A novel approach to collision-induced dissociation (CID) for ion mobility-mass spectrometry experiments

Collision induced dissociation (CID) combined with matrix assisted laser desorption ionization-ion mobility-mass spectrometry (MALDI-IM-MS) is described. In this approach, peptide ions are separated on the basis of mobility in a 15 cm drift cell. Following mobility separation, the ions exit the drift cell and enter a 5 cm vacuum interface with a high field region (up to 1000 V/cm) to undergo collisional activation. Ion transmission and ion kinetic energies in the interface are theoretically evaluated accounting for the pressure gradient, interface dimensions, and electric fields. Using this CID technique, we have successfully fragmented and sequenced a number of model peptide ions as well as peptide ions obtained by a tryptic digest. This instrument configuration allows for the simultaneous determination of peptide mass, peptide-ion sequence, and collision-cross section of MALDI-generated ions, providing information critical to the identification of unknown components in complex proteomic samples.     

Improved PSD and CID on a MALDI TOFMS

The influence of several instrument-operating parameters on the product-ion resolution and mass accuracy in matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) post-source decay (PSD) and collision-induced dissociation (CID) experiments is reported. Voltages commonly applied to the reflectron for PSD and CID experiments were found to be non-ideal; optimization of these voltages resulted in better resolution across each segment of the measured PSD spectrum. Mass resolution, calculated as M/DeltaM (FWHM) for the product-ion peaks, was as high as 2500. Additionally, precursor-ion selection and segment mass range setup were each found to have dramatic influences on product-ion mass accuracy. An understanding of the influence of these variables aided in the interpretation of (a-NH3) and (b - NH3) ions observed in the PSD/CID spectra of a number of peptides. In addition, product ions resulting from coincidence peaks in the precursor-ion selection window were found to be a general problem. With the improvements to resolution and optimization of these mass accuracy variables, the mass accuracy of product ions from MALDI TOF PSD and CID experiments was tested with several reference materials, including the peptides Substance P, bradykinin, angiotensin I, and angiotensin II and the synthetic polymers poly(methyl methacrylate) and polystyrene. The absolute error (Da) for each test material was, on average, below 0.1 Da, demonstrating a significant improvement in mass accuracy using the improved operational parameters and an extension of the use of poly(ethylene glycol) (PEG) as a mass calibrant for the PSD/CID spectra.     

Studying disulfide bond rearrangement by MALDI-RTOF PSD and MALDI-TOF/RTOF high-energy CID (20 keV) experiments of peptides derived from ammodytoxins

Ammodytoxins (Atxs) are presynaptically neurotoxic phospholipases present in Vipera ammodytes ammodytes snake venom. Atxs show a high sequence homology and contain 14 cysteines which form seven biologically relevant disulfide bridges-connecting non-neighboring cysteines. Formic acid cleavage was performed to confirm protein sequences by MALDI RTOF MS and resulted in 95.6% sequence coverage exhibiting only few formylations. Cysteine-containing peptides showed adjacent signals 2 and/or 4 Da lower (according to the number of cysteines present in the peptide) than the theoretical molecular weight indicating disulfide bridge rearrangement. Post-source decay (PSD) and high-energy collision-induced dissociation (CID) at 20 keV experiments showed fragmentation pattern unique for the reduced, thiol group containing and the oxidized, disulfide bridge harboring peptides. Besides typical low-energy fragment ions observed during PSD experiments (a-, b-, y-type ions), additional high-energy fragment ions (c-, x-, w-, d-type and internal fragments) of significant intensity were generated during fragmentation at 20 keV. In the case of charge directing N- and C-termini, x- and w-type ions were also observed during PSD. Good and up to complete sequence coverage was achieved for all studied peptides from Atxs in the case of high-energy CID, whereas PSD lacked information particularly for larger peptides.     

Axial CID and high pressure resonance CID in a miniature ion trap mass spectrometer using a discontinuous atmospheric pressure interface

Axial collision induced dissociation (CID) and high-pressure resonance CID were implemented and compared with normal low-pressure resonance CID in a miniature ion trap mass spectrometer to obtain more complete fragmentation spectra. Axial CID was realized simply by applying a potential to the discontinuous atmospheric pressure interface (DAPI) capillary without performing parent ion isolation before dissociation. High-pressure resonance CID employed a double-introduction pulse scan function, by means of which precursor ions isolated at low-pressure (<10⁻³ torr) were dissociated at high-pressure (0.1 torr-1 torr) with higher excitation energy, so that tandem MS of isolated precursor ions was achieved and extensive fragmentation was obtained. A simple peptide (Leu-enkephalin) and dye molecule (rhodamine B) ionized by ESI were used to investigate both methods and compare them with normal low-pressure resonance CID.     

