Fragmentation of phosphopeptides by atmospheric pressure MALDI and ESI/ion trap mass spectrometry

An investigation of phosphate loss from phosphopeptide ions was conducted, using both atmospheric pressure matrix-assisted laser desorption/ionization (AP MALDI) and electrospray ionization (ESI) coupled to an ion trap mass spectrometer (ITMS). These experiments were carried out on a number of phosphorylated peptides in order to investigate gas phase dephosphorylation patterns associated with phosphoserine, phosphothreonine, and phosphotyrosine residues. In particular, we explored the fragmentation patterns of phosphotyrosine containing peptides, which experience a loss of 98 Da under collision induced dissociation (CID) conditions in the ITMS. The loss of 98 Da is unexpected for phosphotyrosine, given the structure of its side chain. The fragmentation of phosphoserine and phosphothreonine containing peptides was also investigated. While phosphoserine and phosphothreonine residues undergo a loss of 98 Da under CID conditions regardless of peptide amino acid composition, phosphate loss from phosphotyrosine residues seems to be dependent on the presence of arginine or lysine residues in the peptide sequence.     

Studies on the dephosphorylation of phosphotyrosine-containing peptides during post-source decay in matrix-assisted laser desorption/ionization

Phosphorylation of tyrosine residues in proteins is a common regulatory mechanism, although it accounts for less than 1% of the total O-phosphate content in proteins. Whereas aromatic phosphorylation sites can be identified by a number of different analytical techniques, sequence analysis of phosphotyrosine-containing proteins at the low picomole or even femtomole level is still a challenging task. This paper describes the post-source decay in matrix-assisted laser desorption/ionization mass spectrometry of phosphotyrosine-containing model peptides by comparing their fragmentation behavior with sequence-homologous unphosphorylated peptides. Whereas the parent ions showed significant losses of HPO3, all phosphorylated fragment ions of the b- and y-series displayed only minor dephosphorylated signals, which often were not detectable. Surprisingly, one of the studied phosphotyrosine-containing sequences displayed, in addition to the [M + H - 80]+ ion, a more abundant [M + H - 98]+ ion, which could be explained by elimination of phosphoric acid. This dephosphorylation pattern was very similar to the patterns obtained for phosphoserine- and phosphothreonine-containing peptides. Because the dephosphorylation pattern of the parent ion is often used to identify modified amino acids in peptides, we investigated possible dephosphorylation mechanisms in detail. Therefore, we substituted single trifunctional amino acid residues and incorporated deuterated phosphotyrosine residues. After excluding direct elimination of phosphoric acid from tyrosine, we could show that the obtained loss of H3PO4 depends on aspartic acid and arginine residues. Most likely the HPO3 group is transferred to aspartic acid followed by cleavage of phosphoric acid forming a succinimide. On the other hand, arginine appears to induce the H3PO4 loss by protonation of phosphotyrosine leaving a phenyl cation.     

Nonbonded interactions in membrane-active cyclic biopolymers. III. Analogs of valinomycin

Several analogs of valinomycin have been simulated. These are analogs obtained by changing the configuration of the amino acid and hydroxy acid residues one at a time. Nonbonded interactions have been calculated for three conformations of each analog. The electrostatic and polarization contributions to the energy are conformation selective, whereas the Pauli repulsion is the only component which is isomer selective. For the ring chirality considered here the analog of valinomycin having all the three L -valyl residues replaced by D -valyl residues is predicted to be frozen in a “top open” conformation.     

A self-immobilizing and fluorogenic unnatural amino acid that mimics phosphotyrosine

Synthesis of the first self-immobilizing, fluorogenic unnatural amino acid that mimics phosphotyrosine (pTyr) is reported. By using solid-phase peptide synthesis, it was subsequently incorporated into peptide-based probes which found applications in bioimaging and fluorescence-activated cell sorting (FACS).     

