A Pseudo MS<Superscript>3</Superscript> Approach for Identification of Disulfide-Bonded Proteins: Uncommon Product Ions and Database Search

It has previously been reported that disulfide and backbone bonds of native intact proteins can be concurrently cleaved using electrospray ionization (ESI) and collision-induced dissociation (CID) tandem mass spectrometry (MS/MS). However, the cleavages of disulfide bonds result in different cysteine modifications in product ions, making it difficult to identify the disulfide-bonded proteins via database search. To solve this identification problem, we have developed a pseudo MS³ approach by combining nozzle-skimmer dissociation (NSD) and CID on a quadrupole time-of-flight (Q-TOF) mass spectrometer using chicken lysozyme as a model. Although many of the product ions were similar to those typically seen in MS/MS spectra of enzymatically derived peptides, additional uncommon product ions were detected including c_i-1 ions (the i^th residue being aspartic acid, arginine, lysine and dehydroalanine) as well as those from a scrambled sequence. The formation of these uncommon types of product ions, likely caused by the lack of mobile protons, were proposed to involve bond rearrangements via a six-membered ring transition state and/or salt bridge(s). A search of 20 pseudo MS³ spectra against the Gallus gallus (chicken) database using Batch-Tag, a program originally designed for bottom up MS/MS analysis, identified chicken lysozyme as the only hit with the expectation values less than 0.02 for 12 of the spectra. The pseudo MS³ approach may help to identify disulfide-bonded proteins and determine the associated post-translational modifications (PTMs); the confidence in the identification may be improved by incorporating the fragmentation characteristics into currently available search programs.     

Application of MALDI TOF/TOF mass spectrometry and collision-induced dissociation for the identification of disulfide-bonded peptides

This paper describes a method for the fast identification and composition of disulfide-bonded peptides. A unique fragmentation signature of inter-disulfide-bonded peptides is detected using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)/TOF mass spectrometry and high-energy collision-induced dissociation (CID). This fragmentation pattern identifies peptides with an interconnected disulfide bond and provides information regarding the composition of the peptides involved in the pairing. The distinctive signature produced using CID is a triplet of ions resulting from the cleavage of the disulfide bond to produce dehydroalanine, cysteine or thiocysteine product ions. This method is not applicable to intra-peptide disulfide bonds, as the cleavage mechanism is not the same and a triplet pattern is not observed. This method has been successfully applied to identifying disulfide-bonded peptides in a number of control digestions, as well as study samples where disulfide bond networks were postulated and/or unknown.     

Comparison of collision-induced dissociation and electron-induced dissociation of <Emphasis Type="Italic">singly protonated</Emphasis> aromatic amino acids,cystine and related simple peptides using a hybrid linear ion trap–FT-ICR mass spectrometer

The gas-phase fragmentation reactions of singly protonated aromatic amino acids, their simple peptides as well as simple models for intermolecular disulfide bonds have been examined using a commercially available hybrid linear ion trap-Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Low-energy collision-induced dissociation (CID) reactions within the linear ion trap are compared with electron-induced dissociation (EID) reactions within the FT-ICR cell. Dramatic differences are observed between low-energy CID (which occurs via vibrational excitation) and EID. For example, the aromatic amino acids mainly fragment via competitive losses of NH(3) and (H(2)O+CO) under CID conditions, while side-chain benzyl cations are major fragment ions under EID conditions. EID also appears to be superior in cleaving the S-S and S-C bonds of models of peptides containing an intermolecular disulfide bond. Systematic studies involving fragmentation as a function of electron energy reveal that the fragmentation efficiency for EID occurs at high electron energy (more than 10 eV) compared with the low-electron energy (less than 0.2 eV) typically observed for electron capture dissociation fragmentation. Finally, owing to similarities between the types of fragment ions observed under EID conditions and those previously reported in ultraviolet photodissociation experiments and the electron-ionization mass spectra, we propose that EID results in fragmentation via electronic excitation and vibrational excitation. EID may find applications in analyzing singly charged molecular ions formed by matrix-assisted laser desorption ionization.     