In-source CID of guanosine: Gas phase ion-molecule reactions

In-source collision induced dissociation was applied to access second generation ions of protonated guanosine. The in-source gas-phase behavior of [BH2]+-NH3 (m/z 135, C5H3N4O+) was investigated. Adduct formation and reactions with available solvent molecules (H2O and CH3OH) were demonstrated. Several addition/elimination sequences were observed for this particular ion and solvent molecules. Dissociation pathways for the newly formed ions were developed using a QqTOF mass spectrometer, permitting the assignment of elemental compositions of all product ions produced. Reaction schemes were suggested arising from the ring-opened intermediate of the protonated base moiety [BH2]+, obtained from fragmentation of guanosine. The mass spectral data revealed that the in-source CH3OH-reaction product underwent more complex fragmentations than the comparable ion following reaction with H2O. A rearrangement and a parallel radical dissociation pathway were discerned. Apart from the mass spectrometric evidence, the fragmentation schemes are supported by density functional theory calculations, in which the reaction of the ring-opened protonated guanine intermediate with CH3OH and a number of subsequent fragmentations were elaborated. Additionally, an in-source transition from the ring-opened intermediate of protonated guanine to the ring-opened intermediate of protonated xanthine was suggested. For comparison, a low-energy collision induced dissociation study of xanthosine was performed. Its dissociation pathways agreed with our assumption.     

Influence of size on apparent scrambling of sequence during CID of b-type ions

We investigated the influence of peptide size on the apparent loss of sequence during collision-induced dissociation (CID) of b ions using a group of peptides containing from between 4 and 10 residues. Although scrambling of sequence for b ₃⁺ generated from tetrapeptides is minimal, significant formation of nondirect sequence ions (i.e., ions for which scrambling has apparently occurred) was observed for all larger b ions included in the study.     

共线量子散射方法研究D+CID体系

用共线量子散射方法计算D+CID体系的反应几率和传能几率。发现存在很强的振荡现象。体系的碰撞中间态的寿命的计算结果为fs-ps数量级。当某一反应通道打开时,在能量阈值附近通常都伴随着一些长寿命的碰撞中间态。这一点该体系中表现很比较明显。     

Label Scrambling During CID of Covalently Labeled Peptide Ions

Covalent labeling along with mass spectrometry is finding more use as a means of studying the higher order structure of proteins and protein complexes. Diethylpyrocarbonate (DEPC) is an increasingly used reagent for these labeling experiments because it is capable of modifying multiple residues at the same time. Pinpointing DEPC-labeled sites on proteins is typically needed to obtain more resolved structural information, and tandem mass spectrometry after protein proteolysis is often used for this purpose. In this work, we demonstrate that in certain instances, scrambling of the DEPC label from one residue to another can occur during collision-induced dissociation (CID) of labeled peptide ions, resulting in ambiguity in label site identity. From a preliminary study of over 30 labeled peptides, we find that scrambling occurs in about 25% of the peptides and most commonly occurs when histidine residues are labeled. Moreover, this scrambling appears to occur more readily under non-mobile proton conditions, meaning that low charge-state peptide ions are more prone to this reaction. For all peptides, we find that scrambling does not occur during electron transfer dissociation, which suggests that this dissociation technique is a safe alternative to CID for correct label site identification. Graphical Abstract

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基于化学衍生和CID碎裂模式鉴定蛋白精氨酸-ADP-核糖基化的新方法

建立了一种新的基于碰撞诱导解离（CID）碎裂模式鉴定精氨酸-腺苷二磷酸（ADP）-核糖基化多肽的新方法. 首先,在碱性条件下将精氨酸-ADP-核糖基化血管紧张素-Ⅰ转变为鸟氨酸化血管紧张素-Ⅰ,或在磷酸二酯酶和碱性磷酸酶处理下水解为精氨酸核糖基化血管紧张素-Ⅰ,然后对上述2种衍生物进行基于CID碎裂模式的串联质谱分析. 结果表明,与未衍生的精氨酸-ADP-核糖基化血管紧张素-Ⅰ相比,在鸟氨酸化血管紧张素-Ⅰ和精氨酸核糖基化血管紧张素-Ⅰ的质谱图上发现大部分来自于肽骨架碎裂的离子峰,可提供足够的序列信息以确定精氨酸-ADP-核糖基化位点.     

