Mass spectrometric characterization of peptides containing different oxidized tryptophan residues

The term reactive oxygen species refers to small molecules that can oxidize, for example, nearby proteins, especially cysteine, methionine, tryptophan, and tyrosine residues. Tryptophan oxidation is always irreversible in the cell and can yield several oxidation products, such as 5-hydroxy-tryptophan (5-HTP), oxindolylalanine (Oia), kynurenine (Kyn), and N-formyl-kynurenine (NFK). Because of the severe effects that oxidized tryptophan residues can have on proteins, there is a great need to develop generally applicable and highly sensitive techniques to identify the oxidized residue and the oxidation product. Here, the fragmentation behavior of synthetic peptides corresponding to sequences recently identified in three skeletal muscle proteins as containing oxidized tryptophan residues were studied using postsource decay and collision-induced dissociation (CID) in matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)/TOF mass spectrometry (MS) and CID in an electrospray ionization (ESI) double quadrupole TOF-MS. For each sequence, a panel of five different peptides containing Trp, 5-HTP, Kyn, NFK, or Oia residue was studied. It was always possible to identify the modified positions by the y-series and also to distinguish the different oxidation products by characteristic fragment ions in the lower mass range by tandem MS. NFK- and Kyn-containing peptides displayed an intense signal at m/z 174.1, which could be useful in identifying accordingly modified peptides by a sensitive precursor ion scan. Most importantly, it was always possible to distinguish isomeric 5-HTP and Oia residues. In ESI- and MALDI-MS/MS, this was achieved by the signal intensity ratios of two signals obtained at m/z 130.1 and 146.1. In addition, high collision energy CID in the MALDI-TOF/TOF-MS also permitted the identification of these two isomeric residues by their v- and w-ions, respectively.     

Mass spectrometric measurement of endogenous peptides

Mass spectrometric analytical techniques utilizing fast atom bombardment are used to ionize peptides extracted from biological sources, and tandem mass spectrometry methods are used to select a unique amino acid sequence-determining fragment ion for quantification of that peptide at the picomole level. This type of analysis maximizes the molecular specificity.     

Mass spectrometric characterization of lipid-modified peptides for the analysis of acylated proteins

The analysis of acylated proteins by mass spectrometry (MS) has largely been overshadowed in proteomics by the analysis of glycosylated and phosphorylated proteins; however, lipid modifications on proteins are proving to be of increasing importance in biomedical research. In order to identify the marker ions and/or neutral loss fragments that are produced upon collision-induced dissociation, providing a means to identify the common lipid modifications on proteins, peptides containing an N-terminally myristoylated glycine, a palmitoylated cysteine and a farnesylated cysteine were chemically synthesized. Matrix-assisted laser desorption/ionization time-of-flight time-of-flight (MALDI-TOF-TOF), electrospray ionization quadrupole time-of-flight (ESI Q-TOF), and electrospray ionization hybrid triple-quadrupole/linear ion trap (ESI QqQ(LIT)) mass spectrometers were used for the analysis. The peptide containing the N-terminally myristoylated glycine, upon CID, produced the characteristic fragments a1 (240.4 Th) and b1 (268.4 Th) ions as well as a low-intensity neutral loss of 210 Da (C14H26O). The peptides containing a farnesylated cysteine residue fragmented to produce a marker ion at a m/z of 205 Th (C15H25) as well as other intense farnesyl fragment ions, and a neutral loss of 204 Da (C15H24). The peptides containing a palmitoylated cysteine moiety generated neutral losses of 238 Da (C16H30O) and 272 Da (C16H32OS); however, no marker ions were produced. The neutral losses were more prominent in the MALDI-TOF-TOF spectra, whereas the marker ions were more abundant in the ESI QqQ(LIT) and Q-TOF mass spectra.     

Mass spectrometric characterization of gelsolin isoforms

Gelsolin is the most widely expressed member of the actin capping and severing family of proteins. There are two isoforms of gelsolin: isoform 1, a secretory (plasma) protein that is 51 amino acids longer than isoform 2, a cytosolic protein, at the N‐terminus; the first 27 amino acids is a signal sequence. Both isoforms are coded by a single gene and differ as a result of alternative initiation site/splicing. The level of gelsolin in the blood and cerebrospinal fluid (CSF) is altered in many diseases including amyloidoses and other neurodegenerative disorders. Although quantitative analysis of gelsolin has been reported, lack of suitable antibodies makes it impossible to differentiate these two isoforms by immunodetection techniques and no other technique is available. Therefore, ambiguity exists whether gelsolin present in circulation is isoform 1 or also isoform 2 released from lysed cells. We report in this communication a mass spectrometric approach to identify isoform 1 of gelsolin immunopurified from human plasma and CSF. Recombinant isoform 1 was used as reference. Copyright © 2010 John Wiley & Sons, Ltd.     

