Specific tandem mass spectrometric detection of AGE‐modified arginine residues in peptides

Glycation is a non‐enzymatic reaction of protein amino and guanidino groups with reducing sugars or dicarbonyl products of their oxidative degradation. Modification of arginine residues by dicarbonyls such as glyoxal and methylglyoxal results in formation of advanced glycation end‐products (AGEs). In mammals, these modifications impact in diabetes mellitus, uremia, atherosclerosis and ageing. However, due to the low abundance of individual AGE‐peptides in enzymatic digests, these species cannot be efficiently detected by LC‐ESI‐MS‐based data‐dependent acquisition (DDA) experiments. Here we report an analytical workflow that overcomes this limitation. We describe fragmentation patterns of synthetic AGE‐peptides and assignment of modification‐specific signals required for unambiguous structure retrieval. Most intense signals were those corresponding to unique fragment ions with m/z 152.1 and 166.1, observed in the tandem mass spectra of peptides, containing glyoxal‐ and methylglyoxal‐derived hydroimidazolone AGEs, respectively. To detect such peptides, specific and sensitive precursor ion scanning methods were established for these signals. Further, these precursor ion scans were incorporated in conventional bottom‐up proteomic approach based on data‐dependent acquisition (DDA) LC‐MS/MS experiments. The method was successfully applied for the analysis of human serum albumin (HSA) and human plasma protein tryptic digest with subsequent structure confirmation by targeted LC‐MS/MS (DDA). Altogether 44 hydroimidazolone‐ and dihydroxyimidazolidine‐derived peptides representing 42 AGE‐modified proteins were identified in plasma digests obtained from type 2 diabetes mellitus (T2DM) patients. Copyright © 2015 John Wiley & Sons, Ltd.     

An improved method for de novo sequencing of arginine-containing, Nalpha-tris(2,4,6-trimethoxyphenyl)phosphonium-acetylated peptides

An improved method for de novo sequencing of arginine-containing peptides modified with succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-Ac-OSu) is reported. A tagging reagent, TMPP-Ac-OSu, was introduced to improve the sequence analysis of peptides owing to the simplified fragmentation pattern. However, peptides containing arginine residues did not fragment efficiently even after TMPP-Ac modification at their N-termini. This report describes how fragmentation efficiency of TMPP-Ac-modified arginine-containing peptides was significantly improved by modifying the guanidino group on the side chain of arginine with acetylacetone.     

Application of the NG-(2,2,5,7,8-pentamethylchroman-6-sulphonyl) derivative of FMOC-arginine to peptide synthesis

The trifluoroacetic acid labile pentamethylchromanylsulphonyl protecting group for the guanidino group of arginine has been used in conjunction with the base-labile Fmoc Nα protecting group for the synthesis of arginine-containing peptides.     

4(R/S)-Guanidinylprolyl collagen peptides: on-resin synthesis, complexation with plasmid DNA, and the role of peptides in enhancement of transfection

Chimeric collagen peptides containing cationic 4(R/S)-guanidinylproline are synthesized by in situ amidinylation of 4(R/S)-aminoproline residues. These peptides uniquely enhance the transfection efficiency of GFP-encoded plasmid DNA (pRmHa3-GFP) into cells through efficient DNA condensation resulting from nonspecific electrostatic interactions of cationic guanidino groups and localize in subcytoplasmic organelles.     

Epitopic Peptides Identified by LC?CELISA and LC?CMS

A new strategy of a liquid chromatography (LC)-enzyme linked immunosorbent assay (ELISA) combined with liquid chromatography-mass spectrometry (LC?CMS) was developed to identify the epitopic peptides for the antigen in Streptococcus suis. Metallendopeptidase, a potential antigen for S. suis, was digested by trypsin. The tryptic solution was separated by LC under optimal condition and collected into tubes. After it was dried using a cooling vacuum, the immunogenecity of peptides in each tube was investigated by ELISA against its corresponding antibody. All peptides in each tube with positive signals based on ELISA results were identified by LC?CMS. Accordingly, six putative peptides of metallendopeptidase were found and synthesized, all of which possessed high antigenicity recognized by the antibody.     

Herstellung von Argininpeptiden mitN 7,N 8-(1,2-Dihydroxycyclohex-1,2-ylen)-Schutz

A method for the synthesis of arginine peptides is described, in which the side chain guanidine function is blocked through reaction with 1,2-cyclohexanedione in borate buffers.Coupling to the carboxyl group of arginine was achieved by active ester, by dicyclohexylcarbodiimide/1-hydroxybenzotriazole¹, and by the mixed anhydride methods². Neither lactam formation nor acylation of the vicinal hydroxyls of the N⁷, N⁸-(1,2-dihydroxycyclohex-1,2-ylene) guanidino group was observed.Removal of the protecting group is strongly influenced by steric factors. Some side reactions observed during modification of peptides and protein fragments with 1,2-cyclohexanedione are also described.

