phosphopeptides identification with single incubation. Pre-experiments are recommended to decide an optimum peptide-to-TiO(2) beads ratio when it comes to different samples. Interestingly, deficient beads can help identify much more multiphosphorylated peptides than the optimum peptide-to-beads ratio by consecutive incubations. Therefore, if multiphosphorylated peptides identification is desired, deficient beads amount is preferred. In addition, consecutive incubation using deficient beads could be used as a fractionation of phosphopeptides besides as an enrichment method.

Minimal Information About Sample Preparation for Phosphoproteomics

This guideline describes parameters and conditions involved in phosphopeptide sample preparation. It covers from the description and preparation of the cells and tissues to the fractionation and specific enrichment of phosphopeptides for MS analysis. The guideline is prepared in order to easily cope with many of the experimental designs used in phosphoproteomic studies. The document is subdivided as follows:1. General features2. Sample processing3. Protein Purification/Fractionation4. Peptide Purification/Fractionation5. Phosphopeptide enrichment

Minimal Information About Sample Preparation for Phosphoproteomics

AbstractThis guideline describes parameters and conditions involved in phosphopeptide sample preparation. It covers from the description and preparation of the cells and tissues to the fractionation and specific enrichment of phosphopeptides for MS analysis. The guideline is prepared in order to easily cope with many of the experimental designs used in phosphoproteomic studies. The document is subdivided as follows:1. General features2. Sample processing3. Protein Purification/Fractionation4. Peptide Purification/Fractionation5. Phosphopeptide enrichment