Gas-Phase Fragmentation of [M + nH + OH]<Superscript>n•+</Superscript> Ions Formed from Peptides Containing Intra-Molecular Disulfide Bonds

In this study, we systematically investigated gas-phase fragmentation behavior of [M + nH + OH]^n•+ ions formed from peptides containing intra-molecular disulfide bond. Backbone fragmentation and radical initiated neutral losses were observed as the two competing processes upon low energy collision-induced dissociation (CID). Their relative contribution was found to be affected by the charge state (n) of [M + nH + OH]^n•+ ions and the means for activation, i.e., beam-type CID or ion trap CID. Radical initiated neutral losses were promoted in ion-trap CID and for lower charge states where mobile protons were limited. Beam-type CID and dissociation of higher charge states of [M + nH + OH]^n•+ ions generally gave abundant backbone fragmentation, which was highly desirable for characterizing peptides containing disulfide bonds. The amount of sequence information obtained from CID of [M + nH + OH]^n•+ ions was compared with that from CID of disulfide bond reduced peptides. For the 11 peptides studied herein, similar extent of sequence information was obtained from these two methods.     

Collision Induced Dissociation Products of Disulfide-Bonded Peptides: Ions Result from the Cleavage of More Than One Bond

Disulfide bonds are a post-translational modification (PTM) that can be scrambled or shuffled to non-native bonds during recombinant expression, sample handling, or sample purification. Currently, mapping of disulfide bonds is not easy because of various sample requirements and data analysis difficulties. One step towards facilitating this difficult work is developing a better understanding of how disulfide-bonded peptides fragment during collision induced dissociation (CID). Most automated analysis algorithms function based on the assumption that the preponderance of product ions observed during the dissociation of disulfide-bonded peptides result from the cleavage of just one peptide bond, and in this report we tested that assumption by extensively analyzing the product ions generated when several disulfide-bonded peptides are subjected to CID on a quadrupole time of flight (QTOF) instrument. We found that one of the most common types of product ions generated resulted from two peptide bond cleavages, or a double cleavage. We found that for several of the disulfide-bonded peptides analyzed, the number of double cleavage product ions outnumbered those of single cleavages. The influence of charge state and precursor ion size was investigated, to determine if those parameters dictated the amount of double cleavage product ions formed. It was found in this sample set that no strong correlation existed between the charge state or peptide size and the portion of product ions assigned as double cleavages. These data show that these ions could account for many of the product ions detected in CID data of disulfide bonded peptides. We also showed the utility of double cleavage product ions on a peptide with multiple cysteines present. Double cleavage products were able to fully characterize the bonding pattern of each cysteine where typical single b/y cleavage products could not.     

Fragmentation of intra-peptide and inter-peptide disulfide bonds of proteolytic peptides by nanoESI collision-induced dissociation

Characterisation and identification of disulfide bridges is an important aspect of structural elucidation of proteins. Covalent cysteine-cysteine contacts within the protein give rise to stabilisation of the native tertiary structure of the molecules. Bottom-up identification and sequencing of proteins by mass spectrometry most frequently involves reductive cleavage and alkylation of disulfide links followed by enzymatic digestion. However, when using this approach, information on cysteine-cysteine contacts within the protein is lost. Mass spectrometric characterisation of peptides containing intra-chain disulfides is a challenging analytical task, because peptide bonds within the disulfide loop are believed to be resistant to fragmentation. In this contribution we show recent results on the fragmentation of intra and inter-peptide disulfide bonds of proteolytic peptides by nano electrospray ionisation collision-induced dissociation (nanoESI CID). Disulfide bridge-containing peptides obtained from proteolytic digests were submitted to low-energy nanoESI CID using a quadrupole time-of-flight (Q-TOF) instrument as a mass analyser. Fragmentation of the gaseous peptide ions gave rise to a set of b and y-type fragment ions which enabled derivation of the sequence of the amino acids located outside the disulfide loop. Surprisingly, careful examination of the fragment-ion spectra of peptide ions comprising an intramolecular disulfide bridge revealed the presence of low-abundance fragment ions formed by the cleavage of peptide bonds within the disulfide loop. These fragmentations are preceded by proton-induced asymmetric cleavage of the disulfide bridge giving rise to a modified cysteine containing a disulfohydryl substituent and a dehydroalanine residue on the C-S cleavage site.     

