Gas-phase scrambling of disulfide bonds during matrix-assisted laser desorption/ionization mass spectrometry analysis

Evidence for photo-induced radical disulfide bond scrambling in the gas phase during matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is described. The phenomenon was observed during the analysis of tryptic peptides from insulin and was confirmed in the determination of disulfide bonds in the rhamnose-binding lectin SEL24K from the Chinook salmon Oncorhynchus tshawytscha. A possible mechanism for this surprising scrambling is proposed. Despite this finding, the disulfide bond pattern in SEL24K was assigned unambiguously by a multi-enzyme digestion strategy in combination with MALDI mass spectrometry. The pattern was found to be symmetrical in the tandem repeat sequence of SEL24K. To the best of our knowledge, this is the first report of disulfide bond scrambling in the gas phase during MALDI-MS analysis. This observation has important ramifications for unambiguous assignment of disulfide bonds.     

α-Conotoxin ImI incorporating stable cystathionine bridges maintains full potency and identical three-dimensional structure

The two disulfide bonds of α-conotoxin ImI, a peptide antagonist of the α7 nicotinic acetylcholine receptor (nAChR), were systematically replaced with isosteric redox-stable cystathionine thioethers. Regioselective thioether formation was accomplished on solid support through substitution of a γ-chlorohomoalanine by an intramolecular cysteine thiol to produce hybrid thioether/disulfide analogues (2 and 3) as well as a dual cystathionine analogue (4) that were found to be structurally homologous to α-conotoxin ImI by (1)H NMR. The antagonistic activity at the α7 nAChR of cystathionine analogue 3 (pIC(50) = 6.41 ± 0.09) was identical to that of α-conotoxin ImI (1, pIC(50) = 6.41 ± 0.09), whereas those of 2 (pIC(50) = 5.96 ± 0.09) and 4 (pIC(50) = 5.89 ± 0.09) showed a modest decrease. The effect of oxidation of the thioethers to sulfoxides was also investigated, with significant changes in the biological activities observed ranging from a >30-fold reduction (2S═O) to a 3-fold increase (3S═O(B)) in potencies.     

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.     

The contributions of specific amino acid side chains to signal intensities of peptides in matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of complex peptide mixtures is often hampered by signal suppression effects as well as certain intrinsic properties of specific peptides that influence the desorption/ionization behavior. The present systematic study reports on the relationship between the occurrence of certain amino acids in peptides and the intensities of the related ions which appear during MALDI-MS analysis for both tryptic digests of proteins and synthetic peptide mixtures. The analysis of the tryptic digests revealed that the peptide sequences of the most intense peaks detected by MALDI-MS contained significantly higher proportions of arginine, phenylalanine, proline, and leucine than the average values for the measured proteins. The relationship between the relative signal intensities and amino acid compositions of peptides was studied in more detail by the partial least squares (PLS) method using equimolar mixtures of 144 well-characterized synthetic peptides. The regression coefficients clearly indicated that the presence of arginine, phenylalanine, leucine and proline tend to enhance the desorption/ionization process which results in higher MALDI-MS peak intensities. Furthermore, it was shown that the impact of arginine depends strongly on the identity of adjacent amino acids.     

Derivatization by 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate for enhancing the ionization yield of small peptides and glycopeptides in matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry

