Threonine-containing Peptides Research Articles

N- and O-acetylation of threonine residues in the context of proteomics.

The detection of post-translational modifications (PTMs) of proteins is a matter of intensive research. Among all possible pitfalls that may lead to misidentifications, the chemical stability of modified peptides is scarcely questioned. Global proteomic studies devoted to protein acetylation are becoming popular. Thus, we were concerned about the intrinsic stability of O-acetylated peptides because of the O-N acyl transfer reactivity occurring when an amino moiety is present in the vicinity of the acylated hydroxyl group. Here, the behavior of isomeric O- and N-acetylated, N-terminal threonine-containing peptides was explored in a standard proteomic workflow. We demonstrated a strong chemical instability of O-acetylation, which prevents its detection.

Crystal-State Structure Analysis of β-Hydroxy-γ-lactam Constrained Ser/Thr Peptidomimetics

Crystallographic evidence has been obtained in support of the ability of β-hydroxy-α-amino-γ-lactams to induce β-turn conformations within peptides. Two dipeptide model systems of these β-hydroxylated variants of the so-called Freidinger-Veber lactams were prepared from the reaction of N-(Fmoc)oxiranylglycine 2 with methyl m-amino benzoate and methyl lysinate, respectively, and crystallized from benzene or ether. In the solid-state, the lactam moiety was found to adopt dihedral angle geometry similar to extended as well as type II β-turn conformations contingent on the C-terminal amino ester moiety. The β-hydroxy group of the trans-isomer was shown to point away from the turn suggesting its potential for interaction with receptors in conformationally rigid mimics of serine and threonine-containing peptides.

Negative ion dissociation of peptides containing hydroxyl side chains

The dissociation of deprotonated peptides containing hydroxyl side chains was studied by electrospray ionization coupled with Fourier transform ion cyclotron resonance (ESI-FTICR) via sustained off-resonance irradiation collision induced dissociation (SORI-CID). Dissociation under post-source decay (PSD) conditions was performed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF). This work included hexapeptides with one residue of serine, threonine, or tyrosine and five inert alanine residues. During SORI-CID and PSD, dissociation of [M-H](-) yielded c- and y-ions. Side-chain losses of formaldehyde (HCHO) from serine-containing peptides, acetaldehyde (CH(3)CHO) from threonine-containing peptides, and 4-methylene-2,5-cycohexadienone (C(7)H(6)O) from tyrosine-containing peptides were generally observed in the negative ion PSD and SORI-CID spectra. Side-chain loss occurs much less from tyrosine-containing peptides than from serine- and threonine-containing peptides. This is probably due to the bulky side chain of tyrosine, resulting in steric hindrance and poor geometry for dissociation reactions. Additionally, a selective cleavage leading to the elimination of the C-terminal residue from [M-H](-) was observed from the peptides with serine and threonine at the C-terminus. This cleavage does not occur in the dissociation of peptides with an amide group at the C-terminus or peptides with neutral or basic residues at the C-terminus. It also does not occur with tyrosine at the C-terminus. Both the C-terminal carboxylic acid group and the hydroxyl side chain of the C-terminal residue must play important roles in the mechanism of C-terminal residue loss. A mechanism involving both the C-terminal carboxylic acid group and a hydroxyl side chain of serine and threonine is proposed.

Enhanced detection of sulfo‐peptides as onium salts in matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

A new two-component system, consisting of a matrix and an onium salt as comatrix, is described for detection of sulfo-peptides in the positive mode by matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Binary iodonium salts were superior to quaternary phosphonium salts in terms of suppression of desulfation and salt formation with the carboxyl group. Of the iodonium salts examined, bis(4-tert-butylphenyl)iodonium (BTI) hexafluorophosphate and bromide were most effective in giving intensive molecular ion signals in the form of [M(BTI)+BTI](+). The conditions optimized for O-sulfated tyrosine-containing peptides could be applicable for O-sulfated serine- and threonine-containing peptides. In the case of a phospho-peptide, a molecular ion appeared more intensively as a proton adduct than as a BTI adduct.

