Fragmentation of oligosaccharide ions with 157 nm vacuum ultraviolet light

The 157 nm photofragmentation of native and derivatized oligosaccharides was studied in a linear ion trap and in a home-built matrix-assisted laser desorption/ionization (MALDI) tandem time-of-flight (TOF/TOF) mass spectrometer, and the results were compared with collision-induced dissociation (CID) experiments. Photodissociation produces product ions corresponding to high-energy fragmentation pathways; for cation-derivatized oligosaccharides, it yields strong cross-ring fragment ions and provides better sequence coverage than low- and high-energy CID experiments. On the other hand, for native oligosaccharides, CID yielded somewhat better sequence coverage than photodissociation. The ion trap enables CID hybrid MS3 experiments on the high-energy fragment ions obtained from photodissociation.     

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.     

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.     

Sequencing wasp venom peptides by endopeptidase digestion and nested collision-induced dissociation/post-source decay methods

A method incorporating nested collision-induced dissociation/post-source decay (CID/PSD) combined with endopeptidase digestion is described as an approach to determine the sequence of N-terminally modified peptides. The information from immonium and related ions observed in the CID/PSD spectrum was used for the selection of a suitable endopeptidase for the digestion of peptides. Rapid and reliable assignment of peptide sequence was performed by the comparison of CID/PSD spectra of both intact and endopeptidese-digested peptide fragments, since the assignments of the observed fragment ions to either N- or C-terminal ions can thus be carried out unambiguously. This nested CID/PSD method was applied to the sequence determination of two peptides from the solitary wasps Anoplius samariensis and Batozonellus maculifrons (pompilid wasps), which could not be sequenced by the Edman method due to N-terminal modification.     

Internal energy deposition in chemical ionization/tandem mass spectrometry

The efficiency of the collision-induced dissociation (CID) process as a function of the internal energy deposited into the ion during the ionization event was evaluated. (M + H)+ ions of pyrrole, pyrrolidine, pyridine and piperidine (five and six-membered ring heterocyclics) were generated by chemical ionization (CI). The internal energy of the ions was varied by using different reagent gases. Both high-energy (keV) and low-energy (eV) CID were performed on these ions. The experiments showed that the (M + H)+ ions of the five-membered ring compounds, pyrrole and pyrrolidine, have higher fragmentation efficiencies than the six-membered ring compounds, pyridine and piperidine. Fragmentation efficiencies in high-energy CID clearly correlate with the internal energy deposited by the ionization technique. Experiments showed that the low-energy CID process is more sensitive than high-energy CID to changes in internal energy.     

On performing simultaneous electron transfer dissociation and collision-induced dissociation on multiply protonated peptides in a linear ion trap

We propose a tandem mass spectrometry method that combines electron-transfer dissociation (ETD) with simultaneous collision-induced dissociation (CID), termed ETD/CID. This technique can provide more complete sequence coverage of peptide ions, especially those at lower charge states. A selected precursor ion is isolated and subjected to ETD. At the same time, a residual precursor ion is subjected to activation via CID. The specific residual precursor ion selected for activation will depend upon the charge state and m/z of the ETD precursor ion. Residual precursor ions, which include unreacted precursor ions and charge-reduced precursor ions (either by electron-transfer or proton transfer), are often abundant remainders in ETD-only reactions. Preliminary results demonstrate that during an ETD/CID experiment, b, y, c, and z-type ions can be produced in a single experiment and displayed in a single mass spectrum. While some peptides, especially doubly protonated ones, do not fragment well by ETD, ETD/CID alleviates this problem by acting in at least one of three ways: (1) the number of ETD fragment ions are enhanced by CID of residual precursor ions, (2) both ETD and CID-derived fragments are produced, or (3) predominantly CID-derived fragments are produced with little or no improvement in ETD-derived fragment ions. Two interesting scenarios are presented that display the flexibility of the ETD/CID method. For example, smaller peptides that show little response to ETD are fragmented preferentially by CID during the ETD/CID experiment. Conversely, larger peptides with higher charge states are fragmented primarily via ETD. Hence, ETD/CID appears to rely upon the fundamental reactivity of the analyte cations to provide the best fragmentation without implementing any additional logic or MS/MS experiments. In addition to the ETD/CID experiments, we describe a novel dual source interface for providing front-end ETD capabilities on a linear ion trap mass spectrometer.     

