Multi-phosphorylated Peptides Research Articles

Multiphosphorylation-Dependent Recognition of Anti-pS2 Antibodies against RNA Polymerase II C-Terminal Domain Revealed by Chemical Synthesis.

Phosphorylation is a major constituent of the CTD code, which describes the set of post-translational modifications on 52 repeats of a YSPTSPS consensus heptad that orchestrates the binding of regulatory proteins to the C-terminal domain (CTD) of RNA polymerase II. Phospho-specific antibodies are used to detect CTD phosphorylation patterns. However, their recognition repertoire is underexplored due to limitations in the synthesis of long multiphosphorylated peptides. Herein, we describe the development of a synthesis strategy that provides access to multiphosphorylated CTD peptides in high purity without HPLC purification for immobilization onto microtiter plates. Native chemical ligation was used to assemble 12 heptad repeats in various phosphoforms. The synthesis of >60 CTD peptides, 48-90 amino acids in length and containing up to 6 phosphosites, enabled a detailed and rapid analysis of the binding characteristics of different anti-pSer2 antibodies. The three antibodies tested showed positional selectivity with marked differences in the affinity of the antibodies for pSer2-containing peptides. Furthermore, the length of the phosphopeptides allowed a systematic analysis of the multivalent chelate-type interactions. The absence of multivalency-induced binding enhancements is probably due to the high flexibility of the CTD scaffold. The effect of clustered phosphorylation proved to be more complex. Recognition of pSer2 by anti-pSer2-antibodies can be prevented and, perhaps surprisingly, enhanced by the phosphorylation of "bystander" amino acids in the vicinity. The results have relevance for functional analysis of the CTD in cell biological experiments.

Determination of the affinity of biomimetic peptides for uranium through the simultaneous coupling of HILIC to ESI-MS and ICP-MS

Several proteins have been identified in the past decades as targets of uranyl (UO22+) in vivo. However, the molecular interactions responsible for this affinity are still poorly known which requires the identification of the UO22+ coordination sites in these proteins. Biomimetic peptides are efficient chemical tools to characterize these sites. In this work, we developed a dedicated analytical method to determine the affinity of biomimetic, synthetic, multi-phosphorylated peptides for UO22+ and evaluate the effect of several structural parameters of these peptides on this affinity at physiological pH. The analytical strategy was based on the implementation of the simultaneous coupling of hydrophilic interaction chromatography (HILIC) with electrospray ionization mass spectrometry (ESI-MS) and inductively coupled plasma mass spectrometry (ICP-MS). An essential step had been devoted to the definition of the best separation conditions of UO22+ complexes formed with di-phosphorylated peptide isomers and also with peptides of different structure and degrees of phosphorylation. We performed the first separations of several sets of UO22+ complexes by HILIC ever reported in the literature. A dedicated method had then been developed for identifying the separated peptide complexes online by ESI-MS and simultaneously quantifying them by ICP-MS, based on uranium quantification using external calibration. Thus, the affinity of the peptides for UO22+ was determined and made it possible to demonstrate that (i) the increasing number of phosphorylated residues (pSer) promotes the affinity of the peptides for UO22+, (ii) the position of the pSer in the peptide backbone has very low impact on this affinity (iii) and finally the cyclic structure of the peptide favors the UO22+ complexation in comparison with the linear structure. These results are in agreement with those previously obtained by spectroscopic techniques, which allowed to validate the method. Through this approach, we obtained essential information to better understand the mechanisms of toxicity of UO22+ at the molecular level and to further develop selective decorporating agents by chelation.

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.

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.

Dual metal cations coated magnetic mesoporous silica probe for highly selective capture of endogenous phosphopeptides in biological samples.

Forthe first time, dual metal ions (Ti4+-Zr4+) were successfully modified into the channel of magnetic mesoporous silica to obtain an affinity probe for highly selective capture of endogenous phosphopeptides in biological samples. The newly prepared Fe3O4@mSiO2@Ti4+-Zr4+ composites possessed the advantages of ordered mesoporous channels, superparamagnetism, and enhanced affinity properties of dual metal ions of Ti4+ and Zr4+. The phosphopeptide enrichment efficiency of the Fe3O4@mSiO2@Ti4+-Zr4+ composite was investigated, and the result indicated an ultrahigh size exclusive ability (weight ratio of β-casein tryptic digests, BSA, and α-casein protein reached up to 1:1000:1000). Compared tomagnetic affinity probes with single metal ions (Fe3O4@mSiO2@Ti4+, Fe3O4@mSiO2@Zr4+), thecomposite possessed stronger specificity, higher sensitivity, and better efficiency; and more importantly, it showed much enhanced enrichment ability towards both mono- and multi-phosphorylated peptides. Additionally, by utilizing the Fe3O4@mSiO2@Ti4+-Zr4+ affinity probe, a total number of 104 endogenous phosphopeptides including 95 mono-phosphopeptides and 9 multi-phosphopeptides were captured and identified from human saliva, indicating the great potential for the application of the novel probe for the peptidome analysis in the future. Graphic abstract.

