Synthesis of Homoserine Phospho-, H-Phosphono- and Methylphosphonopeptides

Summary Phosphopeptides and mimics thereof are useful tools for the investigation of phosphorylation, an important posttranslational modification of peptides and proteins. In order to investigate different aspects of phosphorylation and dephosphorylation processes, homoserine phospho-, H-phosphono- and methylphosphonopeptides were synthesized. The tetrapeptide H-Gly-Gly-Hse-Ala-OH was used as a model sequence; further, the heptapeptide H-Leu-Arg-Arg-Ala-Hse-Leu-Gly-OH and the octapeptide H-Glu-Ser-Leu-Hse-Ser-Ser-Glu-Glu-OH were synthesized and modified. After selective deprotection of the trityl-protected homoserine residue, phosphorylation or phosphonylation was performed on resin by the global phosphorylation approach using different phosphoamidites. Peptides were analysed by analytical RP-HPLC and electrospray mass spectrometry. All compounds were obtained in yields over 75%. The byproducts observed were both the unmodified peptide and the H-phosphonopeptide in the case of the phosphopeptides, the phosphorylated and the unmodified peptide in the case of the H-phosphonopeptides, and the unmodified peptide in the case of the methylphosphonopeptides. Due to simple purification by RP-HPLC, the method presented gives access to a new class of phosphopeptides and mimics.     

Synthesis of Homoserine Phospho-, H-Phosphono- and Methylphosphonopeptides

Phosphopeptides and mimics thereof are useful tools for the investigation of phosphorylation, an important post-translational modification of peptides and proteins. In order to investigate different aspects of phosphorylation and dephosphorylation processes, homoserine phospho-, H-phosphono- and methylphosphonopeptides were synthesized. The tetrapeptide H-Gly-Gly-Hse-Ala-OH was used as a model sequence; further, the heptapeptide H-Leu-Arg-Arg-Ala-Hse-Leu-Gly-OH and the octapeptide H-Glu-Ser-Leu-Hse-Ser-Ser-Glu-Glu-OH were synthesized and modified. After selective deprotection of the trityl-protected homoserine residue, phosphorylation or phosphonylation was performed on resin by the global phosphorylation approach using different phosphoamidites. Peptides were analysed by analytical RP-HPLC and electrospray mass spectrometry. All compounds were obtained in yields over 75%. The byproducts observed were both the unmodified peptide and the H-phosphonopeptide in the case of the phosphopeptides, the phosphorylated and the unmodified peptide in the case of the H-phosphonopeptides, and the unmodified peptide in the case of the methylphosphonopeptides. Due to simple purification by RP-HPLC, the method presented gives access to a new class of phosphopeptides and mimics.     

Mining phosphopeptide signals in liquid chromatography-mass spectrometry data for protein phosphorylation analysis

Protein phosphorylation is a key post-translational modification that governs biological processes. Despite the fact that a number of analytical strategies have been exploited for the characterization of protein phosphorylation, the identification of protein phosphorylation sites is still challenging. We proposed here an alternative approach to mine phosphopeptide signals generated from a mixture of proteins when liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis is involved. The approach combined dephosphorylation reaction, accurate mass measurements from a quadrupole/time-of-flight mass spectrometer, and a computing algorithm to differentiate possible phosphopeptide signals obtained from the LC-MS analyses by taking advantage of the mass shift generated by alkaline phosphatase treatment. The retention times and m/z values of these selected LC-MS signals were used to facilitate subsequent LC-MS/MS experiments for phosphorylation site determination. Unlike commonly used neutral loss scan experiments for phosphopeptide detection, this strategy may not bias against tyrosine-phosphorylated peptides. We have demonstrated the applicability of this strategy to sequence more, in comparison with conventional data-dependent LC-MS/MS experiments, phosphopeptides in a mixture of alpha- and beta-caseins. The analytical scheme was applied to characterize the nasopharyngeal carcinoma (NPC) cellular phosphoproteome and yielded 221 distinct phosphorylation sites. Our data presented in this paper demonstrated the merits of computation in mining phosphopeptide signals from a complex mass spectrometric data set.     