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.     

A novel salt bridge mechanism highlights the need for nonmobile proton conditions to promote disulfide bond cleavage in protonated peptides under low-energy collisional activation

The gas-phase fragmentation mechanisms of small models for peptides containing intermolecular disulfide links have been studied using a combination of tandem mass spectrometry experiments, isotopic labeling, structural labeling, accurate mass measurements of product ions, and theoretical calculations (at the MP2/6-311 + G(2d,p)//B3LYP/3-21G(d) level of theory). Cystine and its C-terminal derivatives were observed to fragment via a range of pathways, including loss of neutral molecules, amide bond cleavage, and S-S and C-S bond cleavages. Various mechanisms were considered to rationalize S-S and C-S bond cleavage processes, including charge directed neighboring group processes and nonmobile proton salt bridge mechanism. Three low-energy fragmentation pathways were identified from theoretical calculations on cystine N-methyl amide: (1) S-S bond cleavage dominated by a neighboring group process involving the C-terminal amide N to form either a protonated cysteine derivative or protonated sulfenyl amide product ion (44.3 kcal mol(-1)); (2) C-S bond cleavage via a salt bridge mechanism, involving abstraction of the alpha-hydrogen by the N-terminal amino group to form a protonated thiocysteine derivative (35.0 kcal mol(-1)); and (3) C-S bond cleavage via a Grob-like fragmentation process in which the nucleophilic N-terminal amino group forms a protonated dithiazolidine (57.9 kcal mol(-1)). Interestingly, C-S bond cleavage by neighboring group processes have high activation barriers (63.1 kcal mol(-1)) and are thus not expected to be accessible during low-energy CID experiments. In comparison to the energetics of simple amide bond cleavage, these S-S and C-S bond cleavage reactions are higher in energy, which helps rationalize why bond cleavage processes involving the disulfide bond are rarely observed for low-energy CID of peptides with mobile proton(s) containing intermolecular disulfide bonds. On the other hand, the absence of a mobile proton appears to "switch on" disulfide bond cleavage reactions, which can be rationalized by the salt bridge mechanism. This potentially has important ramifications in explaining the prevalence of disulfide bond cleavage in singly protonated peptides under MALDI conditions.     

Structural analysis of leukotriene C<Subscript>4</Subscript> isomers using collisional activation and 157 nm photodissociation

The fragmentation of 5-hydroxy-6-glutathionyl-7,9,11,14-eicosatetraenoic acid [leukotriene C4 or LTC4 (5, 6)] and its isomeric counterpart LTC4 (14, 15) were studied by low and high-energy collisional induced dissociation (CID) and 157 nm photofragmentation. For singly charged protonated LTC4 precursors, photodissociation significantly enhances the signal intensities of informative fragment ions that are very important to distinguish the two LTC4 isomers and generates a few additional fragment ions that are not usually observed in CID experiments. The ion trap enables MSn experiments on the fragment ions generated by photodissociation. Photofragmentation is found to be suitable for the structural identification and isomeric differentiation of cysteinyl leukotrienes and is more informative than low or high-energy CID. We describe for the first time the structural characterization of the LTC4 (14, 15) isomer by mass spectrometry using CID and 157 nm light activation methods.     

Advantages of using nested collision induced dissociation/post-source decay with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: sequencing of novel peptides from wasp venom

We have examined the applicability of the 'nested' collision induced dissociation/post-source decay (CID/PSD) method to the sequencing of novel peptides from solitary wasps which have neurotoxic venom for paralyzing other insects. The CID/PSD spectrum of a ladder peptide derived from an exopeptidase digest was compared with that of the intact peptide. The mass peaks observed only in the CID/PSD spectrum of a ladder peptide were extracted as C-terminal fragment ions. Assignment of C-terminal fragment ions enabled calculation of N-terminal fragment masses, leading to differentiation between N-terminal fragment ions and internal fragment ions. This methodology allowed rapid and sensitive identification by removing ambiguity in the assignment of the fragment ions, and proved useful for sequencing unknown peptides, in particular those available as natural products with a limited supply.     