Highly sensitive detection of phosphopeptides with superparamagnetic Fe3O4@mZrO2 core-shell microspheres-assisted mass spectrometry

Protein phosphorylation is one of the most important post-translational modifications. It is an active research area to study phosphoproteomics for discovery of disease biomarkers and druggable targets. Here we report the development of superparamagnetic Fe3O4@mZrO2 core-shell microspheres with mesoporous structures for highly efficient enrichment of phosphopeptides. We have demonstrated that the mesoporous ZrO2 layer dramatically improves the selective enrichment of phosphopeptides. Our approach allows for in-situ elution and sensitive identification of both mono-phosphorylated and multi-phosphorylated peptides in MALDI-TOF mass spectrometry, with the detection limit of down to the femtomole range. The target phosphopeptides can reliably be enriched for MS analysis from various complex samples including the spiked protein digests and tumor cell lysates. The Fe3O4@mZrO2 core-shell microspheres promise a useful tool for phosphoproteomics by allowing for highly efficient and selective enrichment of the crucial signaling regulators in a low abundance.

Facile liquid-phase deposition synthesis of titania-coated magnetic sporopollenin for the selective capture of phosphopeptides.

Titania-grafted magnetic sporopollenin is synthesized by the liquid-phase deposition (LPD) technique, characterized by SEM, EDX, and nitrogen adsorption porosimetry, and used for the selective enrichment of phosphorylated peptides. The material is low cost because of easier availability of pollens and has rich surface chemistry which enables strong attachment of titania onto magnetic sporopollenin. The material shows higher selectivity of 1:1000 with β-casein spiked in BSA. Higher sensitivity of 10fmol is recorded for phosphopeptides from standard β-casein digest. Twenty phosphorylated peptides are enriched from milk digest and four endogenous phosphopeptides from diluted human serum. The magnetic property of titania-coated magnetic sporopollenin facilitates the fast and effective isolation of phosphopeptides from complex mixtures through external magnet. The material is finally applied to tryptic digest of rat brain cell lysate for phosphopeptide enrichment where 2718 phosphopeptides are identified by using LC-MS/MS with C18 column. Titania-coated magnetic sporopollenin captures both mono-phosphorylated (2489) and multi-phosphorylated peptides (229) due to strong affinity of TiO2 with phosphates. TiO2-coated magnetic material also shows better enrichment efficiency in comparison to commercial TiO2. Graphical abstract.

Immobilization of titanium dioxide/ions on magnetic microspheres for enhanced recognition and extraction of mono- and multi-phosphopeptides.

The authors are presenting a novel strategy for global phosphoproteome recognition in practical samples. It integrates metal oxide affinity chromatography (MOAC) and immobilization metal ion affinity chromatography (IMAC). This resulted in a kind of titanium dioxide/ion-based multifunctional probe (dubbed T2M). The T2M combines the features of MOAC and IMAC including their recognition preferences towards mono- and multi-phosphorylated peptides. Hence, they exhibit an outstanding recognition capability towards global phosphoproteome, high sensitivity (the limit of detection of which is merely 10 fmol) and excellent specificity in MALDI-TOF MS detection. Their performance is further demonstrated by the identification of the phosphoproteome in non-fat milk and human saliva. By combining T2M with nano LC-MS/MS, remarkable results are obtained in the tryptic digestion of healthy eye lens and cataract lens phosphoproteomes. A total of 658 and 162 phosphopeptides, respectively, were identified. This indicates that phosphorylation and the appearance of cataract can be related to each other. Graphical abstract Schematic presentation of thepreparation of titanium dioxide/ion-based multifunctional magnetic nanomaterials (T2M). The T2M based enrichment protocol exhibits outstanding recognition capability towards global phosphoproteome. This protocol shows great prospect for clarifying mechanism of phosphorylation-related diseases via further information acquisition.

A targeted approach for the synthesis of multi-phosphorylated peptides: a tool for studying the role of phosphorylation patterns in proteins.

