Letter: collision-induced dissociation of peptide thioesters: influence of the peptide length on the fragmentation

Five peptide thioesters of increasing length were fragmented under two processes, in-source and in- collision cell fragmentation, using an electrospray source coupled to a triple quadrupole. Comparison of their fragmentations was made in regard to the length. The two fragmentation conditions show that the peptide length has no influence on structural information and that the fragmentation efficiency is higher for the smallest peptides than for the longest. The particularity of these peptide thioesters consists on the neutral loss of ethanethiol. The absence of the a3 fragment ion and the presence of the (a3-17) ion on the CID mass spectra are noted.     

Peptide sequence information derived by pronase digestion and ammonium sulfate in-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

We present the use of Pronase digestion and in-source decay in the presence of ammonium sulfate as complementary techniques to confirm the amino acid sequence of a peptide. Pronase, a commercial preparation from Streptomyces griseus, is a combination of proteolytic enzymes. It produces carboxypeptidase and aminopeptidase ladders using a single Pronase digestion and represents an inexpensive, nonspecific, and fast supplement to traditional sequencing enzymes. However, N-terminal peptidase activity appears dependent on the terminal amino acid residue. We also introduce the use of saturated ammonium sulfate as an "on-slide" sample additive to promote in-source fragmentation of peptides. Use of saturated ammonium sulfate resulted in a simple way to increase peptide backbone fragmentation and essentially produced either a cn or yn ion series. Together these techniques provide useful supplements to existing methods for peptide sequence information.     

Analysis of sulfated peptides using positive electrospray ionization tandem mass spectrometry.

Presented is a method for analyzing sulfated peptides, and differentiating the post-translational modification (PTM) from its isobaric counterpart phosphorylation, using quadrupole time-of-flight (Qq/TOF) mass spectrometry (MS) and positive ion nanoelectrospray MS/MS. A set of commercially available sulfo- and phosphopeptide standards was analyzed via in-source dissociation and MS/MS to generate fragmentation signatures that were used to characterize and differentiate the two modifications. All of the phosphorylated peptides retained their +80 Da modifications under collision-induced decomposition (CID) conditions and peptide backbone fragmentation allowed for the site-specific identification of the modification. In sharp contrast, sulfated peptides lost SO3 from the precursor as the collision energy (CE) was increased until only the non-sulfated form of the peptide was observed. The number of 80 Da losses indicated the number of sulfated sites. By continuing to ramp the CE further, it was possible to fragment the non-sulfated peptides and obtain detailed sequence information. It was not possible to obtain site-specific information on the location of the sulfate moieties using positive ion MS/MS as none of the original precursor ions were present at the time of peptide backbone fragmentation. This method was applied to the analysis of recombinant human B-domain deleted factor VIII (BDDrFVIII), which has six well-documented sulfation sites and several potential phosphorylation sites located in two of the sulfated regions of the protein. Seven peptides with single and multiple +80 Da modifications were isolated and analyzed for their respective PTMs. The fragmentation patterns obtained from the BDDrFVIII peptides were compared with those obtained for the standard peptides; and in all cases the peptides were sulfated. None of the potential phosphorylation sites were found to be occupied, and these results are consistent with the literature.     

Characterization of microcystins using in-source collision-induced dissociation

The efficiency of the in-source collision-induced dissociation (in-source CID) technique for the structural characterization of microcystins (MCYSTs) was evaluated. Microcystins that did not contain arginine underwent facile fragmentation to produce characteristic product ions at relatively low cone voltage and could be fully characterized based on their mass spectra. On the other hand, cyclic peptides possessing arginine residues, such as MCYST-RR, -LR, -YR and nodularin, were considerably more stable under in-source CID conditions and required higher cone voltage to induce fragmentation. This behaviour is explained in terms of the mobile proton model for peptide fragmentation that can be used as an indication for the presence of arginine when unknown microcystins are analyzed. In-source CID was applied to the characterization of microcystins released into water from a Microcystis aeruginosa culture (UTCC299) (UTCC: University of Toronto Culture Collection of Algae and Cyanobacteria). Six microcystins were detected in extracts from UTCC299: I, [D-Asp(3)]MCYST-LR; II, MCYST-LR; III, isomer of MCYST-LR; IV, isomer of methyl MCYST-LR; V, [D-Asp(3), Glu(OCH(3))(6)]MCYST-LR; and VI, [D-Glu(OCH(3))(6)]MCYST-LR. In-source CID provided mass spectral patterns similar to those obtained by CID in the collision cell of the mass spectrometer but was more sensitive for the analysis of microcystins.     