The characterization of glycosylation in proteins by mass spectrometry (MS) is often impeded by strong suppression of ionization of glycopeptides in the presence of non-glycosylated peptides. Glycopeptides with a large carbohydrate part and a short peptide backbone are particularly affected by this problem. To meet the goal of generating mass spectra exhibiting glycopeptide coverages as complete as possible, derivatization of glycopeptides offers a practical way to increase their ionization yield. This paper investigated derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) which is a rapid labeling technique commonly used for fluorescence detection in high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). As test samples we used peptides and glycopeptides obtained by enzymatic digestion of three different glycoproteins, i.e., human antithrombin, chicken ovalbumin, and bovine alpha1-acid-glycoprotein. It was found that AQC derivatization resulted in strongly increased signal intensities when analyzing small peptides and glycopeptides by matrix-assisted laser desorption/ionization (MALDI)-MS. For these compounds the limit of detection could be reduced to low fmol amounts. Without derivatization only glycopeptides containing large peptide backbones were detected by MALDI-MS. This effect was even significant when glycopeptides were pre-separated and enriched by means of lectin affinity chromatography before MALDI-MS analysis and when using electrospray ionization (ESI). This labeling method, applied in combination with MS detection for the first time, was found to be well suited for the enhancement of detection sensitivity for small glycopeptides in MALDI-MS analysis and thus for reducing the need for pre-separation steps.     

Disulfide bond decay during matrix-assisted laser desorption/ionization time-of-flight mass spectrometry experiments

In our laboratory, we have been studying the reductive processes that occur during matrix-assisted laser desorption/ionization (MALDI) experiments. Recently, we have finished an analysis of the DHB matrix effect on the azo group in cyclic peptides. However, deep understanding of disulfide bond behaviour during a mass spectrometry (MS) experiment is much more important in proteomics as its reduction can cause serious errors in protein spectra interpretation. Therefore, we have focused on intra- and intermolecular disulfide bonds as well as disulfide bonds connecting cysteine and 2-thio-5-nitrobenzoic acid (TNB, Ellman's reagent modification) in model peptides during MALDI MS measurements. While the reduction was not observed for intra- and intermolecular cysteine-cysteine disulfide bonds, the disulfide connection between cysteine and TNB was always affected. It was proved that TNB and Ellman's reagent can act as a matrix itself. The results obtained enabled us to propose a reaction mechanism model which is able to describe the phenomena observed during the desorption/ionization process of disulfide-containing molecules.     

A Study of peptide-peptide interaction by matrix-assisted laser desorption/ionization

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to study peptide-peptide interaction. The interaction was seen when 6-aza-2-thiothymine was used as a matrix (pH 5.4), but was disrupted with a more acidic matrix, alpha-cyano-4-hydroxycinnamic acid (pH 2.0). In the present study, we show that dynorphin, an opioid peptide, and five of its fragments that contain two adjacent basic residues (Arg6-Arg7), all interact noncovalently with peptides that contain two to five adjacent acidic residues (Asp or Glu). Two other nonrelated peptides containing two (Arg6-Arg7) or three (Arg1-Lys2-Arg3) adjacent basic amino acid residues were studied and exhibited the same behavior. However, peptides containing adjacent Lys or His did not form noncovalent complexes with acidic peptides. The noncovalent bonding was sufficiently stable that digestion with trypsin only cleaved Arg and Lys residues that were not involved in hydrogen bonding with the acidic residues. In an equimolar mixture of dynorphin, dynorphin fragments (containing the motif RR), and an acidic peptide (minigastrin), the acidic peptide preferentially complexed with dynorphin. If the concentration of minigastrin was increased 10 fold, noncovalent interaction was seen with dynorphin and all its fragments containing the motif RR. In the absence of dynorphin, minigastrin formed noncovalent complexes with all dynorphin fragments. These findings suggest that conformation, equilibrium, and concentration do play a role in the occurrence of peptide-peptide interaction. Observations from this study include: (1) ionic bonds were not disrupted by enzymatic digests, (2) conformation and concentration influenced complex formation, and (3) the complex did not form with fragments of dynorphin or unrelated peptides that did not contain the motifs RR or RKR, nor with a fragment of dynorphin where Arg7 was mutated to a phenylalanine residue. These findings strongly suggest that peptide-peptide interaction does occur, and can be studied by MALDI if near physiologic pH is maintained.     