Protein phosphorylation barcodes, clusters of several phosphorylation sites within a short unfolded region, control many cellular processes. Existing biochemical methods used to study the roles of these barcodes suffer from low selectivity and provide only qualitative data. Chemically synthesized multiphosphopeptide libraries are selective and specific, but their synthesis is extremely difficult using the current peptide synthesis methods. Here we describe a new microwave assisted approach for synthesizing a library of multiphosphopeptides, using the C-terminus of rhodopsin as a proof of concept. Our approach utilizes multiple protocols for synthesizing libraries of multiphosphopeptides instead of the inefficient single protocol methods currently used. Using our approach we demonstrated the synthesis with up to seven phosphorylated amino acids, sometimes next to each other, an accomplishment that was impractical before. Synthesizing the Rhodopsin derived multiphosphopeptide library enabled dissecting the precise phosphorylation barcode required for the recruitment, activation and modulation of the conformation of Arrestin. Since phosphorylation barcodes modulate the activity of hundreds of GPCRs, synthesizing libraries of multiphosphopeptides is the method of choice for studying their molecular mechanisms of action. Our approach provides an invaluable tool for evaluating how protein phosphorylation barcodes regulate their activity.

Zinc Ion-immobilized Magnetic Microspheres for Enrichment and Identification of Multi-phosphorylated Peptides by Mass Spectrometry.

The selective isolation of phosphorylated peptides and subsequent analysis using mass spectrometry is important for understanding how protein kinase and phosphatase signals can precisely modulate the on/off states of signal transduction pathways. However, the isolation and detection of multi-phosphorylated peptides is still limited due to their distinct affinity to various materials and their poor ionization efficiency. Here, we report a highly efficient and selective enrichment of phosphorylated peptides using binuclear Zn2+-dipicolylamine complex-coated magnetic microspheres (ZnMMs). ZnMMs can utilize the rapid and selective isolation/enrichment of phosphorylated peptides and the subsequent mass spectrometric analysis, given the intrinsic magnetic property of magnetic microspheres and the highly selective binding ability of the binuclear Zn2+-dipicolylamine complex to phosphate groups. α-Casein and β-casein were chosen for a proof-of-concept demonstration. We contemplated that phosphopeptides were selectively isolated and enriched from both the tryptic digests of casein proteins and mixed samples with a high degree of sensitivity by facilitating ZnMMs. Especially, ZnMMs showed high efficiency with multi-phosphopeptides, which are in general difficult to be examined by mass analysis on account of their poor ionization efficiency. For the model protein α, β-casein mixture of the tryptic digest, 17 phosphopeptides were identified with ZnMMs and 82% of the enriched phosphopeptides were multi-phosphorylated peptides, indicating that ZnMMs have excellent enrichment efficiency and strong affinity towards multi-phosphorylated peptides.

Dual-Metal Centered Zirconium-Organic Framework: A Metal-Affinity Probe for Highly Specific Interaction with Phosphopeptides.

The highly specific affinity between probes and phosphopeptides is the fundamental interaction for selective identification of phosphoproteomes that uncover the mechanisms of signal transduction, cell cycle, enzymatic regulation, and gene expression in biological systems. In this study, a metal-affinity probe possessing both interactions of metal oxide affinity chromatography (MOAC) and immobilized metal ion affinity chromatography (IMAC) was facilely prepared by immobilizing zirconium(IV) on a zirconium-organic framework of UiO-66-NH2, which holds dual-metal centers of not only the inherent Zr-O cluster but also the immobilized Zr(IV) center. This dual-metal centered zirconium-organic framework (DZMOF) demonstrates as a highly specific metal-affinity probe toward the extraction of phosphopeptides due to the metal-affinity interactions of MOAC and IMAC toward either mono-phosphorylated or multi-phosphorylated peptides. The binding energies of zirconium 3d5/2 and 3d3/2 in this DZMOF are 183.07 and 185.47 eV, respectively, which are higher than those of the intact UiO-66-NH2 (182.84 and 185.17 eV, respectively), confirming the higher metal-affinity interaction between the DZMOF and phosphopeptides. This high metal-affinity probe presents an unprecedented strong performance in anti-nonspecific interference during the capturing of phosphopeptides of β-casein with the molar ratio of β-casein vs bovine serum albumin up to ca. 1:5000. The enrichment of phosphopeptides from a human saliva sample by DZMOF further confirms the great potential of DZMOF in the extraction of low-abundance phosphopeptides for real complex biological samples.