Using accurate mass electrospray ionization-time-of-flight mass spectrometry with in-source collision-induced dissociation to sequence peptide mixtures

Although data-dependent LC-MS-MS with database searching has become au courant for identifying proteins, the technique is constrained by duty-cycle inefficiency and the inability of most tandem mass analyzers to accurately measure peptide product ion masses. In this work, a novel approach is presented for simultaneous peptide fragmentation and accurate mass measurement using in-source collision-induced dissociation (CID) on electrospray ionization (ESI)-time-of-flight (TOF) MS. By employing internal mass reference compounds, mass measurement accuracy within +/-5 ppm for tryptic peptide precursors and +/-10 ppm for most sequence-specific product ions was consistently achieved. Analysis of a complex solution containing several digested protein standards did not adversely affect instrument performance.     

Induced in-source fragmentation for the selective detection of organic bound iodine by liquid chromatography/electrospray mass spectrometry

The detection of unknown organic compounds, e.g., transformation products of known trace pollutants in environmental samples, is a difficult task which can be simplified if the compounds of interest are characterized by a functional group or a heteroatom which can selectively be detected. For the detection of iodinated benzene derivatives, used as X-ray contrast media, we have studied whether such compounds can be selectively detected by negative ion (NI) electrospray ionization via iodide as a product ion of in-source fragmentation, generated by applying a high cone voltage. It was found that selective detection of iodinated benzene derivatives is possible by liquid chromatography/negative ion electrospray ionization (LC/NI-ESI) if the compounds are destroyed before entering the mass spectrometer. To our knowledge this is the first report of the use of NI in-source fragmentation MS for analyses traditionally performed with inductively coupled plasma (ICP)-MS. The novel application of NI in-source fragmentation MS is a tool for those who do not possess the facilities to perform ICP-MS. Furthermore, NI-ESI at varying cone voltages provides both structural information and elemental detection, whereas ICP-MS can only provide elemental detection.     

In-source fragmentation and accurate mass analysis of multiclass flavonoid conjugates by electrospray ionization time-of-flight mass spectrometry

In-source collision-induced dissociation (CID) fragmentation features of multiclass flavonoid glycoconjugates were examined using liquid chromatography electrospray time-of-flight mass spectrometry. Systematic experiments were performed to search for optimal conditions for in-source fragmentation in both positive and negative ion modes. The objective of the study was to attain uniformly appropriate conditions for a wide range of analytes independently of the aglycone, the attached sugar part and the type of bond between the aglycone and the glycan moieties (O- or C-glycosides). Studied substances included representatives of flavonols, flavones, flavanones and anthocyanins and, regarding their glycan parts, mono-, di- and triglycosides with varying distribution of carbohydrate moieties (di-O-glycosides, O-diglycosides, O,C-diglycosides). The breakdown properties of the analytes along with the abundances of the characteristic diagnostic ions required for structural elucidation of complex flavonoid derivatives were evaluated. An optimized value was found for the instrument parameter (fragmentor voltage) affecting the in-source CID fragmentation of the analytes [230 V (ESI+) and 330 V (ESI-)]. Thus, appropriate performance in terms of both highly sensitive full-scan acquisition and fragmentation information was obtained for all the investigated flavonoids. In addition, singularities in the abundance of selected diagnostic ions (e.g. Y(0), Y(1) and Y*) due to variations in the interglycosidic linkage (rutinoside-neohesperidoside) in the glycan part were found and are also evaluated and discussed in detail. The combination of in-source CID fragmentation with high mass accuracy MS detection establishes a working basis for the development of versatile and useful LC-MS methods for wide-scope screening, non-targeted detection and tentative identification of flavonoid derivatives.     