Mass spectrometric characterization of photooxidative protein modifications in Arabidopsis thaliana thylakoid membranes

Oxidative and nitrosative stress leaves footprints in the plant chloroplast in the form of oxidatively modified proteins. Using a mass spectrometric approach, we identified 126 tyrosine and 12 tryptophan nitration sites in 164 nitrated proteolytic peptides, mainly from photosystem I (PSI), photosystem II (PSII), cytochrome b(6) /f and ATP-synthase complexes and 140 oxidation products of tyrosine, tryptophan, proline, phenylalanine and histidine residues. While a high number of nitration sites were found in proteins from four photosynthetic complexes indicating that the nitration belongs to one of the prominent posttranslational protein modifications in photosynthetic apparatus, amino acid oxidation products were determined mostly in PSII and to a lower extent in PSI. Exposure of plants to light stress resulted in an increased level of tyrosine and tryptophan nitration and tryptophan oxidation in proteins of PSII reaction center and the oxygen-evolving complex, as compared to low light conditions. In contrast, the level of nitration and oxidation of these amino acid residues strongly decreased for all light-harvesting proteins of PSII under the same conditions. Based on these data, we propose that oxidative modifications of proteins by reactive oxygen and nitrogen species might represent an important regulatory mechanism of protein turnover under light stress conditions, especially for PSII and its antenna proteins.     

Mass spectrometric analysis of protein phosphorylation

Phosphorylation is one of the most common posttranslational modifications of proteins in eukaryotic cells; it plays an important role in a wide spectrum of biological processes. This makes its study an important task for understanding cell functioning mechanisms. The aim of phosphoproteomics is a global mass spectral analysis of the phosphoprotein composition of cells, i.e., phosphoproteome. Nowadays, new effective methods are actively developed, which succeed not only in the detection of phosphorylated proteins but also in the determination of phosphorylated amino acid residues (phosphorylation sites) and in the quantitative comparison of phosphorylation among several specimens. Despite the analysis of protein phosphorylation remains a complicated problem, the available methods nowadays allow the detection of thousands of phosphorylation sites in the very same experiment. The present review covers the main methods utilized in contemporary phosphoproteomics: phosphoprotein and phosphopeptides enrichment as well as the mass spectrometric analysis of protein phosphorylation.     

Mass spectrometric characterization of phosphorylated peptides using MALDI in‐source decay via redox reactions

Matrix‐assisted laser desorption/ionization in‐source decay (MALDI‐ISD) has been used for characterization of a phosphorylated peptides and proteins because labile phosphate group is not lost during the MALDI‐ISD process. The conventional MALDI‐ISD is initiated by the hydrogen transfer from reducing matrix molecules to peptide backbone, leading to c′‐ and z′‐series ions. In contrast, when an oxidizing chemical 5‐nitrosalicylic acid (5‐NSA) is served as the MALDI‐ISD matrix, a‐ and x‐series ions are specifically generated by hydrogen abstraction from peptide backbone to matrix molecule. The 5‐NSA provides useful complementary information to the conventional MALDI‐ISD for the analysis of amino acid sequencing and site localization of phosphorylation in peptides. The MALDI‐ISD with reducing and oxidizing matrix could be a useful method for the de novo peptide sequencing. Copyright © 2012 John Wiley & Sons, Ltd.     

Mass spectrometric characterization of human skin elastin peptides produced by proteolytic digestion with pepsin and thermitase

This study investigated peptides resulting from the digestion of human skin elastin with pepsin and thermitase. Characterization of the peptides was performed using two complementary mass spectrometric techniques; LC/ESI-ion trap and nano-ESI-qTOF MS. 155 different peptides were identified using a combined database based and de novo sequencing approach resulting in a total sequence coverage of 65.4% calculated on the basis of the precursor tropoelastin (accession number A32707). A potential hydroxylation was found in 29% of the recovered prolines. Furthermore, the absence of amino acids expressed by exon 26A could be confirmed. However, contrary to earlier studies, amino acids expressed by exon 22 seem to exist.     