De novo peptide sequencing by tandem MS using complementary CID and electron transfer dissociation

De novo sequencing of peptides using tandem MS is difficult due to missing fragment ions in the spectra commonly obtained after CID of peptide precursor ions. Complementing CID spectra with spectra obtained in an ion‐trap mass spectrometer upon electron transfer dissociation (ETD) significantly increases the sequence coverage with diagnostic ions. In the de novo sequencing algorithm CompNovo presented here, a divide‐and‐conquer approach was combined with an efficient mass decomposition algorithm to exploit the complementary information contained in CID and ETD spectra. After optimizing the parameters for the algorithm on a well‐defined training data set obtained for peptides from nine known proteins, the CompNovo algorithm was applied to the de novo sequencing of peptides derived from a whole protein extract of Sorangium cellulosum bacteria. To 2406 pairs of CID and ETD spectra contained in this data set, 675 fully correct sequences were assigned, which represent a success rate of 28.1%. It is shown that the CompNovo algorithm yields significantly improved sequencing accuracy as compared with published approaches using only CID spectra or combined CID and ETD spectra.     

Effects of Electron-Transfer Coupled with Collision-Induced Dissociation (ET/CID) on Doubly Charged Peptides and Phosphopeptides

Electron-transfer dissociation (ETD) is a useful peptide fragmentation technique that can be applied to investigate post-translational modifications (PTMs), the sequencing of highly hydrophilic peptides, and the identification of large peptides and even intact proteins. In contrast to traditional fragmentation methods, such as collision-induced dissociation (CID), ETD produces c- and z^·-type product ions by randomly cleaving the N–Cα bonds. The disappointing fragmentation efficiency of ETD for doubly charged peptides and phosphopeptide ions has been improved by ETcaD (supplemental activation). However, the ETD data derived from most database search algorithms yield low confidence scores due to the presence of unreacted precursors and charge-reduced ions within MS/MS spectra. In this work, we demonstrate that eight out of ten standard doubly charged peptides and phosphopeptides can be effortlessly identified by electron-transfer coupled with collision-induced dissociation (ET/CID) using the SEQUEST algorithm without further spectral processing. ET/CID was performed with the further dissociation of the charge-reduced ions isolated from ETD ion/ion reactions. ET/CID had high fragmentation efficiency, which elevated the confidence scores of doubly charged peptide and phosphospeptide sequencing. ET/CID was found to be an effective fragmentation strategy in “bottom-up” proteomic analysis.     

Topoisomer Differentiation of Molecular Knots by FTICR MS: Lessons from Class II Lasso Peptides