Mimicking nature: Phosphopeptide enrichment using combinatorial libraries of affinity ligands

Phosphorylation is a reversible post-translational modification of proteins that controls a plethora of cellular processes and triggers specific physiological responses, for which there is a need to develop tools to characterize phosphorylated targets efficiently. Here, a combinatorial library of triazine-based synthetic ligands comprising 64 small molecules has been rationally designed, synthesized and screened for the enrichment of phosphorylated peptides. The lead candidate (coined A8A3), composed of histidine and phenylalanine mimetic components, showed high binding capacity and selectivity for binding mono- and multi-phosphorylated peptides at pH 3. Ligand A8A3 was coupled onto both cross-linked agarose and magnetic nanoparticles, presenting higher binding capacities (100-fold higher) when immobilized on the magnetic support. The magnetic adsorbent was further screened against a tryptic digest of two phosphorylated proteins (α- and β-caseins) and one non-phosphorylated protein (bovine serum albumin, BSA). The MALDI-TOF mass spectra of the eluted peptides allowed the identification of nine phosphopeptides, comprising both mono- and multi-phosphorylated peptides.

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.

Improving the Phosphoproteome Coverage for Limited Sample Amounts Using TiO2-SIMAC-HILIC (TiSH) Phosphopeptide Enrichment and Fractionation.

Obtaining high phosphoproteome coverage requires specific enrichment of phosphorylated peptides from the often extremely complex peptide mixtures generated by proteolytic digestion of biological samples, as well as extensive chromatographic fractionation prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Due to the sample loss resulting from fractionation, this procedure is mainly performed when large quantities of sample are available. To make large-scale phosphoproteomics applicable to smaller amounts of protein we have recently combined highly specific TiO2-based phosphopeptide enrichment with sequential elution from immobilized metal affinity chromatography (SIMAC) for fractionation of mono- and multi-phosphorylated peptides prior to capillary scale hydrophilic interaction liquid chromatography (HILIC) based fractionation of monophosphorylated peptides. In the following protocol we describe the procedure step by step to allow for comprehensive coverage of the phosphoproteome utilizing only a few hundred micrograms of protein.

Highly specific phosphopeptide enrichment by titanium(IV) cross-linked chitosan composite.

Natural chitosan was applied as supporting material for Ti(IV) based immobilized metal ion affinity chromatographic (IMAC) material (Ti-CTS). Compared with other polymer based IMAC, Ti-CTS can save the cockamamie synthesis procedures and be easy to obtain. The morphology, surface area, pore volume and elemental composition of Ti-CTS were revealed by scanning electron microscopy (SEM), Brunauer-Emmett-Teller (BET) method and X-ray photoelectron spectroscopy (XPS). Tryptic digest products from several standard proteins and two real samples (non-fat milk and serum) were enriched using Ti-CTS to demonstrate the efficiency of this method. The results showed that this composite enables high sensitive and selective phosphopeptide enrichment from casein variants, non-fat milk and human serum. Furthermore, multi-phosphorylated peptides with three serine phospholated sites (S*S*S*) demonstrated high affinity to Ti-CTS. Hence, this method had great potential for future studies of complex phosphoproteomes and especially multi-phosphorylated peptides.

Hydrophilic Nb5+-immobilized magnetic core–shell microsphere – A novel immobilized metal ion affinity chromatography material for highly selective enrichment of phosphopeptides

Rapid and selective enrichment of phosphopeptides from complex biological samples is essential and challenging in phosphorylated proteomics. In this work, for the first time, niobium ions were directly immobilized on the surface of polydopamine-coated magnetic microspheres through a facile and effective synthetic route. The Fe3O4@polydopamine-Nb5+ (denoted as Fe3O4@PD-Nb5+) microspheres possess merits of high hydrophilicity and good biological compatibility, and demonstrated low limit of detection (2fmol). The selectivity was also basically satisfactory (β-casein:BSA=1:500) to capture phosphopeptides. They were also successfully applied for enrichment of phosphopeptides from real biological samples such as human serum and nonfat milk. Compared with Fe3O4@PD-Ti4+ microspheres, the Fe3O4@PD-Nb5+ microspheres exhibit superior selectivity to multi-phosphorylated peptides, and thus may be complementary to the conventional IMAC materials.

On-target titanium dioxide-based enrichment for characterization of phosphorylations in the Adenovirus pIIIa protein

A recently developed titanium dioxide (TiO2) based on-target method for phosphopeptide enrichment and matrix assisted laser desorption-ionization mass spectrometry (MALDI MS) analysis was used to investigate phosphorylations in the Adenovirus type 2 structural protein pIIIa. Lysates of purified virus particles were separated on 1-D SDS-PAGE and the band for the pIIIa protein was excised for tryptic digestion into peptides that were enriched with the on-target method. The enrichment provided by the method clearly improved the detectability of phosphorylated peptides and the results show for the first time evidence for multi-phosphorylated peptides in pIIIa. Moreover, three novel phosphorylations were identified in the protein sequence, even though the precise positions could not be determined. These results illustrate the potential of the method for the characterization of novel phosphoproteomes in biological samples of medical relevance.