Phosphopeptide detection and sequencing by matrix-assisted laser desorption/ionization quadrupole time-of-flight tandem mass spectrometry

A prototype matrix-assisted laser desorption/ionization quadrupole time-of-flight (MALDI-TOF) tandem mass spectrometer was used to sequence a series of phosphotyrosine-, phosphothreonine- and phosphoserine-containing peptides. The high mass resolution and mass accuracy of the instrument allowed the localization of one, three or four phosphorylated amino acid residues in phosphopeptides up to 3.1 kDa. Tandem mass spectra of two different phosphotyrosine peptides permitted amino acid sequence determination and localization of one and three phosphorylation sites, respectively. The phosphotyrosine immonium ion at m/z 216.04 was observed in these MALDI low-energy CID tandem mass spectra. Elimination of phosphate groups was evident from the triphosphorylated peptide but not from the monophosphorylated species. The main fragmentation pathway for the synthetic phosphothreonine-containing peptide and for phosphoserine-containing peptides derived from beta-casein and ovalbumin was the beta-elimination of phosphoric acid with concomitant conversion of phosphoserine to dehydroalanine and phosphothreonine to 2-aminodehydrobutyric acid. Peptide fragment ions of the b- and y-type allowed, in all cases, the localization of phosphorylation sites. Ion signals corresponding to (b-17), (b-18) and (y-17) fragment ions were also observed. The abundant neutral loss of phosphoric acid (-98 Da) is useful for femtomole level detection of phosphoserine-peptides in crude peptide mixtures generated by gel in situ digestion of phosphoproteins.     

Characterization of a phosphorylated peptide and peptoid and peptoid-peptide hybrids by mass spectrometry

Nano-electrospray tandem mass spectrometry (nano-ES-MS/MS) was used to record collision-induced dissociation (CID) spectra of a set of peptoid-peptide hybrids and the complete peptoid derived from the phosphopeptide Ac-pTyr-Glu-Thr-Leu-NH(2) (1). The presence of B and Y'-type fragment ions in the tandem mass spectra of the protonated molecular ions [M + H](+) allowed confirmation of sequence similar to mass spectrometric sequence analysis in peptides. In the isomeric peptoid compounds studied, one or several amino acid residues were replaced by peptoid residues (N-substituted glycine residues), which resulted in characteristic tandem mass spectra with differently increased relative abundances of Y'-and B-type fragment ions. The increment of a particular Y'-ion was directly correlated to the position of a peptoid residue present. In addition to these increased peak intensities, other characteristic peaks were also observed compared with the spectrum of reference peptide 1. When a peptoid phosphotyrosine was incorporated, the presence of this residue was apparent from the occurrence of a relatively intense peak at m/z 187 representing the positively charged side-chain of phosphotyrosine, which was almost absent in the spectrum of the reference peptide 1. Since the threonine side-chain had to be translated into the homo peptoid analog this substitution was apparent from the presence of [M + H](+) and fragment ions 14 mass units higher than observed in the spectrum of the reference phosphopeptide 1. The presence of an NLeu peptoid residue could be confirmed by the specific fragmentation of the immonium ion showing an intense peak in its tandem mass spectrum at m/z 57, which results from the loss of an neutral imine molecule leading to a positively charged [C(4)H(9)](+) ion. By means of these mass spectrometric characteristics, all isomeric peptoid compounds could be distinguished from each other and characterized. The methods used appear to be very useful in future studies of peptoids and peptoid-peptide hybrids.     

A study of the properties of synthetic analogs of the tryptophan-containing fragment 113–121 of the basic protein of myelin

New analogs of fragment 113–121 of the basic protein of myelin were synthesized: Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Arg, in which the phenylalanine in the first position has been replaced by L-alanine (V) and by D-phenylalanine (IX), and also a shortened analog with the deletion of the serine in the second position (XIII) and a lengthened analog with the insertion of glycine between the phenylalanine and tryptophan (XVII). In experiments on guinea pigs, one of the compounds obtained exhibited encephalitogenic activity. The circular dichroism spectra of compounds (I) and (IX), and also of some model compounds, have been studied in order to analyze the contribution of the aromatic amino acid residues to the dichroic absorption.