Synthesis of Phosphopeptides in the Fmoc Mode

The synthesis of phosphopeptides has played a major role in the characterization of protein phosphorylation/dephosphorylation. The current range of synthesis protocols available provides a variety of possible routes by which to approach specific synthetic challenges, and this review article discusses these methods for the preparation of phosphopeptides and provides synthesis notes for each method. Phosphopeptide synthesis is achieved by either introduction of the phosphate group via post-synthetic (‘global’) phosphorylation of a resin-bound peptide or the incorporation of a pre-phosphorylated derivative into the growing peptide chain. Protocols and synthesis notes are provided for the synthesis of phosphoramidites, phosphotyrosyl, -seryl and -threonyl peptides and their mimetics, including thiophosphopeptides. The aim of this review was to provide a synthesis reference guide for Fmoc-based synthesis of both singly and multiply phosphorylated peptides, with particular emphasis given to the most successful and generally applicable methods.     

Mapping and sequence-specific identification of phosphopeptides in unfractionated protein digest mixtures by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

We have demonstrated a procedure for the rapid (minutes), sensitive (less than pmol), and sequence-specific identification of phosphopeptides in unfractionated digests of phosphoproteins using matrix-assisted UV laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry. The mass-dependent identification of one specific 13-residue phosphopeptide (S105-K117), observed among the 153 possible trypsin digest fragments of human beta-casein (211 residues), was confirmed by amino acid sequence analysis of the 33P-labeled peptide after isolation by reverse-phase HPLC. MALDI-TOF was also used to monitor the rate and extent to which an 18-residue N-terminal beta-casein peptide (R1-K18) was phosphorylated in vitro. These results demonstrate that MALDI-TOF may be used (i) to facilitate the identification of sequence-specific sites of protein phosphorylation and dephosphorylation, (ii) to monitor protein and peptide phosphorylation and dephosphorylation reaction rates, even in complex unfractionated mixtures, (iii) to determine the minimum primary structure necessary for the phosphorylation of specific protein surface domains, and (iv) to evaluate the effects of intact protein phosphorylation and dephosphorylation on susceptibility to subsequent proteolytic events.     

Mapping sites of protein phosphorylation by mass spectrometry utilizing a chemical-enzymatic approach: characterization of products from alpha-S1 casein phosphopeptides

A novel chemical-enzymatic approach was developed to facilitate identification of phosphorylation sites in isolated phosphoproteins. ESI-TOF mass spectrometry was used to characterize products from the chemical-enzymatic cleavage of specific phosphorylation sites in bovine alpha-S1 casein and synthetic phosphopeptides containing substitutions at a single phosphorylation site. Further refinements to this approach for identification of protein phosphorylation sites and its utility for the quantification of phosphopeptides by isotope-dilution mass spectrometry are presented.     

Phosphospecific proteolysis for mapping sites of protein phosphorylation

Protein phosphorylation is a dominant mechanism of information transfer in cells, and a major goal of current proteomic efforts is to generate a system-level map describing all the sites of protein phosphorylation. Recent efforts have focused on developing technologies for enriching and quantifying phosphopeptides. Identification of the sites of phosphorylation typically relies on tandem mass spectrometry to sequence individual peptides. Here we describe an approach for phosphopeptide mapping that makes it possible to interrogate a protein sequence directly with a protease that recognizes sites of phosphorylation. The key to this approach is the selective chemical transformation of phosphoserine and phosphothreonine residues into lysine analogs (aminoethylcysteine and beta-methylaminoethylcysteine, respectively). Aminoethylcysteine-modified peptides are then cleaved with a lysine-specific protease to map sites of phosphorylation. A blocking step enables single-site cleavage, and adaptation of this reaction to the solid phase facilitates phosphopeptide enrichment and modification in one step.     