Lasso peptides constitute a class of bioactive peptides sharing a knotted structure where the C-terminal tail of the peptide is threaded through and trapped within an N-terminal macrolactam ring. The structural characterization of lasso structures and differentiation from their unthreaded topoisomers is not trivial and generally requires the use of complementary biochemical and spectroscopic methods. Here we investigated two antimicrobial peptides belonging to the class II lasso peptide family and their corresponding unthreaded topoisomers: microcin J25 (MccJ25), which is known to yield two-peptide product ions specific of the lasso structure under collision-induced dissociation (CID), and capistruin, for which CID does not permit to unambiguously assign the lasso structure. The two pairs of topoisomers were analyzed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) upon CID, infrared multiple photon dissociation (IRMPD), and electron capture dissociation (ECD). CID and ECD spectra clearly permitted to differentiate MccJ25 from its non-lasso topoisomer MccJ25-Icm, while for capistruin, only ECD was informative and showed different extent of hydrogen migration (formation of c•/z from c/z•) for the threaded and unthreaded topoisomers. The ECD spectra of the triply-charged MccJ25 and MccJ25-lcm showed a series of radical b-type product ions ( b￠_n ^· ) \left( {b{\prime}_n^{ \bullet }} \right) . We proposed that these ions are specific of cyclic-branched peptides and result from a dual c/z• and y/b dissociation, in the ring and in the tail, respectively. This work shows the potentiality of ECD for structural characterization of peptide topoisomers, as well as the effect of conformation on hydrogen migration subsequent to electron capture.     

Rapid sequencing and disulfide mapping of peptides containing disulfide bonds by using 1,5-diaminonaphthalene as a reductive matrix

MS/MS is indispensable for the amino acid sequencing of peptides. However, its use is limited for peptides containing disulfide bonds. We have applied the reducing properties of 1,5-diaminonaphthalene (1,5-DAN) as a MALDI matrix to amino acid sequencing and disulfide bond mapping of human urotensin II possessing one disulfide bond, and human guanylin possessing two disulfide bonds. 1,5-DAN was used in the same manner as the usual MALDI matrices without any pre-treatment of the peptide, and MS/MS was performed using a matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometer (MALDI QIT TOFMS). The results demonstrated that MS/MS of the molecular ions reduced by 1,5-DAN provided a series of significant b-/y-product ions. All 11 amino acid residues of urotensin II were identified using 1,5-DAN, while only 5 out of 11 residues were identified using 2,5-dihydroxybenzoic acid (DHB); similarly 11 out of 15 amino acid residues of guanylin were identified using 1,5-DAN, while only three were identified using DHB. In addition, comparison of the theoretical and measured values of the mass differences between corresponding MS/MS product ions using 1,5-DAN and DHB narrowed down the possible disulfide bond arrangement candidates. Consequently, 1,5-DAN as a reductive matrix facilitates rapid amino acid sequencing and disulfide mapping for peptides containing disulfide bonds.     

Tandem MS Analysis of Selenamide-Derivatized Peptide Ions

Our previous study showed that selenamide reagents such as ebselen and N-(phenylseleno)phthalimide (NPSP) can be used for selective and rapid derivatization of protein/peptide thiols in high conversion yield. This paper reports the systematic investigation of MS/MS dissociation behaviors of selenamide-derivatized peptide ions upon collision induced dissociation (CID) and electron transfer dissociation (ETD). In the positive ion mode, derivatized peptide ions exhibit tag-dependent CID dissociation pathways. For instance, ebselen-derivatized peptide ions preferentially undergo Se–S bond cleavage upon CID to produce a characteristic fragment ion, the protonated ebselen (m/z 276), which allows selective identification of thiol peptides from protein digest as well as selective detection of thiol proteins from protein mixture using precursor ion scan (PIS). In contrast, NPSP-derivatized peptide ions retain their phenylselenenyl tags during CID, which is useful in sequencing peptides and locating cysteine residues. In the negative ion CID mode, both types of tags are preferentially lost via the Se–S cleavage, analogous to the S–S bond cleavage during CID of disulfide-containing peptide anions. In consideration of the convenience in preparing selenamide-derivatized peptides and the similarity of Se–S of the tag to the S–S bond, we also examined ETD of the derivatized peptide ions to probe the mechanism for electron-based ion dissociation. Interestingly, facile cleavage of Se–S bond occurs to the peptide ions carrying either protons or alkali metal ions, while backbone cleavage to form c/z ions is severely inhibited. These results are in agreement with the Utah-Washington mechanism proposed for depicting electron-based ion dissociation processes.     