     

Essential of Proline and Valine Residues in the Peptide Derived from Lactoferrin for Angiotensin Converting Enzyme Inhibition

We synthesized Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu, the peptide contained in lactoferrin (Lf), to identify the angiotensin converting enzyme (ACE) inhibition. In an attempt to know the structure‐activity relationship of this peptide, we replaced Pro (the third amino acid residues from N‐terminal) or Val (the fourth amino acid residues from N‐terminal) with Ala (neutral amino acid), Glu (acidic amino acid) or Lys (basic amino acid) to produce six peptides. From the in vitro ACE inhibition (IC₅₀) of these synthesized peptides, the original peptide (Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu) showed higher ACE inhibition than the replaced six peptides. Thus, replacement of Pro at the third amino acid residues or Val at the fourth position with Ala, Glu or Lys revealed the ACE inhibition to be lower than the original form of Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu. Otherwise, we added one peptide at the C‐terminal of Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu and found both products with an addition of Val (Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu‐Val) or Ile (Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu‐Ile) showing a lower ACE inhibition than the original one. The ACE inhibitions produced by both replaced peptides were without significance. Also, deletion of the last peptide at the C‐terminal (Leu‐Arg‐Pro‐Val‐Ala‐Ala) failed to produce a marked change of ACE inhibition as compared to the original one. These results suggest that Pro and Val are essential in the peptide for inhibition of ACE activity.     

An Aminated GDP-Fucose Analog Useful in the Fucosyltransferase Catalyzed Addition of Biological Probes onto Oligosaccharide Chains1

Abstract

An analog (1) of GDP-fucose, where C-6 is derivatized with an eight-atom spacer terminating in a primary amino group, was chemically synthesized. This amino group in sugar nucleotide 1 can be acylated using an N-hydroxysuccinimide ester of biotin and it can be coupled to another molecule that also contains an amino group using squaric acid diethyl ester as the coupling reagent. In this way, biotin and a blood group A-active trisaccharide were linked to C-6 of fucose in GDP-fucose. Both complex sugar nucleotides thus prepared were active as donors for a human milk fucosyltransferase, which transferred the derivatized α-linked fucose residue to a glycoside of N-acetyllactosamine, thus labeling this sequence with either biotin or the blood-group A trisaccharide. Compound 1 is proposed as a general and versatile reagent which should permit the addition of biological probes to the sugar chains of cell surface glycoproteins or glycolipids.     

Total chemical synthesis of a biologically active and homogeneous analog of Human Growth Hormone [Nle14,125,170,Glu29,91,Gln74,Asn107,Asp109]hGH-NH2 by sequential native chemical ligation

We report herein, for the first time, a sequential total chemical synthesis of the Human Growth Hormone analog [Nle^14,125,170,Glu^29,91,Gln⁷⁴,Asn¹⁰⁷,Asp¹⁰⁹]hGH-NH₂, composed of a 191 amino acid residue polypeptide chain containing two disulfide bonds and nine modifications in the natural sequence. Sequential native chemical ligation of three discrete segments of 52, 52 and 87 amino acid residues gave the target full-length polypeptide chain. Subsequent folding with concomitant formation of the native disulfide bonds afforded a correctly folded homogeneous analog which is biologically active.     

Total chemical synthesis of crambin

Crambin is a small (46 amino acids) protein isolated from the seeds of the plant Crambe abyssinica. Crambin has been extensively used as a model protein for the development of advanced crystallography and NMR techniques and for computational folding studies. We set out to establish synthetic access to crambin. Initially, we synthesized the 46 amino acid polypeptide by native chemical ligation of two distinct sets of peptide segments (15 + 31 and 31 + 15 residues). The synthetic polypeptide chain folded in good yield to give native crambin containing three disulfide bonds. The chemically synthesized crambin was characterized by LC-MS and by 2D-NMR. However, the 31-residue peptide segments were difficult to purify, and this caused an overall low yield for the synthesis. To overcome this problem, we synthesized crambin by the native chemical ligation of three segments (15 + 16 + 15 residues). Total synthesis using the ligation of three segments gave more than a 10-fold increase in yield and a protein product of exceptionally high purity. This work demonstrates the efficacy of chemical protein synthesis by the native chemical ligation of three segments and establishes efficient synthetic access to the important model protein crambin for experimental studies of protein folding and stability.     