An automated peptide and protein thiazolidine coupling chemistry for biosensor immobilization giving a unique N-terminal orientation

1 Abbreviations used: Pms, 2-(phenyl(methyl)sulfonio)ethyloxycarbonyl; mAb, monoclonal antibody; EGFR, epidermal growth factor receptor; Peptide and protein sensor chips and microarrays are important tools for biorecognition analysis for the study of both the functions and interactions of biomolecules [1– 4]. They can also be employed in high-throughput assays that require only minute amounts of analyte [5–7]. The success of such studies is dependent on the preparation of appropriately derivatized surfaces. The attachment of peptides and proteins in a specific orientation rather than in a random manner can result in a substantial increase in assay sensitivity and selectivity [1,8]. A number of methods to achieve this have been reported previously. Proteins and peptides containing single free sulfhydryl groups have been immobilized in a defined orientation by thiol capture [1,4,9–11]. A traceless version of the Staudinger ligation, in which an azido-peptide or azido-protein is reacted with a phosphinothioester-derived slide to generate a functional peptide microarray, has been used [12]. We have previously described a simple means of immobilizing N-terminal serineand threonine-containing peptides and proteins onto surfaces bearing streptavidin [13]. Derivatization of the peptide or protein following conversion into an N-terminal carbonyl precursor was achieved by covalent attachment of a water-soluble dipeptide containing a lysine residue bearing an N-cysteine-derived 1,2-aminothiol and an N-biotin moiety. However, each of these immobilization strategies has both advantages and disadvantages, with the latter including the need for a conveniently located functional group or amino acid within the peptide and protein to enable subsequent immobilization in an orientation that will allow

Novel rearranged ions observed for protonated peptides via metastable decomposition in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

C-terminal rearrangement ions [b n-1 + H 2O] (where n refers to the total number of residues of peptides) are frequently observed for peptides which contain basic amino acid(s), especially arginine, at or near their N termini in low- and high-energy collision-induced dissociation or post-source decay (PSD) spectra. Here we report a novel rearrangement, associated with PSD for serine- or threonine-containing peptides that are susceptible to C-terminal rearrangement. Based on PSD analyses of serine- or threonine-containing bradykinin and its analogs, which have been ethyl-esterified or 18O labeled at their C termini, the [ b k + H 2O] (where k denotes the position adjacent to the left of the Ser/Thr residue) ion is generally thought to be formed by the transfer of the hydroxyl moiety of a serine or threonine residue to the carbonyl group of the residue to its left accompanied by the loss of the remaining C-terminal portion of the peptide. When the Ser/Thr is at or near the C terminus, the present [ b k + H 2O] ion could be formed via two pathways, i.e., the Ser/Thr-related rearrangement and the conventional C-terminal rearrangement, which has been clearly verified by 18O labeling at the C terminus. In addition, the ions which are formally designated as [ y m b l + H 2O], where y m b l denotes a b-type internal ion, are also briefly described.

Intracytoplasmic growth and virulence of Listeria monocytogenes auxotrophic mutants

The intracellular growth of several auxotrophic mutants of Listeria monocytogenes was examined in cell culture, and virulence was evaluated in mice by intravenous injection of log-phase bacteria. L. monocytogenes transposon insertion mutants requiring either uracil, phenylalanine, glycine, proline, or nicotinic acid for growth were fully virulent and grew similarly to the parental strain as shown by their growth rates in cell culture. Those requiring all three aromatic amino acids (phenylalanine, tryptophan, and tyrosine) or adenine were 1.5 log10 less virulent than the wild type. A threonine auxotroph, which showed enhanced growth in the presence of threonine-containing peptides as compared with that in the presence of free threonine, was approximately 1 log10 less virulent than the wild type. When host cells were deprived of specific amino acids required by both the host cell and L. monocytogenes, the bacteria continued to grow intracellularly. These studies suggest that the cytoplasm of eucaryotic cells behaves like rich medium, facilitating the growth of an intracellular bacterial pathogen with complex growth requirements. In addition, results related to amino acid deprivation during intracellular growth and specific extracellular growth requirements of a threonine auxotroph suggest that L. monocytogenes may utilize intracellular peptides as a source of amino acids.