Evaluation of protocols for reproducible electrospray in-source collisionally induced dissociation on various liquid chromatography/mass spectrometry instruments and the development of spectral libraries

Mass spectral libraries provide a tool for identifying unknown compounds using both molecular weight and fragmentation information. Mass spectrometers with electrospray ionisation (ESI) and atmospheric chemical ionisation (ApCI) sources have the capability to produce data of this type using in-source collisionally induced dissociation (CID), and in-source CID libraries can be created. Due to the variation in electrospray source design from different instrument manufacturers, the production of reproducible in-source CID spectra that can be used in libraries for all instrument types is not a trivial task. To date, the evaluation of the production of in-source CID libraries has tended to focus on similar instruments from one manufacturer. The studies have also tended to focus on specific compound classes, with a limited molecular weight range.This report describes the findings from the investigation of protocols for the creation of mass spectral libraries using ESI in-source CID on six instruments from four different manufacturers. The overall goal was to create a spectral library for the identification of unknowns. The library could then be applied across all manufacturers' electrospray instruments.Two different experimental protocols were attempted. The first used a tuning compound to establish standard ESI source conditions, with fixed fragmentation potentials. The second involved the attenuation of the [M + H](+) ion to a known degree. A diverse range of compounds (pharmaceutical, photographic, pesticides) was tested to establish the reproducibility of the spectra on the six instruments. Both protocols produced spectra on the various instruments that in many cases were very similar. In other examples, the spectra differed not only in their relative ion abundances, but also in terms of the spectral content. Important observations regarding the effect of ion source design are also reported.The degree of spectral reproducibility was calculated off-line by comparing the five most abundant ions (20% for each ion that matches) from each spectrum on each instrument. This approach was adopted, as we do not possess a software package that met our requirements for spectral comparison. Match factors (% fit) were calculated by comparing each spectrum against the spectra recorded for the same compound and then for all other compounds, on each instrument. The % fit values derived by the off-line approach gave a clear view of the spectral reproducibility from instrument to instrument and also discriminated the spectra of the various compounds from each other. The applicability of this approach was tested using a blind trial in which several compounds were presented as unknowns, their in-source CID spectra recorded and the five-ion approach used for identification.     

High mass accuracy in-source collision-induced dissociation tandem mass spectrometry and multi-step mass spectrometry as complementary tools for fragmentation studies of quaternary ammonium herbicides

Fragmentation studies using both an ion-trap mass analyzer and a hybrid quadrupole time-of-flight (Q-TOF) mass spectrometer were performed in order to establish the fragmentation pathways of organic molecules. A general strategy combining MSn data (n = 1-4) in an ion-trap analyzer with tandem mass spectrometry and in-source collision-induced dissociation tandem mass spectrometry (CID MS/MS) in a Q-TOF instrument was applied. The MSn data were used to propose a tentative fragmentation pathway following genealogical relationships. When several assignments were possible, MS/MS and in-source CID MS/MS (Q-TOF) allowed the elemental compositions of the fragments to be confirmed. Quaternary ammonium herbicides (quats) were used as test compounds and their fragmentation pathways were established. The elemental composition of the fragments was confirmed using the TOF analyzer with relative errors <0.0023 Da. Some fragments previously reported in the literature were reassigned taking advantage of the high mass resolution and accuracy of the Q-TOF instrument, which made it possible to solve losses where nitrogen was involved.     

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.     

New example of proline-induced fragmentation in electrospray ionization mass spectrometry of peptides

The positive ion electrospray ionization (ESI+) mass spectra of peptides usually display only protonated molecules provided that soft ionization conditions are applied (low cone voltage to prevent in-source dissociations). Such ions can be multiply charged depending on the molecular weight of the studied compounds. We have experienced an unexpected behavior during the ESI analysis of a modified peptide of relatively high mass (3079 Da). A specific fragmentation occurred even under soft energetic conditions, leading to a mass spectrum containing multiply charged molecular and fragment ions. The selective rupture involved the amide bond between the glutamic acid and proline residues (E-P sequence). The successive replacement of each amino acid by an alanine residue (positional scanning study) was undertaken to assess which part of the sequence induced such selective and abundant fragmentation on multiply charged species. The succession P-P was evidenced as the minimum unit giving rise to the first peptide bond rupture in the sequence X-P-P. Any acidic amino acid at the X position (X = D, E) favored the fragmentation by an intramolecular interaction. Such proline-induced fragmentation occurring readily in the source differed from the literature data on the specific behavior of proline-containing peptides where bond ruptures occur solely in dissociation conditions.     