Oxidized carbon nanotubes as matrix for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of biomolecules

Oxidized carbon nanotubes (CNTs), which can form a stable homogeneous suspension in water close to a solution phase, were synthesized and used for matrix-assisted desorption/ionization mass spectrometric (MALDI-MS) analysis of biomolecules. Infrared (IR) spectra, transmission electron microscopy (TEM) and particle size analysis were used for the characterization of the oxidized CNTs. The results indicate that the physical structure of the CNTs was not damaged, but carboxylate groups were introduced onto the surface of the CNTs. In addition, impurities including amorphous carbon, which is one of the main reasons for ion source contamination, were destroyed by the oxidization. The carboxyl groups on the oxidized surface of the CNTs can not only provide an additional proton source, but can also increase the surface polarity and solubility of the CNTs, making it easier to manipulate which is important for MALDI analysis of some biomolecules, especially larger peptides and proteins. The oxidized CNTs were successfully applied to the analysis of neutral oligosaccharides, peptides, and insulin, and thus promise to be an efficient matrix for MALDI-MS analysis of biomolecules.     

Probing peptide libraries from Conus achatinus using mass spectrometry and cDNA sequencing: identification of delta and omega-conotoxins

The peptide library present in the venom of the piscivorous marine snail Conus achatinus has been probed using a combination of mass spectrometry and cDNA sequencing methods. Matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) analysis, before and following global reduction/alkylation of peptide mixtures, permits the rapid classification of individual components on the basis of the number of disulfide bonds. Mass fingerprinting and the reverse phase HPLC retention times permit a further deconvolution of the library in terms of peptide size and hydrophobicity. Sequencing of cDNA derived using O-superfamily specific primers yielded five complete conotoxin precursor sequences, ranging in polypeptide length from 75-87 residues containing six Cys residues at the C-terminus. Sequence analysis permits classification of the five putative mature peptides (Ac 6.1 to Ac 6.5) as delta, omega, and omega-like conotoxins. The presence of these predicted peptides in crude venom was established by direct matrix assisted laser desorption ionization tandem mass spectrometry (MALDI-MS/MS) sequencing following trypsin digestion of the peptide mixture after global reduction/alkylation. The determination of partial peptide sequences and comparison with the predicted sequences resulted in the identification of four of the five predicted conotoxins. The characterization of posttranslationally modified analogs, which are hydroxylated at proline or amidated at the C-terminus is also demonstrated. Crude venom analysis should prove powerful in studying both inter- and intra-species variation in peptide libraries.     

Efficient enrichment and desalting of protein digests using magnetic mesocellular carbon foams in matrix-assisted laser desorption/ionization mass spectrometry

We demonstrate that magnetic mesocellular carbon foams (Mag-MCF-C) can be effectively used for enrichment and desalting of protein digests or peptides in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The large mesocellular pores and surface area of Mag-MCF-C are likely to mainly contribute to high efficiency in enrichment and desalting of protein digests. The magnetic property of Mag-MCF-C enabled easy and simple enrichment and desalting process comprising adsorption, washing, and separation steps by using an external magnet. Following elution from Mag-MCF-C by using a matrix solution (CHCA in 70% ACN/0.1% TFA), the peptides were subjected to MALDI-MS analysis. As a result, MALDI mass spectra of peptides or tryptic protein digests were distinct even at a peptide concentration as low as 50 pM. The use of Mag-MCF-C resulted in significantly improved sequence coverage for protein identification when compared to other conventional methods. Mag-MCF-C will find applications in mass spectrometric analysis of low abundance peptides or protein digests with high sensitivity.     