Mass spectrometric amino acid sequence determination in arginine-containing peptides

Methods of converting arginine residues into ornithine, N^δ-pyrimidylornithine, N^δ-imidazolidinonidenornithine residues in peptides have been developed. The modified peptides undergo the amino acid type of fragmentation, so that mass spectrometry can now be used for amino acid sequence determination in arginine-containing peptides.     

Mass spectrometric evidence for mechanisms of fragmentation of charge-derivatized peptides

Mass spectrometry of charged derivatives of peptides has been a growing area of interest in the past decade. Fragmentation of charged derivatives of peptides is believed to be different from than that of protonated peptides when analyzed by collisionally activated dissociation-tandem mass spectrometry (CAD-MS/MS). The charged derivatives fragment by charge-remote fragmentation mechanisms, which are usually classified as high-energy (HE)-CAD processes. Our objective in the present study is to investigate the mechanism of fragmentation of charged derivatives of peptides when analyzed by matrix-assisted laser desorption/ionization-postsource decay-mass spectrometry (MALDI-PSD-MS) and electrospray ionization (ESI)-CAD-MS/MS (ion trap), which involve low-energy processes. Three major types of hydrogens (alpha, beta, and amide) are available for migration during the formation of the *a(n) ions (the predominant ion series produced from these charged derivatives). To pinpoint which of the three hydrogens is involved in the formation of the *a(n) ions, deuterium-labeled peptide derivatives with labels at specific sites were synthesized and analyzed by MALDI-PSD-MS and ESI-CAD-MS/MS. Our results suggest that the amide hydrogen of the residue at which the cleavage occurs shifts during the formation of *a(n); this observation serves as evidence for the mechanism proposed earlier by Liao et al. for fragmentation of such charged derivatives. The results also help elucidate the structure of the *a(n) ions, *b(n) ions, and others formed during cleavage at the proline residue, as well as the ions formed during loss of the C-terminal residue from these charged derivatives.     

Selenium-sulfur analogs. Mass spectrometric comparison of substituted selenazoles and thiazoles

The electron impact mass spectra of a series of thiazoles and selenazoles were compared. No major differences in the mass spectral behavior between the sulfur and selenium containing compounds were observed. Isotopic labelling with deuterium permitted deconvolution of ion peaks of identical mass and composition, but arising from different fragmentation pathways.     

Mass spectrometric characterization of two novel inflammatory peptides from the venom of the social wasp Polybia paulista

The social wasp P. paulista is relatively common in southeast Brazil causing many medically important stinging incidents. The seriousness of these incidents is dependent on the amount of venom inoculated by the wasps into the victims, and the characteristic envenomation symptoms are strongly dependent on the types of peptides present in the venom. In order to identify some of these naturally occurring peptides available in very low amounts, an analytical protocol was developed that uses a combination of reversed-phase and normal-phase high-performance liquid chromatography (HPLC) of wasp venom for peptide purification, with matrix-assisted laser desorption/ionization time-of-flight post-source decay mass spectrometry (MALDI-Tof-PSD-MS) and low-energy collision-induced dissociation (CID) in a quadrupole time-of-flight tandem mass spectrometry (QTof-MS/MS) instrument for peptide sequencing at the sub-picomole level. The distinction between Leu and Ile was achieved both by observing d-type fragment ions obtained under CID conditions and by comparison of retention times of the natural peptides and their synthetic counterparts (with different combinations of I and/or L at N- and C-terminal positions). To distinguish the isobaric residues K and Q, acetylation of peptides was followed by Q-Tof-MS analysis. The primary sequences obtained were INWLKLGKMVIDAL-NH(2) (MW 1611.98 Da) and IDWLKLGKMVMDVL-NH(2) (MW 1658.98 Da). Micro-scale bioassay protocols characterized both peptides as presenting potent hemolytic action, mast cell degranulation, and chemotaxis of polymorphonucleated leukocyte (PMNL) cells. The primary sequences and the bioassay results suggest that these toxins constitute members of a new sub-class of mastoparan toxins, directly involved in the occurrence of inflammatory processes after wasp stinging.     