High-energy collision-induced dissociation of small polycyclic aromatic hydrocarbons

High-energy collision-induced dissociation (CID) experiments on polycyclic aromatic hydrocarbons (PAHs) having 2-6 rings, naphthalene, anthracene, phenanthrene, fluoranthene, pyrene and coronene, were performed, and the relative abundances of their fragment ions were investigated as a function of collision energy. The results revealed that the PAHs except naphthalene showed a bimodal-type distribution of positive fragmentation ions, which is closely similar to the fragment-ion distribution reported for the CID of three-dimensional fullerene, C(60)(+) and C(70)(+). The three-ring isomers of anthracene and phenanthrene and the four-ring isomers of fluoranthene and pyrene can be distinguishable in their spectra under an electron ionization energy of 70 eV, but the high-energy CID spectra of the three- and four-ring isomers were almost identical. The fragmentation corresponding to fragment ions in the low-mass region of the bimodal CID spectra could be interpreted by the simple statistical model that fragment ions are formed by random evaporation from the molecular ions after a considerable structural rearrangement, 'phase transition', occurring at some high-energy state.     

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.     

Study of the dissociation of a charge-reduced phosphopeptide formed by electron transfer from an alkali metal target

Doubly protonated phosphopeptide (YGGMHRQET(p)VDC) ions obtained by electrospray ionization were collided with Xe and Cs targets to give singly and doubly charged positive ions via collision-induced dissociation (CID). The resulting ions were analyzed and detected by using an electrostatic analyzer (ESA). Whereas doubly charged fragment ions resulting from collisionally activated dissociation (CAD) were dominant in the CID spectrum with the Xe target, singly charged fragment ions resulting from electron transfer dissociation (ETD) were dominant in the CID spectrum with the Cs target. The most intense peak resulting from ETD was estimated to be associated with the charge-reduced ion with H2 lost from the precursor. Five c-type fragment ions with amino acid residues detached consecutively from the C-terminal were clearly observed without a loss of the phosphate group. These ions must be formed by N--Calpha bond cleavage, in a manner similar to the cases of electron capture dissociation (ECD) and ETD from negative ions. Although the accuracy in m/z of the CID spectra was about +/-1 Th because of the mass analysis using the ESA, it is supposed from the m/z values of the c-type ions that these ions were accompanied by the loss of a hydrogen atom. Four z-type (or y--NH3, or y--H2O) ions analogously detached consecutively from the N-terminal were also observed. The fragmentation processes took place within the time scale of 4.5 micros in the high-energy collision. The present results demonstrated that high-energy ETD with the alkali metal target allowed determination of the position of phosphorylation and the amino acid sequence of post-translational peptides.     

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.     

Comparison of CID,ETD and metastable atom‐activated dissociation (MAD) of doubly and triply charged phosphorylated tau peptides

The fragmentation behavior of the 2+ and 3+ charge states of eleven different phosphorylated tau peptides was studied using collision‐induced dissociation (CID), electron transfer dissociation (ETD) and metastable atom‐activated dissociation (MAD). The synthetic peptides studied contain up to two known phosphorylation sites on serine or threonine residues, at least two basic residues, and between four and eight potential sites of phosphorylation. CID produced mainly b‐/y‐type ions with abundant neutral losses of the phosphorylation modification. ETD produced c‐/z‐type ions in highest abundance but also showed numerous y‐type ions at a frequency about 50% that of the z‐type ions. The major peaks observed in the ETD spectra correspond to the charge‐reduced product ions and small neutral losses from the charge‐reduced peaks. ETD of the 2+ charge state of each peptide generally produced fewer backbone cleavages than the 3+ charge state, consistent with previous reports. Regardless of charge state, MAD achieved more extensive backbone cleavage than CID or ETD, while retaining the modification(s) in most cases. In all but one case, unambiguous modification site determination was achieved with MAD. MAD produced 15–20% better sequence coverage than CID and ETD for both the 2+ and 3+ charge states and very different fragmentation products indicating that the mechanism of fragmentation in MAD is unique and complementary to CID and ETD. Copyright © 2012 John Wiley & Sons, Ltd.     