De novo sequencing of novel neuropeptides directly from Ascaris suum tissue using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight

Direct analysis of tissue by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) allows for the rapid profiling of biological molecules with minimal loss of sample or degradation and reduced likelihood of chemical modification. However, there are still considerable challenges to overcome due to the complexity of tissue and the low quantity of endogenous peptide in a single cell. These problems are exacerbated in the nematode Ascaris suum because of the small size of individual neurons and the paucity of peptide per cell. In an effort to address these difficulties, the recently developed matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) technology was used in combination with an on-target derivatization in order to sequence novel neuropeptides directly from Ascaris nervous tissue. Direct MALDI-TOF/TOF analysis of Ascaris tissue provided the complete amino acid sequences for a previously characterized neuropeptide as well as for three novel peptides with homologues found in other nematodes. These results demonstrate a method for the rapid characterization of sub-femtomolar amounts of peptide directly from tissue using MALDI-TOF/TOF.     

Screening for disulfide-rich peptides in biological sources by carboxyamidomethylation in combination with differential matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Peptides with biological functions often contain disulfide bridges connecting two cysteine residues. In an attempt to screen biological fluids for peptides containing cysteine residues, we have developed a sensitive and specific method to label cysteines selectively and detect the resulting molecular mass shift by differential mass spectrometry. First, reduction of disulfide bridges and carboxyamidomethylation of free thiols is adjusted to quantitatively achieve cysteine alkylation for complex peptide extracts. In a second step, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) before and after chemical derivatization is performed, followed by differential analysis to determine shifted peaks; shifted peaks belong to cysteine-containing peptides, other peaks remain unchanged. The number of cysteines can then be determined by the resulting molecular mass shift. Free, reduced cysteines are shifted by 57 u, two oxidized cysteines involved in disulfide bridges (cystine) result in a shift to higher mass per disulfide bridge of 116 u. Disulfide bridges connecting different amino acid chains like insulin break up during reduction. In this case, two peaks with lower molecular masses result from a single one in the unmodified sample. With this technique, we were able to identify cysteine-containing peptides and short fragments of proteins present in human blood filtrate.     

Context-dependence of the contribution of disulfide bonds to beta-hairpin stability

Incorporation of disulfide bonds to stabilize protein and peptide structures is not always a successful strategy. To advance current knowledge on the contribution of disulfide bonds to beta-hairpin stability, a previously reported beta-hairpin-forming peptide was taken as a template to design a series of Cys-containing peptides. The conformational behavior of these peptides in their oxidized, disulfide-cyclized peptides, and reduced, linear peptides, was investigated on the basis of NMR parameters: NOEs, and 1H and 13C chemical shifts. We found that the effect of disulfide bonds on beta-hairpin stability depends on its location within the beta-hairpin structure, being very small or even destabilizing when connecting two hydrogen-bonded facing residues. When the disulfide bond is linking non-hydrogen-bonded facing residues, we estimated that its contribution to the free-energy change of beta-hairpin folding is approximately -1.0 kcal mol(-1). This value is larger than those reported for most beta-hairpin-stabilizing cross-strand side-chain-side-chain interactions, except for some aromatic-aromatic interactions, in particular the Trp-Trp one, and the cation-pi interaction between Trp and the non-natural methylated Arg/Lys. As disulfide bonds are frequently used to stabilize peptide conformations, our conclusions can be useful for peptide, peptidomimetic, and protein design, and may even extend to other chemical cross-links.     

A fast atom bombardment and matrix-assisted laser desorption/ionization mass spectrometry study of doubly charged porphyrins

In this mass spectrometry (MS) study of doubly charged porphyrin salts, fast atom bombardment (FAB) and matrix-assisted laser desorption/ionization (MALDI) MS techniques are utilized to examine several unique ionic species. The predominant transformation of preformed doubly charged ions in the desorption/ionization mechanism of FAB and MALDI is the result of deprotonation reactions to form singly charged ions of the type [M(2+) - H(+)](+) and of one-electron reductions to form radical cations [M(2+) + e(-)](+.). The dependence of this phenomenon and the formation of a number of additional ionic species on the different matrices and the FAB-matrix additive benzoquinone is examined. The significant analogous behavior of doubly charged porphyrins in FAB- and MALDI-MS leads to the conclusion that one-electron reductions are of distinct relevance in the desorption/ionization mechanism of MALDI.     