Analysis of protein phosphorylation by mass spectrometry

The reversible phosphorylation of proteins is recognized as an essential post-translational modification regulating cell signaling and ultimately function of biological systems. Detection of phosphopeptides and localization of phosphorylation sites remains quite a challenge, even if the protein is purified to near homogeneity. Mass spectrometry has become a vital technique that is routinely utilized for the identification of proteins from whole cell lysates. Nonetheless, due to the minimal amount of phosphorylation found on proteins, enrichment steps for isolating phosphopeptides from complex mixtures have been the focus of many research groups world-wide. In this review, we describe some current methods for the enrichment of phosphopeptides that are compatible with mass spectrometry for assignment of phosphorylation sites. Phosphorylation modifications on proteins and peptides are either directly isolated by solid-phase approaches or chemically modified for selective isolation and/or improved characterization by mass spectrometry. These strategies hold the potential for rapid and sensitive profiling of phosphoproteins from a variety of sources and cellular conditions.     

Comparison of different IMAC techniques used for enrichment of phosphorylated peptides.

Four commercially available immobilized metal ion affinity chromatography (IMAC) methods for phosphopeptide enrichment were compared using small volumes and concentrations of phosphopeptide mixtures with or without extra-added bovine serum albumin (BSA) nonphosphorylated peptides. Addition of abundant tryptic BSA peptides to the phosphopeptide mixture increases the demand for selective IMAC capture. While SwellGel gallium Discs, IPAC Metal Chelating Resin, and ZipTipMC Pipette Tips allow for the possibility of enriching phosphopeptides, the Gyrolab MALDI IMAC1 also presents the possibility of verifying existing phosphopeptides after a dephosphorylation step. Phosphate-containing peptides are identified through a mass shift between phosphorylated and dephosphorylated spectra of 80 Da (or multiples of 80 Da). This verification is useful if the degree of phosphorylation is low in the sample or if the ionization is unfavorable, which often is the case for phosphopeptides. A peptide mixture in which phosphorylated serine, threonine, and tyrosine were represented was diluted in steps and thereafter enriched using the four different IMAC methods prior to analyses with matrix assisted laser desorption/ionization mass spectrometry. The enrichment of phosphopeptides using SwellGel Gallium Discs or Gyrolab MALDI IMAC1 was not significantly affected by the addition of abundant BSA peptides added to the sample mixture, and the achieved detection limits using these techniques were also the lowest. All four of the included phosphopeptides were detected by MALDI-MS only after enrichment using the Gyrolab MALDI IMAC1 compact disc (CD) and detection down to low femtomole levels was possible. Furthermore, selectivity, reproducibility, and detection for a number of other phosphopeptides using the IMAC CD are reported herein. For example, two phosphopeptides sent out in a worldwide survey performed by the Proteomics Research Group (PRG03) of the Association of Biomolecular Resource Facilities (ABRF) were detected and verified by means of the 80 Da mass shift achieved by on-column dephosphorylation.     

Dephosphorylation specificities of protein phosphatase for cardiac troponin I, troponin T, and sites within troponin T