Statistical evaluation of electrospray tandem mass spectra for optimized peptide fragmentation

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the method of choice for the analysis of complex peptide mixtures. It combines the separation power of nanoflow LC with highly specific sequence analysis, allowing automated peptide sequencing with high resolution and throughput. For peptide fragmentation, the current experimental setup uses predefined parameters based on the mass-to-charge ratio of the individual precursor. Suitable parameters are typically established by empirical evaluation of fragment spectra of individual peptides used as standards. As a result, nonoptimal fragment spectra are obtained if peptides show fragmentation behavior different from these standards, which often result in the loss of sequence-specific fragment ion information. Here we describe a statistical approach for the systematic evaluation of the quality of individual peptide fragment spectra based on the calculation of their arithmetic mean and standard deviation. The method utilizes the dependence of these parameters on the difference in electric potential across the collision cell to determine the value that results in maximum information content. We show that the method is applicable to fragment spectra generated from a variety of multiply-charged tryptic peptides, over a wide concentration range, and on different types of mass analyzers. We also show how this novel approach can be used to define optimized collision energy settings over a wide mass-to-charge range.     

Improving peptide fragmentation by N-terminal derivatization with high proton affinity

An improved method of de novo peptide sequencing based on mass spectrometry using novel N-terminal derivatization reagents with high proton affinity has been developed. The introduction of a positively charged group into the N-terminal amino group of a peptide is known to enhance the relative intensity of b-ions in product ion spectra, allowing the easy interpretation of the spectra. However, the physicochemical properties of charge derivatization reagents required for efficient fragmentation remain unclear. In this study, we prepared several derivatization reagents with high proton affinity, which are thought to be appropriate for peptide fragmentation under low-energy collision-induced dissociation (CID) conditions, and examined their usefulness in de novo peptide sequencing. Comparison of the effects on fragmentation among three derivatization reagents having a guanidino or an amidino moiety, which differ in proton affinity, clearly indicated that there was an optimal proton affinity for efficient fragmentation of peptides. Among reagents tested in this study, derivatization with 4-amidinobenzoic acid brought about the most effective fragmentation. This derivatization approach will offer a novel de novo peptide sequencing method under low-energy CID conditions.     

N-Terminal peptide aldehydes as electrophiles in combinatorial solid phase synthesis of novel peptide isosteres

N-Terminal peptide aldehydes were synthesized on a solid support and utilized as electrophiles in nucleophilic reactions in order to furnish novel and diverse peptide isosteres. The aldehyde moiety of the peptide was synthesized by coupling a protected aldehyde building block to the peptide and deprotecting it quantitatively in less than 3 min. It was found that protection of the two succeeding amide nitrogens was necessary in order to avoid reaction between the aldehyde and backbone amides. The N-terminal peptide aldehydes were successfully reacted in the following way: (a) reductive amination with a large variety of amines, leading to N-alkyl-gamma-aminobutyric peptide isosteres positioned centrally in the peptide; (b) reductive amination with amino esters, leading to N-terminal 2,5-diketopiperazine peptides; (c) Horner-Wadsworth-Emmons olefination, leading to unsaturated peptide isosteres positioned centrally in the peptide; and (d) Pictet-Spengler condensations, leading to tetrahydro-beta-carbolines either positioned centrally in a peptide or fused with a diketopiperazine ring in the N-terminus of the peptide.     

Sequencing cyclic peptide inhibitors of mammalian ribonucleotide reductase by electrospray ionization mass spectrometry.