Herrn Professor Dr.Hermann Stetter zum 65. Geburtstag gewidmet.     

Benzylic rearrangement stable isotope labeling for quantitation of guanidino and ureido compounds in thyroid tissues by liquid chromatography-electrospray ionization mass spectrometry

Benzylic rearrangement stable isotope labeling (BRSIL) was explored to quantify the guanidino and ureido compounds (GCs and UCs). This method employed a common reagent, benzil, to label the guanidino and ureido groups through nucleophilic attacking then benzylic migrating. The use of BRSIL was investigated in the analysis of five GCs (creatine, l-arginine, homoarginine, 4-guanidinobutyric acid, and methylguanidine) and two UCs (urea and citrulline). The labeling was found simple and specific. The introduction of bi-phenyl group and the generation of nitrogen heterocyclic ring in the benzil-d0/d5 labeled GCs and UCs improved the retention behaviors in liquid chromatography (LC) and increased the sensitivity of electrospray ionization mass spectrometry (ESI MS) detection. The fragment ion pairs of m/z 182/187 and m/z 210/215 from the benzil-d0/d5 tags facilitated the discovery of potential GCs and UCs candidates residing in biological matrices. The use of BRSIL combined with LC-ESI MS was applied for simultaneously quantitation of GCs and UCs in thyroid tissues. It was demonstrated that nine GCs and UCs were detected, six of which were further quantified based on corresponding standards. It was concluded that five GCs and UCs (l-arginine, homoarginine, 4-guanidinobutyric acid, methylguanidine, and citrulline) were statistically significantly different (p < 0.05) between the para-carcinoma and carcinoma thyroid tissue samples.     

Evaluation of protein quantification using standard peptides containing single conservative amino acid replacements

Structural analogs are evaluated as peptide internal standards for protein quantification with liquid chromatography‐multiple reaction monitoring mass spectrometry (LC‐MRM); specifically, single conservative amino acid replacements (SCAR) are performed to create tagged standards that differ by the addition or subtraction of a single methylene group in one amino acid side chain. Because the performance of stable isotope‐labeled standards (SIS) has been shown to be superior to structural analogs, differences in both development and quantitative performance between assays based on SIS and SCAR peptides are explored. To establish an assay using the structural analogs, analysis of endogenous, SCAR and SIS peptides was performed to examine their ion signal, fragmentation patterns and response in LC‐MRM. Performance of SCAR and SIS peptides was compared for quantification of epidermal growth factor receptor from lung cancer cell lysates and immunoglobulin M in the serum of multiple myeloma patients. Copyright © 2012 John Wiley & Sons, Ltd.     

Fluorescence derivatization of bioactive peptides for high-performance liquid chromatography

New fluorescence derivatization techniques are introduced for the quantitative determination of arginine- or tyrosine-containing peptides by high-performance liquid chromatography with fluorescence detection. The methodology offers enhancement of both sensitivity and specificity. It is thus suitable for trace (0.1–10 pmol) analysis of the bioactive peptides such as angiotensins and enkephalins.     

Synthesis of 17,18-diornithine-beta-corticotropin-(1-24)-tetracosapeptide, a biologically active analogue of adrenocorticotropic hormone

β-Corticotropin (ACTH) possesses a striking sequence of basic amino-acids-Lys-Lys-Arg-Arg- (No. 15–18) which is apparently necessary for the corticotropic activity: synthetic N-terminal peptides containing this sequence are active, those lacking for example the two arginines as in the hexadecapeptide II [2] [3] [4] are essentially inactive. We have synthesized 17,18-diornithine-β-corticotropin-(1–24)-tetracosapeptide (III), an analogue of the highly active β-corticotropin-(1–24)-tetracosapeptide (I), in which the arginine residues 17 and 18 are replaced by ornithine. Quite unexpectedly this analogue is as highly potent in vitro (SAFFRAN & SCHALLY ) and in vivo (s.c. application, SAYERS test) as the tetracosapeptide with the ‘natural’ sequence. This equal potency holds also for lipotropic and melanotropic activity. We may conclude that the two guanidino groups of the arginines in pos. 17 and 18 are not essential for biological activity, but that they may be replaced by amino groups, as in ornithine. Due to the replacement of the guanidino groups by the more easily protectable amino group, the diornithine analogue III is more readily synthesized than the original diarginine polypeptide I.     