Protein dephosphorylation by protein phosphatase 1 (PP1), acting in concert with protein kinase C (PKC) and protein kinase A (PKA), is a pivotal regulatory mechanism of protein phosphorylation. Isolated rat cardiac myofibrils phosphorylated by PKC/PKA and dephosphorylated by PP1 were used in determining dephosphorylation specificities, Ca(2+)-stimulated Mg(2+)ATPase activities, and Ca(2+) sensitivities. In reconstituted troponin (Tn) complex, PP1 displayed distinct substrate specificity in dephosphorylation of TnT preferentially to TnI, in vitro. In situ phosphorylation of cardiomyocytes with calyculin A, a protein phosphatase inhibitor, resulted in an increase in the phosphorylation stiochiometry of TnT (0.3 to 0.5 (67%)), TnI (2.6 to 3.6 (38%)), and MLC2 (0.4 to 1.7 (325%)). These results further confirmed that though MLC2 is the preferred target substrate for protein phosphatase in the thick filament, the Tn complex (TnI and TnT) from thin filament and C-protein in the thick filament are also protein phosphatase substrates. Our in vitro dephosphorylation experiments revealed that while PP1 differentially dephosphorylated within TnT at multiple sites, TnI was uniformly dephosphorylated. Phosphopeptide maps from the in vitro experiments show that TnT phosphopeptides at spots 4A and 4B are much more resistant to PP1 dephosphorylation than other TnT phosphopeptides. Mg(2+)ATPase assays of myofibrils phosphorylated by PKC/PKA and dephosphorylated by PP1 delineated that while PKC and PKA phosphorylation decreased the Ca(2+)-stimulated Mg(2+)ATPase activities, dephosphorylation antagonistically restored it. PKC and PKA phosphorylation decreased Ca(2+) sensitivity to 3.6 microM and 5.0 microM respectively. However, dephosphorylation restored the Mg(2+)ATPase activity of PKC (99%) and PKA (95%), along with the Ca(2+) sensitivities (3.3 microM and 3.0 microM, respectively).     

Laser-induced dissociation of phosphorylated peptides using matrix assisted laser desorption/ionization tandem time-of-flight mass spectrometry

Reversible phosphorylation is one of the most important posttranslational modifications of cellular proteins. Mass spectrometry is a widely used technique in the characterization of phosphorylated proteins and peptides. Similar to nonmodified peptides, sequence information for phosphopeptides digested from proteins can be obtained by tandem mass analysis using either electrospray ionization or matrix assisted laser desorption/ionization (MALDI) mass spectrometry. However, the facile loss of neutral phosphoric acid (H₃PO₄) or HPO₃ from precursor ions and fragment ions hampers the precise determination of phosphorylation site, particularly if more than one potential phosphorylation site or concensus sequence is present in a given tryptic peptide. Here, we investigated the fragmentation of phosphorylated peptides under laser-induced dissociation (LID) using a MALDI-time-of-flight mass spectrometer with a curved-field reflectron. Our data demonstrated that intact fragments bearing phosphorylated residues were produced from all tested peptides that contain at least one and up to four phosphorylation sites at serine, threonine, or tyrosine residues. In addition, the LID of phosphopeptides derivatized by N-terminal sulfonation yields simplified MS/MS spectra, suggesting the combination of these two types of spectra could provide an effective approach to the characterization of proteins modified by phosphorylation.     

Universal and confident phosphorylation site localization using phosphoRS

An algorithm for the assignment of phosphorylation sites in peptides is described. The program uses tandem mass spectrometry data in conjunction with the respective peptide sequences to calculate site probabilities for all potential phosphorylation sites. Tandem mass spectra from synthetic phosphopeptides were used for optimization of the scoring parameters employing all commonly used fragmentation techniques. Calculation of probabilities was adapted to the different fragmentation methods and to the maximum mass deviation of the analysis. The software includes a novel approach to peak extraction, required for matching experimental data to the theoretical values of all isoforms, by defining individual peak depths for the different regions of the tandem mass spectrum. Mixtures of synthetic phosphopeptides were used to validate the program by calculation of its false localization rate versus site probability cutoff characteristic. Notably, the empirical obtained precision was higher than indicated by the applied probability cutoff. In addition, the performance of the algorithm was compared to existing approaches to site localization such as Ascore. In order to assess the practical applicability of the algorithm to large data sets, phosphopeptides from a biological sample were analyzed, localizing more than 3000 nonredundant phosphorylation sites. Finally, the results obtained for the different fragmentation methods and localization tools were compared and discussed.     