Identification of Proteins and Phosphoproteins Using Pulsed Q Collision Induced Dissociation (PQD)

Pulsed Q collision induced dissociation (PQD) was developed to facilitate detection of low-mass reporter ions from labeling reagents (e.g., iTRΑQ) in peptide quantification using an LTQ mass spectrometer (MS). Despite the large number of linear ion traps worldwide, the use and optimization of PQD for protein identification have been limited, in part due to less effective ion fragmentation relative to the collision induced dissociation (CID). PQD expands the m/z coverage of fragment ions to the lower m/z range by circumventing the typical low mass cut-off of an ion trap MS. Since database searching relies on the matching between theoretical and observed spectra, it is not clear how ion intensity and peak number might affect the outcomes of a database search. In this report, we systematically evaluated the attributes of PQD mass spectra, performed intensity optimization, and assessed the benefits of using PQD on the identification of peptides and phosphopeptides from an LTQ. Based on head-to-head comparisons between CID (higher intensity) and PQD (better m/z coverage), peptides identified using PQD generally have Xcorr scores lower than those using CID. Such score differences were considerably diminished by the use of 0.1% m-nitrobenzyl alcohol (m-NBA) in mobile phases. The ion intensities of both CID and PQD were adversely affected by increasing m/z of the precursor, with PQD more sensitive than CID. In addition to negating the 1/3 rule, PQD enhances direct bond cleavage and generates patterns of fragment ions different from those of CID, particularly for peptides with a labile functional group (e.g., phosphopeptides). The higher energy fragmentation pathway of PQD on peptide fragmentation was further compared to those of CID and the quadrupole-type activation in parallel experiments.     

Determination of polychlorodibenzo-p-dioxins and polychlorodibenzofurans by gas chromatography/tandem mass spectrometry and gas chromatography/triple mass spectrometry in a quadrupole ion trap

The determination of tetra- to octachlorodibenzo-p-dioxins and tetra- to octachlorodibenzofurans (PCCD/Fs) by high-resolution gas chromatography/tandem mass spectrometry (HRGC/MS/MS) and high-resolution gas chromatography/triple mass spectrometry (HRGC/MS(3)) in a quadrupole ion trap, equipped with an external ion source, is presented. MS/MS involves a typical four-step process, namely ionization, parent ion isolation, collision-induced dissociation (CID) and mass analysis of the daughter ions. For the MS(3) experiment, the MS/MS scan function is used with the addition of selected daughter ion isolation, their CID and the mass analysis of second-generation product ions called 'grand-daughter ions.' For both methods, the energies necessary for the CID of the 17 PCDD/Fs were determined and optimized using multiple scan functions with different CID amplitudes. The CID efficiency, defined as the signal ratio of fragment ions detected from the major dissociation channels to molecular ions isolated, was 1.15-2.40 V for parent ion dissociation (MS/MS) and 1.05-1.50 V for daughter ion dissociation (MS(3)) and for all the chloro congeners. The same sensitivity (1 pg microl(-1)) can be reached with both the MS/MS and MS(3) methods and linear responses were obtained between 1 and 100 pg microl(-1) injected.     

Free radical-induced site-specific peptide cleavage in the gas phase: Low-energy collision-induced dissociation in ESI- and MALDI mass spectrometry