Context-dependent fluorescence detection of a phosphorylated tyrosine residue by a ribonucleopeptide

Tools for selective recognition and sensing of specific phosphorylated tyrosine residues on the protein surface are essential for understanding signal transduction cascades in the cell. A stable complex of RNA and peptide, a ribonucleopeptide (RNP), provides effective approaches to tailor RNP receptors and fluorescent RNP sensors for small molecules. In vitro selection of an RNA-derived pool of RNP afforded RNP receptors specific for a phosphotyrosine residue within a defined amino-acid sequence Gly-Tyr-Ser-Arg. The RNP receptor for the specific phosphotyrosine residue was successfully converted to a fluorescent RNP sensor for sequence-specific recognition of a phosphorylated tyrosine by screening a pool of fluorescent phosphotyrosine-binding RNPs generated by a combination of the RNA subunits of phosphotyrosine-binding RNPs and various fluorophore-modified peptide subunits. The phosphotyrosine-binding RNP receptor and fluorescent RNP sensor constructed from the RNP receptor not only discriminated phosphotyrosine against tyrosine, phosphoserine, or phosphothreonine, but also showed specific recognition of amino acid residues surrounding the phosphotyrosine residue. A fluorescent RNP sensor for one of the tyrosine phosphorylation sites of p100 coactivator showed a binding affinity to the target site ~95-fold higher than the other tyrosine phosphorylation site. The fluorescent RNP sensor has an ability to function as a specific fluorescent sensor for the phosphorylated tyrosine residue within a defined amino-acid sequence in HeLa cell extracts.     

Preparation of pseudo glycoamino acid and its application to glycopeptide synthesis

A pseudo glycoamino acid composed of a 1,3-diol structure and an amino acid was synthesized. This amino acid analog can act as an alternative acceptor to an amino acid containing GlcNAc for transglycosylation by Endo-M. A pseudo glycopeptide was synthesized using the pseudo glycoamino acid by a solid phase procedure. We attempted transglycosylation of N-glycan to the peptide using Endo-M.     

Phosphorylation of an alpha-synuclein peptide fragment enhances metal binding

By using Tb3+ as a luminescent probe, we demonstrate that the phosphorylation state of a 14-residue peptide fragment of alpha-synuclein, a protein implicated in Parkinson's Disease, dramatically affects the metal ion affinity of the peptide. Whereas the unphosphorylated peptide and its phosphoserine analogue show weak Tb3+ binding, its phosphotyrosine analogue shows tight 1:1 binding as well as 2:1 and 3:1 Tb:peptide adducts. Our data suggest that the phosphorylated amino acid must be appropriately positioned among additional ligating residues to establish this phosphorylation-dependent metal binding.     

Cholic acid-based fluorescent probes for enantioselective recognition of trifunctional amino acids

The ditopic fluorescent photoinduced electron transfer (PET) amino acid sensory probes and were designed and synthesized from cholic acid. To confer the probes with specific binding ability, an amidothiourea moiety and a cyclic diamino-chiral receptive site were introduced on the C17 side chain and the C7 and C12 hydroxyl pendants, respectively. In acetonitrile, the probes demonstrated differential binding toward trifunctional amino acids like serine, lysine, threonine and tyrosine against other simple amino acids. Enantioselectivities (KD/KL) of up to 8.9 and sensitivities in the micromolar range with the probes were observed for trifunctional amino acids.     