Gas-phase fragmentations of anionic complexes of serine- and threonine-containing peptides

The (M + Ca − 3H) − and (M − H) − anions of 11 tetra- and larger peptides that contain either Ser or Thr are studied by using fast atom bombardment (FAB) and collision-induced decomposition (CID). The Ser- and Thr-containing negative ions decompose to lose either 30 u (Ser) or 44 u (Thr) in the ion source, metastably, and upon CID. The CID of the FAB-formed (M + Ca − 3H − X) − or (M − H − X) − fragment ions, in which X = 30 or 44 u, is the same as the CID of (M + Ca − 3H) − or (M − H) − anions of peptides in which the Ser and Thr residues are replaced with Gly. Two possible mechanisms for the decompositions are discussed, and theoretical molecular mechanical calculations provide further evidence regarding the mechanisms.

Solid-phase synthesis of O-phosphorothioylserine- and -threonine-containing peptides as well as of O-phosphoserine- and -threonine-containing peptides

This paper describes the synthesis of O-phosphorothioylserine, -threonine, and -tyrosine by sulfurization of the intermediate phosphite triester using phenylacetyl disulfide. Several deprotection procedures were investigated to find the optimal conditions necessary for obtaining the deprotected phosphorothioate amino acids. This method could then be extended to the preparation of O-phosphorothioylserine- and -threonine-containing pentapeptides on a solid phase. After completion of the synthesis of the peptides on the solid support, the amino acid residue containing the free hydroxyl function was phosphitylated using N,N-diisopropylbis(4-chlorobenzyl)phosphoramidite and subsequently sulfurized using phenylacetyl disulfide. Silylitic cleavage of the protecting groups, using thiophenol and m-cresol as scavengers, gave the pure deprotected phosphorothioylated peptides . The corresponding phosphopeptides were obtained when the phosphitylated peptides attached to the solid support were oxidized instead of sulfurized using tert-butylhydroperoxyde

Substrate recognition determinants for rhodopsin kinase: studies with synthetic peptides, polyanions, and polycations.

Rhodopsin kinase phosphorylates serine- and threonine-containing peptides from bovine rhodopsin's carboxyl-terminal sequence. Km's for the peptides decrease as the length of the peptide is increased over the range 12-31 amino acids, reaching 1.7 mM for peptide 318-348 from the rhodopsin sequence. The Km for phosphorylation of rhodopsin is about 10(3) lower than that for the peptides, which suggests that binding of rhodopsin kinase to its substrate, photolyzed rhodopsin, involves more than just binding to the carboxyl-terminal peptide region that is to be phosphorylated. A synthetic peptide from the rhodopsin sequence that contains both serines and threonines is improved as a substrate by substitution of serines for the threonines, suggesting that serine residues are preferred as substrates. Analogous 25 amino acid peptides from the human red or green cone visual pigment, a beta-adrenergic receptor, or M1 muscarinic acetylcholine receptors are better substrates for bovine rhodopsin kinase than is the peptide from bovine rhodopsin. An acidic serine-containing peptide from a non-receptor protein, alpha s1B-casein, is also a good substrate for rhodopsin kinase. However, many basic peptides that are substrates for other protein kinases--histone IIA, histone IIS, clupeine, salmine, and a neurofilament peptide--are not phosphorylated by rhodopsin kinase. Polycations such as spermine or spermidine are nonessential activators of phosphorylation of rhodopsin or its synthetic peptide 324-348. Polyanions such as poly(aspartic acid), dextran sulfate, or poly(adenylic acid) inhibit the kinase. Poly(L-aspartic acid) is a competitive inhibitor with respect to rhodopsin (KI = 300 microM) and shows mixed type inhibition with respect to ATP.

Two-step fragmentation of threonine-containing peptides

Two-step fragmentation of threonine-containing peptides