Reference-facilitated phosphoproteomics: fast and reliable phosphopeptide validation by microLC-ESI-Q-TOF MS/MS

Recent advances in instrument control and enrichment procedures have enabled us to quantify large numbers of phosphoproteins and record site-specific phosphorylation events. An intriguing problem that has arisen with these advances is to accurately validate where phosphorylation events occur, if possible, in an automated manner. The problem is difficult because MS/MS spectra of phosphopeptides are generally more complicated than those of unmodified peptides. For large scale studies, the problem is even more evident because phosphorylation sites are based on single peptide identifications in contrast to protein identifications where at least two peptides from the same protein are required for identification. To address this problem we have developed an integrated strategy that increases the reliability and ease for phosphopeptide validation. We have developed an off-line titanium dioxide (TiO(2)) selective phosphopeptide enrichment procedure for crude cell lysates. Following enrichment, half of the phosphopeptide fractionated sample is enzymatically dephosphorylated, after which both samples are subjected to LC-MS/MS. From the resulting MS/MS analyses, the dephosphorylated peptide is used as a reference spectrum against the original phosphopeptide spectrum, in effect generating two peptide spectra for the same amino acid sequence, thereby enhancing the probability of a correct identification. The integrated procedure is summarized as follows: 1) enrichment for phosphopeptides by TiO(2) chromatography, 2) dephosphorylation of half the sample, 3) LC-MS/MS-based analysis of phosphopeptides and corresponding dephosphorylated peptides, 4) comparison of peptide elution profiles before and after dephosphorylation to confirm phosphorylation, and 5) comparison of MS/MS spectra before and after dephosphorylation to validate the phosphopeptide and its phosphorylation site. This phosphopeptide identification represents a major improvement as compared with identifications based only on single MS/MS spectra and probability-based database searches. We investigated an applicability of this method to crude cell lysates and demonstrate its application on the large scale analysis of phosphorylation sites in differentiating mouse myoblast cells.     

Phosphopeptide substrates of a phosphoprotein phosphatase from rat liver

The substrate specificity of a preparation of phosphoprotein phosphatase (Mr = 32 000) from rat liver was investigated. Phosphopeptides based on the structure Leu-Arg-Arg-Ala-Ser(P)-Val-Ala-Glx-Leu and Ala-Arg-Thr-Lys-Arg-Ser-Gly-Ser(P)-Val-Tyr-Glu-Pro-Leu-Lys were used. These phosphopeptides correspond to the phosphorylation sites of rat liver pyruvate kinase (type L) and the beta subunit of rabbit muscle phosphorylase b kinase, respectively. A decrease in the apparent Km values and a concomitant increase in Vmax values was observed when the number of amino acyl residues after the phosphoseryl residue in the respective phosphopeptides were increased from 2 to 4, 5, or 6. Most of the phosphopeptides investigated generally showed apparent Km values higher than the values obtained with phosphopyruvate kinase. Ala-Ser(P)-Val-Ala and Gly-Ser(P)-Val-Tyr appeared to be the shortest phosphopeptides that could be dephosphorylated rapidly. These findings support the hypothesis that a small part of the phosphoprotein may be sufficient to fulfill the minimal requirements for its dephosphorylation.     

Mitochondrial phosphoproteome revealed by an improved IMAC method and MS/MS/MS

IMAC in combination with mass spectrometry is a promising approach for global analysis of protein phosphorylation. Nevertheless this approach suffers from two shortcomings: inadequate efficiency of IMAC and poor fragmentation of phosphopeptides in the mass spectrometer. Here we report optimization of the IMAC procedure using (32)P-labeled tryptic peptides and development of MS/MS/MS (MS3) for identifying phosphopeptide sequences and phosphorylation sites. The improved IMAC method allowed recovery of phosphorylated tryptic peptides up to approximately 77% with only minor retention of unphosphorylated peptides. MS3 led to efficient fragmentation of the peptide backbone in phosphopeptides for sequence assignment. Proteomics of mitochondrial phosphoproteins using the resulting IMAC protocol and MS3 revealed 84 phosphorylation sites in 62 proteins, most of which have not been reported before. These results revealed diverse phosphorylation pathways involved in the regulation of mitochondrial functions. Integration of the optimized batchwise IMAC protocol with MS3 offers a relatively simple and more efficient approach for proteomics of protein phosphorylation.     