MALDI-TOF/TOF CID study of 4-alkyl-substituted polystyrene fragmentation reactions

MALDI-TOF/TOF CID experiments were conducted on a variety of hydrogen-terminated poly(4-methylstyrene), hydroxylated poly(t-butylstyrene), and polystyrene precursor ions: n = 10, 15, 20, 25, and 30, where the number of repeat units n corresponds to the oligomer mass number. The influences of structure, molecular weight, and effective collision kinetic energy on degradation mechanisms were examined to test the generality of our multi-chain fragmentation model developed for polystyrene. Each depolymerization mechanism is presented in detail with experimental and computational data to justify/rationalize its occurrence and effective kinetic energy dependence. These processes show the complex interrelationship between the various pathways along with preferred production of secondary radicals, which suppresses the appearance of primary radicals. Additionally, Py-GC/MS experimental data are presented, for comparison of the multimolecular free radical reactions in pyrolysis with the unimolecular fragmentation reactions of MS/MS. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of erythropoietin biosimilars using mass spectrometric CID and HCD techniques

Recombinant human erythropoietin (rHuEPO) is the first cloned hematopoietic growth factor available for pharmaceutical treatment, especially anemia, since 1988. Unfortunately, the ability of rHuEPO in boosting erythropoiesis, and thus aerobic capacity has led to its abuse in endurance sport. Besides, the expiry of the original rHuEPO patent has resulted in many biosimilars being produced which led to special requirements regarding quality control. As a consequence, there is a huge demand for all rHuEPOs to be well characterized for the ease of identification, differentiation, and detection. Glycoproteomic mass spectrometry, the most current promising approach for rHuEPOs analysis, was employed to characterize 4 rHuEPOs including epoetin-α, epoetin-β, darbepoetin-α and Mircera. Via nanoLC-ESI-MS/MS, distinct glycoproteomic profiles of each rHuEPO have been achieved for differential analysis. With two different fragmentation methods, collision-induced dissociation (CID) and higher-energy collision dissociation (HCD), maximum of 75%, 70%, 70%, and 77% protein sequence coverages were attained for epoetin-α, epoetin-β, darbepoetin-α and Mircera, respectively. From the peptides/glycopeptides mixture, similar and unique peptides/glycopeptides (biomarkers) for each rHuEPO have been identified. With the discovery of high quality and unique biomarkers, a more standardized and efficient method for quality control and rHuEPO abuse detection can be developed.     

The role of mobile protons in negative ion CID of oligosaccharides

Carbohydrates of all classes consist of glycoform mixtures built on common core units. Determination of compositions and structures of such mixtures relies heavily on tandem mass spectrometric data. Analysis of native glycans is often necessary for samples available in very low quantities and for sulfated glycan classes. Negative tandem mass spectrometry (MS) provides useful product ion profiles for neutral oligosaccharides and is preferred for acidic classes. In previous work from this laboratory, site-specific influences of sialylation on product ion profiles in the negative mode were elucidated. The present results show how the interplay of two other acidic groups, uronic acids and sulfates, determines product ion patterns for chondroitin sulfate oligosaccharides. Unsulfated chondroitin oligosaccharides dissociate to form C-type ions almost exclusively. Chondroitin sulfate oligosaccharides produce abundant B- and Y-type ions from glycosidic bond cleavage with C- and Z-types in low abundances. These observations are explained in terms of competing proton transfer reactions that occur during the collisional heating process. Mechanisms for product ion formation are proposed based on tandem mass spectra and the abundances of product ions as a function of collision energy.     