Improved matrix-assisted laser desorption/ionization mass spectrometric analysis of tryptic hydrolysates of proteins following guanidination of lysine-containing peptides

Analysis of tryptic digests of proteins using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry commonly results in superior detection of arginine-containing peptides compared with lysine-containing counterparts. The effect is attributable in part to the greater stability of the arginine-containing peptide ions associated with the sequestration of the single ionizing proton on the arginine side-chain. Reaction of peptides with O-methylisourea resulted in conversion of lysine to homoarginine residues with consequent improved detection during MALDI-MS. Analysis of the underivatized tryptic digest of the yeast protein, enolase, revealed peptides representing 20% of the protein; the corresponding figure after derivatization was 46%.     

Phosphoric acid enhances the performance of Fe(III) affinity chromatography and matrix-assisted laser desorption/ionization tandem mass spectrometry for recovery, detection and sequencing of phosphopeptides

An integrated analytical strategy for enrichment, detection and sequencing of phosphorylated peptides by matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS) is reported. o-Phosphoric acid was found to enhance phosphopeptide ion signals in MALDI-MS when used as the acid dopant in 2,5-dihydroxybenzoic acid (2,5-DHB) matrix. The effect was largest for multiply phosphorylated peptides, which exhibited an up to ten-fold increase in ion intensity as compared with standard sample preparation methods. The enhanced phosphopeptide response was observed during MALDI-MS analysis of several peptide mixtures derived by proteolytic digestion of phosphoproteins. Furthermore, the mixture of 2,5-DHB and o-phosphoric acid was an excellent eluant for immobilized metal affinity chromatography (IMAC). Singly and multiply phosphorylated peptide species were efficiently recovered from Fe(III)-IMAC columns, reducing sample handling for phosphopeptide mapping by MALDI-MS and subsequent phosphopeptide sequencing by MALDI-MS/MS. The enhanced response of phosphopeptide ions in MALDI facilitates MS/MS of large (>3 kDa) multiply phosphorylated peptide species and reduces the amount of analyte needed for complete characterization of phosphoproteins.     

Investigation of calmodulin-peptide interactions using matrix-assisted laser desorption/ionization mass spectrometry

In this report, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to study the binding interactions between calmodulin and two target peptides (melittin and substance P). Various matrix conditions were tested and the less acidic matrix DHAP and THAP were found to favor the survival of the intact calcium-calmodulin as well as the calmodulin-peptide complexes. However, the application of direct MALDI-MS to detect the intact complexes turned out to be very difficult due to the dissociation of the complexes and the formation of nonspecific aggregates. In contrast, the specific binding of the target peptides to calmodulin could be easily deduced using intensity-fading (IF) MALDI-MS. Compared with the nonbinding control, clear reduction in the ion abundances of the target peptides was observed with the addition of calmodulin. Relative binding affinities of different peptides towards the protein could also be estimated using IF-MALDI-MS. This study may extend the application of IF-MALDI-MS in the analysis of noncovalent complexes and offer a perspective into the utility of MALDI-MS as an alternative approach to study the peptides binding to calmodulin.     

Proteomics of gluten: mapping of the 1Bx7 glutenin subunit in Chinese Spring cultivar by matrix-assisted laser desorption/ionization