Mass spectrometric approaches for the identification of anthracycline analogs produced by actinobacteria

Anthracyclines are a well‐known chemical class produced by actinobacteria used effectively in cancer treatment; however, these compounds are usually produced in few amounts because of being toxic against their producers. In this work, we successfully explored the mass spectrometry versatility to detect 18 anthracyclines in microbial crude extract. From collision‐induced dissociation and nuclear magnetic resonance spectra, we proposed structures for five new and identified three more anthracyclines already described in the literature, nocardicyclins A and B and nothramicin. One new compound 8 (4‐[4‐(dimethylamino)‐5‐hydroxy‐4,6‐dimethyloxan‐2‐yl]oxy‐2,5,7,12‐tetrahydroxy‐3,10‐dimethoxy‐2‐methyl‐3,4‐dihydrotetracene‐1,6,11‐trione) was isolated and had its structure confirmed by ¹H nuclear magnetic resonance. The anthracyclines identified in this work show an interesting aminoglycoside, poorly found in natural products, 3‐methyl‐rhodosamine and derivatives. This fact encouraged to develop a focused method to identify compounds with aminoglycosides (rhodosamine, m/z 158; 3‐methyl‐rhodosamine, m/z 172; 4′‐O‐acethyl‐3‐C‐methyl‐rhodosamine, m/z 214). This method allowed the detection of four more anthracyclines. This focused method can also be applied in the search of these aminoglycosides in other microbial crude extracts. Additionally, it was observed that nocardicyclin A, nothramicin and compound 8 were able to interact to DNA through a DNA‐binding study by mass spectrometry, showing its potential as anticancer drugs. Copyright © 2016 John Wiley & Sons, Ltd.     

Mass spectrometric characterization of poly(ethylene terephthalate-co-p-oxybenzoate)

Copolyesters containing ethylene terephthalate and p-oxybenzoate units in mole ratios of ca. 80:20, 70:30, 40:60, and 20:80, respectively, were examined by direct pyrolysis–mass spectrometry to obtain information about the thermal stabilities and the sequences distributions of the two types of units as a function of composition. Thermal stability as measured by rate of volatilization increased with increasing oxybenzoate content, as did the amount of char residue formed. The electron impact mass spectrum of the volatile fragments showed the formation of predominantly linear fragments with carboxyl and vinyl end groups. Dimer, trimer, and tetramer fragments containing either or both types of units could be identified and their relative amounts could be estimated from peak intensities. The results were consistent with those expected for random distributions of the two units, i.e., for statistical copolymers.     

Synthesis and biological evaluation of a series of novel salicylanilides as inhibitors of EGFR protein tyrosine kinases

The synthesis and biological evaluation of two series of salicylanilide derivatives on the EGFR and ErbB-2 tyrosine kinases inhibitory activities were conducted.Of the tested compounds those having an additional aryl group substituted on the anilino ring were active on the EGFR tyrosine kinase inhibition(7a-c and 13a,13c,13d,13f).The inhibitory activities were all in the low micromolar or submicromolar range.In addition,compound 13a was found to have dual inhibitory activities both on EGFR and ErbB-2 tyrosine kinases(1.654±1.280 and 7.134±1.265μmol/L).     

Mass spectrometric study of novel estra derivatives of amino acids and peptides

The mass spectra of novel estra derivatives of amino acids and peptides were studied using electron impact and fast atom bombardment ionization, high-resolution data and mass-analysed ion kinetic energy spectrometry. The characteristic peaks of the basic nucleus of estrogenic steroids, the amino acid sequence of the peptide chain and partial skeletal rearrangement of some of these compounds were observed.     

Mass spectrometric detection of biotinylated peptides captured by avidin functional affinity electrophoresis

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to detect biotinylated peptides captured by avidin functional affinity electrophoresis (AFAEP). Peptide samples loaded onto AFAEP were heated with sodium dodecyl sulfate to ensure that the peptides are negatively charged, and thus migrate electrophoretically toward the cathode through the embedded avidin zone in the middle of the gel. To detect the biotinylated peptides, the band containing the avidin-biotinylated peptide complexes was excised from a 7.5% w/v native polyacrylamide gel, and biotinylated peptides were extracted with aqueous 95% v/v formamide (pH 8.2), aqueous 6 M guanidine HCl (pH 1.5), or water, at temperatures from 4 to 95 degrees C for periods from 5 min to 24 h. It was observed that all three solvents are capable of extracting biotinylated peptides and avidin from the gel, but the best results were obtained with aqueous 95% v/v formamide (pH 8.2) at 65 degrees C for 20 min. However, some AFAEP-captured biotinylated peptides are not stable and are extensively modified by formamide during extraction at too high a temperature or too long an extraction time.