Protein identification is routinely accomplished by peptide sequencing using mass spectrometry (MS) after enzymatic digestion. Site-specific chemical modification may improve peptide ionization efficiency or sequence coverage in mass spectrometry. We report herein that amino group of lysine residue in peptides can be selectively modified by reaction with a peroxycarbonate and the resulting lysine peroxycarbamates undergo homolytic fragmentation under conditions of low-energy collision-induced dissociation (CID) in electrospray ionization (ESI) and matrix-assisted laser desorption and ionization (MALDI) MS. Selective modification of lysine residue in peptides by our strategy can induce specific peptide cleavage at or near the lysine site. Studies using deuterated analogues of modified lysine indicate that fragmentation of the modified peptides involves apparent free-radical processes that lead to peptide chain fragmentation and side-chain loss. The formation of a-, c-, or z-types of ions in MS is reminiscent of the proposed free-radical mechanisms in low-energy electron capture dissociation (ECD) processes that may have better sequence coverage than that of the conventional CID method. This site-specific cleavage of peptides by free radical- promoted processes is feasible and such strategies may aid the protein sequencing analysis and have potential applications in top-down proteomics.     

Sequencing of T-superfamily conotoxins from <Emphasis Type="Italic">Conus virgo</Emphasis>: Pyroglutamic acid identification and disulfide arrangement by MALDI mass spectrometry

De novo mass spectrometric sequencing of two Conus peptides, Vi1359 and Vi1361, from the vermivorous cone snail Conus virgo, found off the southern Indian coast, is presented. The peptides, whose masses differ only by 2 Da, possess two disulfide bonds and an amidated C-terminus. Simple chemical modifications and enzymatic cleavage coupled with matrix assisted laser desorption ionization (MALDI) mass spectrometric analysis aided in establishing the sequences of Vi1359, ZCCITIPECCRI-NH(2), and Vi1361, ZCCPTMPECCRI-NH(2), which differ only at residues 4 and 6 (Z = pyroglutamic acid). The presence of the pyroglutamyl residue at the N-terminus was unambiguously identified by chemical hydrolysis of the cyclic amide, followed by esterification. The presence of Ile residues in both the peptides was confirmed from high-energy collision induced dissociation (CID) studies, using the observation of w(n)- and d(n)-ions as a diagnostic. Differential cysteine labeling, in conjunction with MALDI-MS/MS, permitted establishment of disulfide connectivity in both peptides as Cys2-Cys9 and Cys3-Cys10. The cysteine pattern clearly reveals that the peptides belong to the class of T-superfamily conotoxins, in particular the T-1 superfamily.     

Development of a tandem time‐of‐flight mass spectrometer with an electrospray ionization ion source

A new tandem time‐of‐flight mass spectrometer with an electrospray ionization ion source ‘ESI‐TOF/quadTOF’ was designed and constructed to achieve the desired aim of structural elucidation via high‐energy collision‐induced dissociation (CID), and the simultaneous detection of all fragment ions. The instrument consists of an orthogonal acceleration‐type ESI ion source, a linear TOF mass spectrometer, a collision cell, a quadratic‐field ion mirror and a microchannel plate detector. High‐energy CID spectra of doubly protonated angiotensin II and bradykinin were obtained. Several fragment ions such as a‐, d‐, v‐ and w‐type ions, characteristic of high‐energy CID, were clearly observed in these spectra. These high‐energy CID fragment ions enabled confirmation of the complete sequence, including leucine–isoleucine determinations. It was demonstrated that high‐energy CID of multiply protonated peptides could be achieved in the ESI‐TOF/quadTOF. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of collision-induced dissociation mechanisms and cross-sections in organophosphorus compounds by atmospheric pressure ionization tandem mass spectrometry

Nineteen organophosphorus compounds (OPCs) of the generic classes (dialkyl alkyl′-phosphonates (twelve compounds) and tri-alkyl phosphates (seven compounds) were investigated by atmospheric pressure ionization (API) tandem mass spectrometry (MS/MS). All OPCs showed consistent and comparable collision-induced dissociation (CID) pathways allowing for a generalized scheme for dissociation. When the alkyl groups are equal to or larger than ethyl, CID mechanisms are dominated by McLafferty rearrangements and neutral losses are stable molecules in all cases except cleavage of P? C or O? C bonds to form alkyl fragment ions. CID cross-sections for the OPCs were determined with excellent reproducibility and a good correlation was found with a simple model. Inferences with respect to ionic structures were made from observed trends in the cross-section measurements. Limitations in API-MS/MS instruments of this design are found in the energy distribution of the ionization and ion sampling events.     