Mammalian ribonucleotide reductase (mRR) is a potential target for cancer intervention. A series of lactam-bridged cyclic peptide inhibitors (1-9) of mRR have been synthesized and tested in previous work. These inhibitors consist of cyclic and linear regions, causing their mass spectral characterization to be a challenge. We determined the fragmentation mechanism of cyclic peptides 1-9 using an ion-trap mass spectrometer equipped with an ESI source. Low-energy collision-induced dissociation of sodiated cyclic peptides containing linear branches follows a general pathway. Fragmentation of the linear peptide region produced mainly a and b ions. The ring peptide region was more stable and ring opening required higher collision energy, mainly occurring at the amide bond adjacent to the lactam bridge. The sodium ion, which bound to the carbonyl oxygen of the lactam bridge, acted as a fixed charge site and directed a charge-remote, sequence-specific fragmentation of the ring-opened peptide. Amino acid residues were cleaved sequentially from the C-terminus to the N-terminus. Our findings have established a new way to sequence cyclic peptides containing a lactam bridge based on charge-remote fragmentation. This methodology will permit unambiguous identification of high-affinity ligands within cyclic peptide libraries.     

Intriguing Mass Spectrometric Behavior of Guanosine Under Low Energy Collision-Induced Dissociation: H<Subscript>2</Subscript>O Adduct Formation and Gas-Phase Reactions in the Collision Cell

An in-depth study of the fragmentation pathway of guanosine was conducted by using an in-source collision-induced dissociation high-mass accuracy tandem mass spectrometry experiment. The equivalent of MS4 data, a level of information normally achieved on ion trap instruments, was obtained on a Q-TOF mass spectrometer. The combination of the features of high-resolution, accuracy, and in-source CID permitted the unambiguous elucidation of the different fragmentation pathways. Furthermore the elemental compositions of the product ions generated were assigned and their mutual genealogical relationships established. Formerly proposed dissociation pathways of guanosine were revisited and elaborated on more deeply. Furthermore, the presence of H2O in the collision cell of several tandem MS instruments was demonstrated and its effect on the product ion spectra investigated. The neutral gain of H2O by particular fragments of guanosine was experimentally proven by using argon, saturated with H2(18)O, as the collision gas. Data indicating the occurrence of more complex reactions in the collision cell as a result of the presence of H2O were produced, specifically relating to neutral gain/neutral loss sequences. In silico calculations supported the experimental observation of neutral gain by guanosine fragments and predicted a similar behavior for adenosine. The latter was subsequently experimentally confirmed.     

In-source collision induced dissociation of inorganic explosives for mass spectrometric signature detection and chemical imaging

The trace detection, bulk quantification, and chemical imaging of inorganic explosives and components was demonstrated utilizing in-source collision induced dissociation (CID) coupled with laser desorption/ionization mass spectrometry (LDI-MS). The incorporation of in-source CID provided direct control over the extent of adduct and cluster fragmentation as well as organic noise reduction for the enhanced detection of both the elemental and molecular ion signatures of fuel-oxidizer mixtures and other inorganic components of explosive devices. Investigation of oxidizer molecular anions, specifically, nitrates, chlorates, and perchlorates, identified that the optimal in-source CID existed at the transition between fragmentation of the ionic salt bonds and molecular anion bonds. The chemical imaging of oxidizer particles from latent fingerprints was demonstrated, including both cation and anion components in positive and negative mode mass spectrometry, respectively. This investigation demonstrated LDI-MS with in-source CID as a versatile tool for security fields, as well as environmental monitoring and nuclear safeguards, facilitating the detection of elemental and molecular inorganic compounds at nanogram levels.     