Citric Acid-Assisted Two-Step Enrichment with TiO2 Enhances the Separation of Multi- and Monophosphorylated Peptides and Increases Phosphoprotein Profiling

Phosphopeptide enrichment is essential for large-scale phosphoprotein profiling. Titanium dioxide (TiO2) is widely used in phosphopeptide enrichment, but it is limited in the isolation of multiphosphorylated peptides due to their strong binding. In this study, we found that citric acid greatly affects the binding of mono- and multiphosphopeptides with TiO2, which can be used for stepwise phosphopeptide separation coupled with mass spectrum (MS) identification. We first loaded approximately 1 mg of peptide mixture of HeLa cell digests onto TiO2 beads in highly concentrated citric acid (1 M). Then the flow-through fraction was diluted to ensure low concentration of citric acid (50 mM) and followed by loading onto another aliquot of TiO2 beads. The two eluted fractions were subjected to nanoLC-MS/MS analysis. We identified 1,500 phosphorylated peptides, of which 69% were multiphosphorylated after the first enrichment. After the second enrichment, 2,167 phosphopeptides, of which 92% were monophosphorylated, were identified. In total, we successfully identified 3,136 unique phosphopeptides containing 3,973 phosphosites utilizing this strategy. Finally, more than 37% of the total phosphopeptides and 2.6-fold more of the multiphosphorylated peptides were identified as compared to the frequently used DHB/TiO2 enrichment strategy. Combining SCX with CATSET, we identified 14,783 phosphopeptides and 15,713 phosphosites, of which 3,678 were unrecorded in PhosphoSitePlus database. This two-step separation procedure for sequentially enriching multi- and monophosphorylated peptides by using citric acid is advantageous in multiphosphorylated peptide separation, as well as for more comprehensive phosphoprotein profiling.

Mass spectrometric analysis of mono- and multi-phosphopeptides by selective binding with NiZnFe2O4 magnetic nanoparticles

Selective isolation of mono- and multi-phosphorylated peptides is important for understanding how a graded protein kinase or phosphatase signal can precisely modulate the on and off states of signal transduction pathways. Here we report that metal ions at exposed octahedral sites of nano-ferrites, including Fe3O4, NiFe₂O₄, ZnFe₂O₄ and NiZnFe₂O₄, have distinctly selective coordination abilities with mono- and multi- phosphopeptides. Due to their intrinsic magnetic properties and high surface area to volume ratios, these nanoparticles enable the rapid isolation of mono- and multi-phosphopeptides by an external magnetic field. Model phosphoprotein α-casein and two synthesized mono- and di-phosphopeptides have been chosen for proof-of-principle demonstrations, and these nanoparticles have also been applied to phosphoproteome profiling of zebrafish eggs. It is shown that NiZnFe₂O₄ is highly selective for multi-phosphopeptides. In contrast, Fe₃O₄, NiFe₂O₄ and ZnFe₂O₄ can bind with both mono- and multi-phosphopeptides with relatively stronger affinity towards mono-phosphopeptides.

Combinatorial Use of Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC) and Strong Cation Exchange (SCX) Chromatography for In-Depth Phosphoproteome Analysis

In large-scale phosphoproteomics studies, fractionation by strong cation exchange (SCX) or electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) is commonly used to reduce sample complexity, fractionate phosphopeptides from their unmodified counterparts, and increase the dynamic range for phosphopeptide identification. However, these procedures do not succeed to separate, both singly and multiply phosphorylated peptides due to their inverse physicochemical characteristics. Hence, depending on the chosen method only one of the two peptide classes can be efficiently separated. Here, we present a novel strategy based on the combinatorial separation of singly and multiply phosphorylated peptides by SCX and ERLIC for in-depth phosphoproteome analysis. In SCX, mostly singly phosphorylated peptides are retained and fractionated while not-retained multiply phosphorylated peptides are fractionated in a subsequent ERLIC approach (SCX-ERLIC). In ERLIC, multiply phosphorylated peptides are fractionated, while not-retained singly phosphorylated peptides are separated by SCX (ERLIC-SCX). Compared to single step fractionations by SCX, the combinatorial strategies, SCX-ERLIC and ERLIC-SCX, yield up to 48% more phosphopeptide identifications as well as a strong increase in the number of detected multiphosphorylated peptides. Phosphopeptides identified in two subsequent, complementary fractionations had little overlap (5%) indicating that ERLIC and SCX are orthogonal methods ideally suited for in-depth phosphoproteome studies.