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.     

Electrospray ionization tandem mass spectrometry sequencing of novel skin peptides from Ranid frogs containing disulfide bridges

Tandem mass spectrometry sequencing, as well as Edman sequencing of peptides belonging to the Rana genus, represents a difficult task due to the presence of a disulfide bridge at the C-terminus and their rather high molecular masses (over 2000 Da). The present study throws light upon the sequence of three rather long peptides (more than 20 amino acid residues each) isolated from the skin secretion of Russian frogs, Rana ridibunda and Rana arvalis. This novel aspect involves the fact that the sequences (including two sequences established de novo) were determined exclusively by means of mass spectrometry. A combination of electron capture dissociation (ECD) and collision-induced dissociaiton (CID) data accompanied by exact mass measurements (LTQ Fourier transform ion cyclotron resonance mass spectrometer) facilitated reaching the goal. To overcome the difficulty dealing with disulphide bridges ("Rana box"), reduction of the S-S bond with dithiotreitol followed by derivatization of Cys residues with iodoacetamide was used. The sequence was determined using combined spectral data on y and b series of fragment ions. A multiple mass spectrometry (MS(3)) experiment was also used to elucidate the sequence inside the "Rana box" after cysteine derivatization. Exact mass measurements were used to differentiate between Lys and Gln residues, while characteristic losses of 29 and 43 Da (d and w fragment ions) in CID and ECD experiments allowed us to distinguish between Ile and Leu isomeric acids.     

Comparison of laser-induced dissociation and high-energy collision-induced dissociation using matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) for peptide and protein identification

The fragmentation of peptides under laser-induced dissociation (LID) as well as high-energy collision-induced dissociation (CID) conditions has been investigated. The effect of the different fragmentation mechanisms on the formation of specific fragment ion types and the usability of the resulting spectra, e.g. for high-throughput protein identification, has been evaluated. Also, basic investigations on the influence of the matrix, as well as laser fluence, on the fragment ion formation and the consequences in the spectral appearance are discussed. The preconditions for obtaining 'pure' CID spectra on matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) instruments are evaluated and discussed as well as the differences between LID and CID in the resulting fragment ion types. While containing a wealth of information due to additional fragment ions in comparison with LID, CID spectra are significantly more complex than LID spectra and, due to different fragmentation patterns, the CID spectra are of limited use for protein identification, even under optimized parameter settings, due to significantly lower scores for the individual spectra. Conditions for optimal results regarding protein identification using MALDI-TOF/TOF instruments have been evaluated. For database searches using tandem mass spectrometric data, the use of LID as fragmentation technique in combination with parameter settings supporting the use of internal fragment ions turned out to yield the optimal results.     

On-Line Electrochemical Reduction of Disulfide Bonds: Improved FTICR-CID and -ETD Coverage of Oxytocin and Hepcidin

Particularly in the field of middle- and top-down peptide and protein analysis, disulfide bridges can severely hinder fragmentation and thus impede sequence analysis (coverage). Here we present an on-line/electrochemistry/ESI-FTICR-MS approach, which was applied to the analysis of the primary structure of oxytocin, containing one disulfide bridge, and of hepcidin, containing four disulfide bridges. The presented workflow provided up to 80 % (on-line) conversion of disulfide bonds in both peptides. With minimal sample preparation, such reduction resulted in a higher number of peptide backbone cleavages upon CID or ETD fragmentation, and thus yielded improved sequence coverage. The cycle times, including electrode recovery, were rapid and, therefore, might very well be coupled with liquid chromatography for protein or peptide separation, which has great potential for high-throughput analysis.