Quantitation of human glutathione S-transferases in complex matrices by liquid chromatography/tandem mass spectrometry with signature peptides

Direct quantitation of glutathione S-transferase (GST) isoforms [alpha (GST-A) and micro (GST-M)] in human liver cytosol was achieved by liquid chromatography/tandem mass spectrometry (LC/ESI-MS/MS) analysis of signature peptides of GST-A and GST-M and their corresponding stable isotopic peptide internal standards via multiple reaction monitoring (MRM). The selection of signature peptides was performed via trypsin digestion of commercially available cDNA-expressed GST-A1 and GST-M1, followed by LC/ESI-MS/MS with an ion trap mass spectrometer and sequencing with the TurboSEQUEST application. Quantitative analysis of the selected signature peptides in the multi-reaction monitoring (MRM) mode was performed using a triple-quadruple mass spectrometer. A series of human cytosol samples was quantitatively analyzed for levels of GST-A and GST-M. The total level of GST-A and GST-M obtained from this LC/ESI-MS/MS method was well correlated with the total level of GST determined by the 1-chloro-2,4-dinitrobenzene (CDNB) method.     

Effects of an arginine-selective tagging procedure on the fragmentation behavior of peptides studied by electrospray ionization tandem mass spectrometry (ESI-MS/MS)

The presence of arginine as the naturally occurring amino acid with the highest gas-phase basicity strongly influences the fragmentation behavior of peptides undergoing collision-induced dissociation. Using a derivatization procedure recently developed in our group, based on a reversible reaction of the guanidino group with 2,3-butanedione and an arylboronic acid, we examined how this label affects the fragmentation patterns of labeled versus unlabeled peptides in MS/MS experiments. As part of this fundamental study, two groups of model peptides (angiotensins and bradykinins) as well as tryptic peptides were labeled according to our protocol and subjected to collision-induced dissociation (CID) in both a triple quadrupole and a quadrupole ion trap instrument. It was found that for angiotensins containing an AspArg sequence, C-terminal cleavage at Asp that occurs for native peptides was completely inhibited in Arg-labeled peptides. For bradykinins and peptides obtained from tryptic digests of standard proteins, some sample peptides were little affected by the tagging of arginine residues. Others, in contrast, exhibited an almost total loss of nonspecific backbone cleavage and their fragment ion spectra were dominated by loss of the arginine tag. These and other experimental results are discussed in view of the nature of the arginine tag and the concept of proton mobility.     

Quantitative peptidomics of mouse pituitary: comparison of different stable isotopic tags

Determining the relative levels of neuropeptides in two samples is important for many biological studies. An efficient, sensitive and accurate technique for relative quantitative analysis involves tagging the peptides in the two samples with isotopically distinct labels, pooling the samples and analyzing them using liquid chromatography/mass spectrometry (LC/MS). In this study, we compared two different sets of isotopic tags for analysis of endogenous mouse pituitary peptides: succinic anhydride with either four hydrogens or deuteriums and [3-(2,5-dioxopyrrolidin-1-yloxycarbonyl)propyl]trimethylammonium chloride with either nine hydrogens or deuteriums. These two labels react with amines and impart either a negative charge (succinyl) or a positive charge (4-trimethylammoniumbutyryl (TMAB)). Every endogenous mouse pituitary peptide labeled with the light TMAB reagent eluted from the C18 reversed-phase column at essentially the same time as the corresponding peptide labeled with the heavy reagent. Most of the peptides labeled with succinyl groups also showed co-elution of the heavy- and light-labeled forms on LC/MS. The mass difference between the heavy and light TMAB reagents (9 Da per label) was larger than that of the heavy and light succinyl labels (4 Da per label), and for some peptides the larger mass difference provided more accurate determination of the relative abundance of each form. Altogether, using both labels, 82 peptides were detected in Cpe(fat/fat) mouse pituitary extracts. Of these, only 16 were detected with both labels, 41 were detected only with the TMAB label and 25 were detected only with the succinyl label. A number of these peptides were de novo sequenced using low-energy collisional tandem mass spectrometry. Whereas the succinyl group was stable to the collision-induced dissociation of the peptide, the TMAB-labeled peptides lost 59 Da per H9 TMAB group. Several peptides identified in this analysis represent previously undescribed post-translational processing products of known pituitary prohormones. In conclusion, both succinyl and TMAB isotopic labels are useful for quantitative peptidomics, and together these two labels provide more complete coverage of the endogenous peptides. Copyright (c) 2005 John Wiley & Sons, Ltd.     