Aza-amino acid scanning of secondary structure suited for solid-phase peptide synthesis with fmoc chemistry and aza-amino acids with heteroatomic side chains

Aza-peptides, peptide analogues in which the alpha-carbon of one or more of the amino acid residues is replaced with a nitrogen atom, exhibit a propensity for adopting beta-turn conformations. A general Fmoc-protection protocol for the stepwise solid-phase synthesis of aza-peptides has now been developed based on the activation of N'-alkyl fluoren-9-ylmethyl carbazates with phosgene for coupling the aza-amino acid residues. This method has proven effective for introducing aza-amino acid residues with aliphatic (Ala, Leu, Val, and Gly) and aromatic (Phe, Tyr, and Trp) side chains. Acid promoted loss of aromatic side chains was noted with aza-Trp and aza-Tyr residues during peptide cleavage and suppressed by temperature control in the case of the latter. In addition, aza-peptides with heteroatomic side chain residues (Lys, Orn, Arg, and Asp) were conveniently synthesized using this protocol. Partial aza-amino acid scans were performed on three biologically active peptides: the potent tetrapeptide melanocortin receptor agonist, Ac-His-d-Phe-Arg-Trp-NH2; the growth hormone secretagogue hexapeptide, GHRP-6, His-d-Trp-Ala-Trp-d-Phe-Lys-NH2; and the human calcitonin gene-related peptide (hCGRP) antagonist, FVPTDVGPFAF-NH2. This practical procedure for aza-amino acid scanning using Fmoc-based solid-phase synthesis should find general utility for probing the existence and importance of beta-turn conformations in bioactive peptides.     

The role of phosphorylated residues in peptide-peptide noncovalent complexes formation

Electrospray mass spectrometry (ESI-MS) has become the tool of choice for the study of noncovalent complexes. Our previous work has highlighted the role of phosphorylated amino acid residues in the formation of noncovalent complexes through electrostatic interaction with arginine residues’ guanidinium groups. In this study, we employ tandem mass spectrometry to investigate the gas-phase stability and dissociation pathways of these noncovalent complexes. The only difference in the three phosphopeptides tested is the nature of the phosphorylated amino acid residue. In addition the absence of acidic residues and an amidated carboxyl terminus insured that the only negative charge came from the phosphate, which allowed for the comparison of the noncovalent bond between arginine residues and each of the different phosphorylated residues. Dissociation curves were generated by plotting noncovalent complex ion intensities as a function of the nominal energy given to the noncovalent complex ion before entering the collision cell. These results showed that noncovalent complexes formed with phosphorylated tyrosine were the most stable, followed by serine and threonine, which had similar stability.     

蜂毒肽类似物的合成和生物活性研究

Melittin(GIGAVLKVLTTGLPALISWIKRKRQQ-NH₂)是蜂毒中含26个氨基酸残基的多肽,具有抗菌和溶血等生物活性,是典型的阳离子抗菌肽.本文设计合成了蜂毒肽C端15残基肽片段(GLPALISWIKRKRQQ-NH₂)及其类似物(15残基).研究了Melittin及这些合成肽的抗菌活性、溶血活性、疏水性及二级结构.结果表明,合成的类似物的溶血活性明显降低,抗菌活性基本保留,且与其疏水性相关.类似物中与碱性氨基酸簇(KRKR)距离较远的残基的疏水性对其抗菌活性有较大的贡献.多肽溶血与抗菌机理不同.类似物的抗菌活性和溶血活性与其二级结构(α-螺旋结构)没有明显的相关性.     

Photolabile and paramagnetic reagents for the investigation of transmembrane signaling events.

To investigate the dynamics of membrane processes that may be integral components of specific transmembrane signaling events we have synthesized several novel paramagnetic probes and their photoreactive counterparts. The structure of these probes was designed to (1) restrict "flipping" across the membrane bilayer; (2) contain paramagnetic or photoreactive moieties that could be placed at specific depths within the bilayer; (3) provide information about membrane structure as well as dynamics of protein movement; and (4) in the case of the photoreactive probes, be of high specific radioactivity. The molecules described in this paper consist of amino acid, dipeptide, or carbohydrate groups attached to arylazide- or nitroxide-bearing fatty acids. The synthesis and initial characterization of these membrane probes is described.