Schiff base derivatives of peptide esters: Relative abundance of N-terminal,C-terminal and ‘internal’ fragments as a function of the blocking group

Electron-impact (EI) mass spectrometry of peptide derivatives is usually interpreted in terms of fragmentation where the charge resides on the N-terminal fragments and to a lesser degree on the less common, charged C-terminal fragments. Substituted and unsubstituted benzylidene, cinnamylidene, α- and β-naphthylidene derivatives of a reference tripeptide, valileala, gave both N- and C-terminal fragments as well as molecular ions. The order of increasing ion current (normalized) in C-terminal fragments was: acetylacetonyl, 4-dimethylaminonaphthylidene, p-dimethyl-aminobenzylidene, 3-pyridylmethylidene, p-diethylaminocinnamylidene, benzylidene, 2-hydroxy-naphthylidene, 4-pyridylmethylidene, p-nitrobenzylidene, p-methoxybenzylidene, p-cyanobenzylidene, cinnamylidene, p-dimethylaminocinnamylidene, β-indolylmethylidene, β-naphthylidene, 2-pyridylmethylidene and α-naphthylidene. The order for this value among the N-terminal fragments is significantly different, however (Day, Falter, Lehman and Hamilton, J. Org. Chem. in press). In addition to N- and C-terminal fragments, many spectra contain internal fragments, arising from loss of fragments from both ends, which provide sequence information. These fragments are found in the mass spectra of Schiff bases formed from various aromatic aldehydes with peptide esters. The interpretation of the latter pattern is facilitate in some cases by deuterium labeling at the α-carbon of the N-terminal amino acid residue of peptides. Such a pattern provides sequence information supplemental to that available involving N- and C-terminal fragmentations. In derivatives of hexaglycine, tetraphenylalanine and tryptophylmethionylaspartyl (β-OEt) phenylalanine amide, for example, substantial sequence information was contained in the internal fragments; in some cases the sequence could be deduced only if the internal fragments were utilized. The 4-dimethylamino-naphthylidene derivatives have proven to be the most useful to date in terms of volatility, tendency to maximize cleavage into N-terminal fragments, intensity of molecular ions and generation of useful mass spectra of certain peptide esters refractory to mass spectrometry in the form of any other derivative investigated.     

Structural analysis of oligosaccharides by a combination of electrospray mass spectrometry and bromine isotope tagging of reducing-end sugars with 2-amino-5-bromopyridine

Model reducing-end oligosaccharides were successfully labeled by a brominated aromatic amine reagent, 2-amino-5-bromopyridine (ABP), through reductive amination. Using either a combination of liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) with in-source fragmentation or liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS), sequence information corresponding to the model oligosaccharides was revealed with little ambiguity via the diagnostic unique twin peaks arising from the bromine isotopes, for both the molecular ions of the derivatized oligosaccharides and their fragments. No fragment ions arising from loss of the bromine atom were observed.     

Negative Ion In-Source Decay Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for Sequencing Acidic Peptides

Matrix-assisted laser desorption/ionization (MALDI) in-source decay was studied in the negative ion mode on deprotonated peptides to determine its usefulness for obtaining extensive sequence information for acidic peptides. Eight biological acidic peptides, ranging in size from 11 to 33 residues, were studied by negative ion mode ISD (nISD). The matrices 2,5-dihydroxybenzoic acid, 2-aminobenzoic acid, 2-aminobenzamide, 1,5-diaminonaphthalene, 5-amino-1-naphthol, 3-aminoquinoline, and 9-aminoacridine were used with each peptide. Optimal fragmentation was produced with 1,5-diaminonphthalene (DAN), and extensive sequence informative fragmentation was observed for every peptide except hirudin(54–65). Cleavage at the N–Cα bond of the peptide backbone, producing c′ and z′ ions, was dominant for all peptides. Cleavage of the N–Cα bond N-terminal to proline residues was not observed. The formation of c and z ions is also found in electron transfer dissociation (ETD), electron capture dissociation (ECD), and positive ion mode ISD, which are considered to be radical-driven techniques. Oxidized insulin chain A, which has four highly acidic oxidized cysteine residues, had less extensive fragmentation. This peptide also exhibited the only charged localized fragmentation, with more pronounced product ion formation adjacent to the highly acidic residues. In addition, spectra were obtained by positive ion mode ISD for each protonated peptide; more sequence informative fragmentation was observed via nISD for all peptides. Three of the peptides studied had no product ion formation in ISD, but extensive sequence informative fragmentation was found in their nISD spectra. The results of this study indicate that nISD can be used to readily obtain sequence information for acidic peptides.

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