Rapid sequencing of a peptide containing a single disulfide bond using high-energy collision-induced dissociation

A peptide containing a single disulfide bond was sequenced using high-energy collision-induced dissociation (HE-CID) in conjunction with a high mass resolution time-of-flight tandem mass spectrometer equipped with a matrix-assisted laser desorption/ionization source. This mass spectrometer, which has spiral ion trajectory, allowed both high mass resolution and high precursor ion selectivity. It is difficult to obtain sufficient product ions from peptides containing disulfide bonds using HE-CID due to the single collision in the gas phase. To compensate for insufficient dissociation, the disulfide bond was cleaved via an in-source reduction process using 1,5-diaminonaphthalene, a reducing matrix. After applying the reduction in the ionization, subsequent sequencing using HE-CID provided the detailed structural information of the peptide containing the single disulfide bond.     

Novel natural peptides from Hyla arborea schelkownikowi skin secretion

Hyla arborea schelkownikowi is one of the leaf frog species inhabiting the southern territories of Russia and the former USSR. This frog species is a member of the Hylidae Rafinesque, 1815 batrachians family. The present study deals with the previously uninvestigated peptidome of the Hyla arborea schelkownikowi skin secretion. Nano‐electrospray ionization Fourier transform mass spectrometry (nanoESI‐FTMS) of the skin secretion, in the intact form and after acetylation, was selected as the general method of analysis. Electron‐capture dissociation (ECD) and collision‐induced dissociation (CID) fragmentation were both employed, while de novo sequencing was performed by manual interpretation of the MS data. The suppression of the cyclization of b‐ions in the mass spectrometer by the acetylation reaction proved to be very efficient for the de novo sequencing of short peptides. Ten skin peptides were found and all of them, except for bradykinin, had not previously been reported. Six of the peptides belong to the tryptophyllins and related peptides, while three peptides are similar to the aureins. Copyright © 2010 John Wiley & Sons, Ltd.     

Fragmentation behavior of Amadori‐peptides obtained by non‐enzymatic glycosylation of lysine residues with ADP‐ribose in tandem mass spectrometry

Mono‐ and poly‐adenosine diphosphate (ADP)‐ribosylation are common post‐translational modifications incorporated by sequence‐specific enzymes at, predominantly, arginine, asparagine, glutamic acid or aspartic acid residues, whereas non‐enzymatic ADP‐ribosylation (glycation) modifies lysine and cysteine residues. These glycated proteins and peptides (Amadori‐compounds) are commonly found in organisms, but have so far not been investigated to any great degree. In this study, we have analyzed their fragmentation characteristics using different mass spectrometry (MS) techniques. In matrix‐assisted laser desorption/ionization (MALDI)‐MS, the ADP‐ribosyl group was cleaved, almost completely, at the pyrophosphate bond by in‐source decay. In contrast, this cleavage was very weak in electrospray ionization (ESI)‐MS. The same fragmentation site also dominated the MALDI‐PSD (post‐source decay) and ESI‐CID (collision‐induced dissociation) mass spectra. The remaining phospho‐ribosyl group (formed by the loss of adenosine monophosphate) was stable, providing a direct and reliable identification of the modification site via the b‐ and y‐ion series. Cleavage of the ADP‐ribose pyrophosphate bond under CID conditions gives access to both neutral loss (347.10 u) and precursor‐ion scans (m/z 348.08), and thereby permits the identification of ADP‐ribosylated peptides in complex mixtures with high sensitivity and specificity. With electron transfer dissociation (ETD), the ADP‐ribosyl group was stable, providing ADP‐ribosylated c‐ and z‐ions, and thus allowing reliable sequence analyses. Copyright © 2010 John Wiley & Sons, Ltd.