Identification of bifucosylated glycoforms using low‐energy CID spectra

We have used tandem mass spectrometry (MS/MS)‐based analysis of glycopeptides in order to identify the composition and structure of rare glycoforms. The results illustrate utility of low‐energy MS/MS for structure identification. We have shown the presence of bifucosylated and trifucosylated glycoforms in human α‐1‐acid glycoprotein (AGP), a major plasma glycoprotein. Fucosylation in the case of AGP always occurs on the antennae; core fucosylation was not observed.     

Study on the CID Fragmentation Pathways of Deprotonated 4’-Monophosphoryl Lipid A

Lipid A, the membrane-bound phosphoglycolipid component of bacteria, is held responsible for the clinical syndrome of gram-negative sepsis. In this study, the fragmentation behavior of a set of synthetic lipid A derivatives was studied by electrospray ionization multistage mass spectrometry (ESI-MSⁿ), in conjunction with tandem mass spectrometry (MS/MS), using low-energy collision-induced dissociation (CID). Genealogical insight about the fragmentation pathways of the deprotonated 4’-monophosphoryl lipid A structural analogs led to proposals of a number of alternative dissociation routes that have not been reported previously. Each of the fragment ions was interpreted using various possible mechanisms, consistent with the principles of reactions described in organic chemistry. Specifically, the hypothesized mechanisms are: (i) cleavage of the C-3 primary fatty acid leaves behind an epoxide group attached to the reducing sugar; (ii) cleavage of the C-3’ primary fatty acid (as an acid) generates a cyclic phosphate connected to the nonreducing sugar; (iii) cleavage of the C-2’ secondary fatty acid occurs both in acid and ketene forms; iv) the C-2 and C-2’ primary fatty acids are eliminated as an amide and ketene, respectively; (v) the ^0,2A₂ cross-ring fragment contains a four-membered ring (oxetanose); (vi) the ^0,4A₂ ion is consecutively formed from the ^0,2A₂ ion by retro-aldol, retro-cycloaddition, and transesterification; and (vii) formations of H₂PO₄⁻ and PO₃⁻ are associated with the formation of sugar epoxide. An understanding of the relation between ^0,2A₂ and ^0,4A₂-type sugar fragments and the different cleavage mechanisms of the two ester-linked primary fatty acids is invaluable for distinguishing lipid A isomers with different locations of a single ester-linked fatty acid (i.e., at C-3 or C-3’). Thus, in addition to a better comprehension of lipid A fragmentation processes in mass spectrometers, our observations can be applied for a more precise elucidation of naturally occurring lipid A structures.     

Threshold CID investigation of isomeric Cu(I) azabox complexes

An improved method for deconvoluting energy-resolved collision-induced dissociation cross sections yields ligand binding energies for organometallic complexes with substantially less prior information than before. Application to isomeric 2:1 complexes of azabox ligands with Cu(I) gives consistent results for the binding energies of the ligands to homo- and heterochiral complexes with pseudo-enantiomeric ligands for cases where previous deconvolution methods had failed to give satisfactory results.     

Collision cell pressure effect on CID spectra pattern using triple quadrupole instruments: a RRKM modeling

Control of the ion internal energy in mass spectrometry is needed to establish a workable mass spectral library. The purpose of this study is to understand and to compare the pressure effects on the collision‐induced dissociation (CID) spectrum pattern recorded using triple quadrupole instruments. The monoprotonated Leucine enkephalin [YGGFL, H⁺] was used as a thermometer molecule to calibrate the electrospray ionization (ESI) and the CID internal energies deposited on the molecular species and the time scale of ion decompositions. The survival yield and the ratio of a₄/b₄ fragment ions were mainly monitored. The energy uptake for the ESI source geometry used in our study has no impact on the CID spectrum fingerprint. The collision cell pressure for the [YGGFL, H⁺] has a major influence on the SY curves slope and on the experimental time scale. To demonstrate the pressure effect on internal energy distribution, three models (threshold, thermal and collisional) based on RRKM theory were built using the Masskinetics software. As a result, the limit of each model is discussed, and the investigation demonstrates that the thermal model, using truncated Maxwell‐Boltzmann internal energy distribution, is well‐suited for simulating the experimental data at high pressure widely used in the analytical conditions. Copyright © 2013 John Wiley & Sons, Ltd.     