The verification of the cDNA-deduced sequence of the high molecular weight glutenin subunit 1Bx7 in Chinese Spring cultivar was achieved by direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis of the tryptic fragments. The published sequence of the 1Bx7 subunit contains 5 Lys and 15 Arg residues but, due to the presence of three Arg-Pro bonds, which are generally resistant to cleavage by trypsin, or cleaved to a very limited extent by trypsin, 19 peptides can be predicted. The identification of the tryptic fragments was achieved by direct MALDI-MS analysis by using three different matrices (DHB, SA and HCCA) in combination with the most compatible sample preparation procedures in order to obtain the maximum sequence coverage. MALDI analysis of the 1Bx7 tryptic digest resulted in the identification of the expected peptides and additional fragments arising from non-specific cleavages; the fragments that were not detected are peptides with low mass (from 147.2 to 317.4), so we obtained a sequence coverage of 98.8%. The results reported here also indicated that the sequence of the 1Bx7 subunit from cv. Chinese Spring is different from the cDNA-deduced sequence reported in the literature; in particular, a possible insertion of the hexapeptide QPGQGQ within the sequence Gln630-Tyr725 was suggested. Finally, it is possible to rule out glycosylation of the 1Bx7 subunit, or any other post-translational modification, to within the detection limits of the method.     

Investigation of some biologically relevant redox reactions using electrochemical mass spectrometry interfaced by desorption electrospray ionization

Recently we have shown that, as a versatile ionization technique, desorption electrospray ionization (DESI) can serve as a useful interface to combine electrochemistry (EC) with mass spectrometry (MS). In this study, the EC/DESI-MS method has been further applied to investigate some aqueous phase redox reactions of biological significance, including the reduction of peptide disulfide bonds and nitroaromatics as well as the oxidation of phenothiazines. It was found that knotted/enclosed disulfide bonds in the peptides apamin and endothelin could be electrochemically cleaved. Subsequent tandem MS analysis of the resulting reduced peptide ions using collision-induced dissociation (CID) and electron-capture dissociation (ECD) gave rise to extensive fragment ions, providing a fast protocol for sequencing peptides with complicated disulfide bond linkages. Flunitrazepam and clonazepam, a class of nitroaromatic drugs, are known to undergo reduction into amines which was proposed to involve nitroso and N-hydroxyl intermediates. Now in this study, these corresponding intermediate ions were successfully intercepted and their structures were confirmed by CID. This provides mass spectrometric evidence for the mechanism of the nitro to amine conversion process during nitroreduction, an important redox reaction involved in carcinogenesis. In addition, the well-known oxidation reaction of chlorpromazine was also examined. The putative transient one-electron transfer product, the chlorpromazine radical cation (m/z 318), was captured by MS, for the first time, and its structure was also verified by CID. In addition to these observations, some features of the DESI-interfaced electrochemical mass spectrometry were discussed, such as simple instrumentation and the lack of background signal. These results further demonstrate the feasibility of EC/DESI-MS for the study of the biology-relevant redox chemistry and would find applications in proteomics and drug development research.     

Matrix-assisted laser desorption/ionization time-of-flight and nano-electrospray ionization ion trap mass spectrometric characterization of 1-cyano-2-substituted-benz[f]isoindole derivatives of peptides for fluorescence detection

A series of hexa- to decapeptides (molecular mass range 800-1200) were labeled with naphthalene-2,3-dicarboxaldehyde, which preferentially reacts with the primary amino groups of a peptide. A highly stable peptide conjugate is formed, which allows selective analysis by fluorescence at excitation and emission wavelengths of 420 and 490 nm, respectively. After removal of unreacted compounds, the peptide conjugates were characterized by matrix-assisted laser desorption/ionization (MALDI) time-of-flight and nano-electrospray ionization (ESI) ion trap mass spectrometry. They readily form both [M + H]+ ions by MALDI and both [M + H]+ and [M + 2H]2+ ions by ESI. Furthermore, the fragmentation behavior of the N-terminally tagged peptides, exhibiting an uncharged N-terminus, was investigated applying post-source decay fragmentation with a curved field reflector and collision-induced dissociation with a quadrupole ion trap. Fragmentation is dominated in both cases by series of a-, b- and y-type ions and [M + H - HCN]+ ions. Peptide bonds adjacent to the fluorescence label were less susceptible to cleavage than the bonds of the non-derivatized peptide ions. In general, the resulting fragment ion patterns were less complex than those of the underivatized peptides.