Protein identification by accurate mass matrix-assisted laser desorption/ionization imaging of tryptic peptides

The spatial distribution of proteins in tissue sections can be used to identify potential markers for pathological processes. Tissue sections are often subjected to enzymatic digestion before matrix‐assisted laser desorption/ionization (MALDI) imaging. This study is targeted at improving the on‐tissue identification of tryptic peptides by accurate mass measurements and complementary off‐line liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) analysis. Two adjacent mouse brain sections were analyzed in parallel. The first section was spotted with trypsin and analyzed by MALDI imaging. Direct on‐tissue MS/MS experiments of this section resulted in the identification of 14 peptides (originating from 4 proteins). The second tissue section was homogenized, fractionated by ultracentrifugation and digested with trypsin prior to LC/ESI‐MS/MS analysis. The number of identified peptides was increased to 153 (corresponding to 106 proteins) by matching imaged mass peaks to peptides which were identified in these LC/ESI‐MS/MS experiments. All results (including MALDI imaging data) were based on accurate mass measurements (RMS <2 ppm) and allow a confident identification of tryptic peptides. Measurements based on lower accuracy would have led to ambiguous or misleading results. MS images of identified peptides were generated with a bin width (mass range used for image generation) of Δm/z = 0.01. The application of accurate mass measurements and additional LC/MS measurements increased both the quality and the number of peptide identifications. The advantages of this approach for the analysis of biological tissue sections are demonstrated and discussed in detail. Results indicate that accurate mass measurements are needed for confident identification and specific image generation of tryptic peptides in tissue sections. Copyright © 2011 John Wiley & Sons, Ltd.     

Synthesis, Characterization and the Effect of Glucose-lowering of Guanidino Acid Oxovanadium（Ⅳ） Complexes

Two guanidino acid oxovanadium(Ⅳ) complexes have been synthesized. Preliminary tests in vivo have shown that the two title complexes all display lowering glucose activity in vivo to STZ-rats. The effect of glucose-lowering of guanidino acetic acid oxovanadium(Ⅳ) complex in vivo is higher than that of guanidino propanoic acid oxovanadium(Ⅳ) complex.     

An Amidinohydrolase Provides the Missing Link in the Biosynthesis of Amino Marginolactone Antibiotics

Desertomycin A is an aminopolyol polyketide containing a macrolactone ring. We have proposed that desertomycin A and similar compounds (marginolactones) are formed by polyketide synthases primed not with γ‐aminobutanoyl‐CoA but with 4‐guanidinylbutanoyl‐CoA, to avoid facile cyclization of the starter unit. This hypothesis requires that there be a final‐stage de‐amidination of the corresponding guanidino‐substituted natural product, but no enzyme for such a process has been described. We have now identified candidate amidinohydrolase genes within the desertomycin and primycin clusters. Deletion of the putative desertomycin amidinohydrolase gene dstH in Streptomyces macronensis led to the accumulation of desertomycin B, the guanidino form of the antibiotic. Also, purified DstH efficiently catalyzed the in vitro conversion of desertomycin B into the A form. Hence this amidinohydrolase furnishes the missing link in this proposed naturally evolved example of protective‐group chemistry.     

Liquid chromatography/tandem mass spectrometry characterization of oxidized amyloid beta peptides as potential biomarkers of Alzheimer's disease

Alzheimer's disease is characterized by the deposition of senile plaques that consist primarily of amyloid beta peptides. There is substantial evidence that amyloid beta is oxidized in vivo, which has led to the suggestion that oxidative stress is an important mediator of Alzheimer's disease. Metal-catalyzed oxidation can mimic in vivo oxidation of amyloid beta because the metal ion binds to the amino acid residues at the site of oxidation, which then deliver reactive oxygen species to that site. Based on electrospray mass spectrometry, it has been suggested that metal-catalyzed oxidation occurs on histidines-13 and -14. Unfortunately, the amyloid beta peptides provide complex spectra, so it is difficult to definitively characterize the sites of oxidation. Trypsin digestion of both native and oxidized amyloid beta1-16 and amyloid beta1-40 resulted in the formation of tryptic peptides corresponding to amyloid beta6-16, which could be separated by liquid chromatography (LC). Sites of oxidation were then unequivocally characterized as histidine-13 and histidine-14 by LC/tandem mass spectrometric (MS/MS) analysis of the tryptic peptides. The ability to analyze the specific amyloid beta6-16 tryptic fragments derived from full-length amyloid beta peptides will make it possible to determine whether oxidation in vivo occurs at specific histidine residues and/or at other amino acid residues such as methionine-35. Using methodology based on LC/MS/MS it will also be possible to analyze the relative amounts of oxidized peptides and native peptide in cerebrospinal fluid from patients with Alzheimer's disease as biomarkers of oxidative stress.     