Comparison of LID versus CID activation modes in tandem mass spectrometry of peptides

We report our contribution to the systematic investigation of peptide fragmentations performed on high‐performance Tof equipment, operating in MS and MS/MS modes, such as ESI‐QqTof and MALDI‐Tof/Tof instruments that are commonly available today in proteomic laboratories. Whereas the former analyzer's configuration provides low‐energy collision‐induced dissociations (CID), the latter allows tunable activation methods of the selected parent ion to induce either metastable laser‐induced dissociations (LID) or high‐energy CID (‘gas on spectra LID’). Fragmentation of the monoprotonated ion of 53 peptides (FW 807–2853 g/mol) was undertaken upon low‐energy CID on an ESI‐QTof mass spectrometer (Waters) as well as high‐energy CID and LID conditions on a MALDI Ultraflex mass spectrometer (Bruker). Systematic comparison of MS/MS spectra provided useful information on the performance of each piece of equipment for efficient peptide sequencing and also insights into the observed fragmentation behaviors. Copyright © 2008 John Wiley & Sons, Ltd.     

Electrospray low energy CID and MALDI PSD fragmentations of protonated sulfinamide cross-linked peptides

Murine S100A8 (A8) is a major cytoplasmic neutrophil protein and is converted to novel oxidation products containing Cys-epsilon amino-Lys sulfinamide cross-links and Met-sulfoxide by the neutrophil oxidant HOCl. Seven products were separated using RP-HPLC, with electrospray ionization mass spectrometry (ESI-MS) masses after deconvolution of 10,354, 10,388, +/- 1, and 20,707, +/- 3 Da, and all were resistant to reduction by dithiothreitol. The major products with masses of 10,354 Da contained Cys41-Lys34/35 intramolecular cross-links. Additional isomeric products with identical masses (10,354 Da) were isolated and peptide mapping and ESI/MS indicated that Cys41 forms covalent sulfinamide cross-links with either Lys6, Lys76, Lys83, or Lys87 present in A8. Electrospray low energy collisionally induced (CID) spectra of multiply-charged AspN digest peptides with sulfinamide cross-links contained characteristic fragmentations that corresponded to simple cleavage of the nitrogen-sulfur bond with charge retention on either of the fragment ions, allowing conformation of cross-linked peptides. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) post source decay spectra of [M + H] + ions of the same sulfinamide-containing cross-linked peptides fragment similarly, but additional facile fragmentation reactions corresponding to formation of a protonated peptide containing de-hydroalanine were attributed to cleavage of the carbon-sulfur bond. In addition, lose of methanesulfenic acid from Met-sulfoxide was observed. A sulfinamide-containing adduct was isolated after incubation of the A8/HOCl reaction mixture with Lys or alpha N-acetyl Lys with masses of 10,500 or 10,542 Da. ESI/MS/MS and MALDI/post decay source (PSD) analysis of A8(32)-(57)-sulfinamide showed the same characteristic fragmentations as those in the sulfinamide cross-linked peptides, confirming the Cys41-Lys sulfinamide cross-link and suggesting that peptide-peptide sulfinamides may all fragment similarly, allowing ready identification of these cross-links in proteins from more complex biological materials.     

"Fast excitation" CID in a quadrupole ion trap mass spectrometer

Collision-induced dissociation (CID) in a quadrupole ion trap mass spectrometer is usually performed by applying a small amplitude excitation voltage at the same secular frequency as the ion of interest. Here we disclose studies examining the use of large amplitude voltage excitations (applied for short periods of time) to cause fragmentation of the ions of interest. This process has been examined using leucine enkephalin as the model compound and the motion of the ions within the ion trap simulated using ITSIM. The resulting fragmentation information obtained is identical with that observed by conventional resonance excitation CID. "Fast excitation" CID deposits (as determined by the intensity ratio of the a(4)/b(4) ion of leucine enkephalin) approximately the same amount of internal energy into an ion as conventional resonance excitation CID where the excitation signal is applied for much longer periods of time. The major difference between the two excitation techniques is the higher rate of excitation (gain in kinetic energy) between successive collisions with helium atoms with "fast excitation" CID as opposed to the conventional resonance excitation CID. With conventional resonance excitation CID ions fragment while the excitation voltage is still being applied whereas for "fast excitation" CID a higher proportion of the ions fragment in the ion cooling time following the excitation pulse. The fragmentation of the (M + 17H)(17+) of horse heart myoglobin is also shown to illustrate the application of "fast excitation" CID to proteins.