磷酸化蛋白质组学的新进展及其在肝脏生理和病理机制中的应用

蛋白质磷酸化是最常见的蛋白质翻译后修饰形式。由于蛋白质的磷酸化形式可以被磷酸酶和磷酸激酶进行可逆的调控,所以在众多的生命活动过程中蛋白质的磷酸化修饰起着重要的调控作用,因此对生物体内蛋白质磷酸化修饰的系统研究对于揭示生命科学的奥秘显得十分重要。近年来,随着质谱技术和生物信息学软件以及磷酸化肽段富集方法的发展,利用质谱对生物体内蛋白质磷酸化修饰研究的技术逐渐成熟。肝脏作为人体最重要的代谢和免疫器官,深入研究肝脏细胞内蛋白质磷酸化修饰形式对于理解其功能具有重要指导意义。目前,迅速发展的磷酸化蛋白质组学技术已经被广泛应用到肝脏功能的生物学研究中。这些研究加深了人们对肝脏的生理及病理状态的分子生物学机制的了解。本文综述了当前磷酸化蛋白质组学的研究进展和磷酸化蛋白质组学在肝脏中的研究。     

Efficient enrichment of phosphopeptides by magnetic TiO₂-coated carbon-encapsulated iron nanoparticles

Titanium dioxide (TiO₂) has been widely used for phosphopeptide enrichment. Several approaches have been reported to produce magnetic TiO₂ affinity probes. In this report, we present a facile approach to immobilize TiO₂ onto poly(acrylic acid)‐functionalized magnetic carbon‐encapsulated iron nanoparticles as affinity probes for efficient enrichment of phosphopeptides. By using the new magnetic TiO₂ affinity probes, denoted as TiO₂‐coated Fe@CNPs, rapid and effective MALDI‐TOF MS profiling of phosphopeptides was demonstrated in different model systems such as tryptic digests of β‐casein, and complex β‐casein/BSA mixture. The TiO₂‐coated Fe@CNPs out‐performed the commercial TiO₂‐coated magnetic beads for detection of phosphopeptides from tryptic digests of β‐casein/BSA mixture with a molar ratio of 1:100. The new TiO₂‐coated magnetic probes were also proven to be applicable for real life samples. The magnetic TiO₂‐coated Fe@CNPs were employed to selectively isolate phosphopeptides from tryptic digests of HeLa cell lysates and out‐performed the commercial magnetic TiO₂ beads in the number of identified phosphopeptides and phosphorylation sites. In a 200‐μg equivalent of HeLa cell lysates, we identified 1415 unique phosphopeptides and 1093 phosphorylation sites, indicating the good performance of the new approach.     

Identification of phosphorylation sites in the polo-like kinases Plx1 and Plk1 by a novel strategy based on element and electrospray high resolution mass spectrometry

A novel strategy for the determination of protein phosphorylation sites is described and applied to the polo-like kinases Plx1 (Xenopus laevis) and Plk1 (Homo sapiens). The strategy comprises the sequential application of the following techniques: proteolytic digestion, capillary liquid chromatography (LC)-inductively coupled plasma mass spectrometry with phosphorus detection, capillary LC-electrospray mass spectrometry and electrospray tandem mass spectrometry. In this approach, phosphopeptides are generated, their elution time in capillary LC is determined, candidate phosphopeptides at the corresponding elution times are identified, and positive identification and sequencing of phosphopeptides is performed in the last step of the analysis. Using this technique, Ser25/26, Ser326, and Ser340 were identified as phosphorylation sites in recombinant Plx1, and Ser340 was identified as the major phosphorylation site in a kinase-dead mutant of Plx1 expressed in okadaic acid-treated Sf9 insect cells. A site corresponding to Ser326 in Plx1 was also shown to be phosphorylated in the human polo-like kinase Plk1 (Ser335). Element mass spectrometry with phosphorus detection provides a quantitative phosphorylation profile of all phosphorylation sites accessible by LC.