Novel Dual Two‐Dimensional Liquid Chromatography Online Coupled to Ultraviolet Detector,Fluorescence Detector,Ion‐Trap Mass Spectrometer for Short Peptide Amino Acid Sequence Determination with Bottom‐Up Strategy

A novel dual two‐dimensional (2D) high‐performance liquid chromatography (LC) setup coupled online to an ultraviolet (UV) detector, fluorescence (FL) detector, and ion‐trap mass spectrometer (MS) has been developed for determining the amino acid sequence of short peptides using a novel bottom‐up strategy. Short peptides were electrothermally hydrolyzed to shorter peptides and amino acid enantiomers. The first 2D LC‐UV and FL system was used to separate and identify the produced parent and daughter short peptides and amino acid isomers and enantiomers in the hydrolysate; the second 2D LC‐MS was used to identify the presence of cysteine and obtain the molecular mass signals and N‐terminal peptide fragment ion signals for parent and daughter short peptides. The identified amino acid enantiomers are used to form any possible short peptides by permutation and combination in an order from dipeptide to a tripeptide, to a tetrapeptide, and to even higher short peptides. The correct short peptides are confirmed by comparing the molecular weights of the constituent amino acid enantiomers and the molecular weights of identified short peptides together, with the characteristic N‐terminal peptide fragment ion signals. The amino acid sequence of the dipeptide ester aspartame and the tripeptide glutathione was successfully determined by this method.     

Effects of peptide acetylation and dimethylation on electrospray ionization efficiency

Peptide acetylation and dimethylation have been widely used to derivatize primary amino groups (peptide N‐termini and the ε‐amino group of lysines) for chemical isotope labeling of quantitative proteomics or for affinity tag labeling for selection and enrichment of labeled peptides. However, peptide acetylation results in signal suppression during electrospray ionization (ESI) due to charge neutralization. In contrast, dimethylated peptides show increased ionization efficiency after derivatization, since dimethylation increases hydrophobicity and maintains a positive charge on the peptide under common LC conditions. In this study, we quantitatively compared the ESI efficiencies of acetylated and dimethylated model peptides and tryptic peptides of BSA. Dimethylated peptides showed higher ionization efficiency than acetylated peptides for both model peptides and tryptic BSA peptides. At the proteome level, peptide dimethylation led to better protein identification than peptide acetylation when tryptic peptides of mouse brain lysate were analyzed with LC‐ESI‐MS/MS. These results demonstrate that dimethylation of tryptic peptides enhanced ESI efficiency and provided up to two‐fold improved protein identification sensitivity in comparison with acetylation. Copyright © 2016 John Wiley & Sons, Ltd.     

The relative influence of phosphorylation and methylation on responsiveness of peptides to MALDI and ESI mass spectrometry

Qualitative and quantitative analysis of post‐translational protein modifications by mass spectrometry is often hampered by changes in the ionization/detection efficiencies caused by amino acid modifications. This paper reports a comprehensive study of the influence of phosphorylation and methylation on the responsiveness of peptides to matrix‐assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry. Using well‐characterized synthetic peptide mixtures consisting of modified peptides and their unmodified analogs, relative ionization/detection efficiencies of phosphorylated, monomethylated, and dimethylated peptides were determined. Our results clearly confirm that the ion yields are generally lower and the signal intensities are reduced with phosphopeptides than with their nonphosphorylated analogs and that this has to be taken into account in MALDI and ESI mass spectrometry. However, the average reduction of ion yield caused by phosphorylation is more pronounced with MALDI than with ESI. The unpredictable impact of phosphorylation does not depend on the hydrophobicity and net charge of the peptide, indicating that reliable quantification of phosphorylation by mass spectrometry requires the use of internal standards. In contrast to phosphorylation, mono‐ and dimethylated peptides frequently exhibit increased signal intensities in MALDI mass spectrometry (MALDI‐MS). Despite minor matrix‐dependent variability, MALDI methods are well suited for the sensitive detection of dimethylated arginine and lysine peptides. Mono‐ and dimethylation of the arginine guanidino group did not significantly influence the ionization efficiency of peptides in ESI‐MS. Copyright © 2009 John Wiley & Sons, Ltd.