An investigation of the energetics of peptide ion dissociation by laser desorption chemical ionization fourier transform mass spectrometry

The energy dependence of competing fragmentation pathways of protonated peptide molecules is studied via laser desorption—chemical ionization in a Fourier transform ion cyclotron resonance spectrometer. Neutral peptide molecules are desorbed by the technique of substrate-assisted laser desorption, followed by post-ionization with a proton transfer reagent ion species. The chemical ionization reaction activates the protonated peptide molecules, which then fragment in accordance with the amount of excess energy that is deposited. Chemical ionization forms a protonated molecule with a narrower distribution of activation energy than can be formed by activation methods such as collision activated dissociation. Furthermore, the upper limit of the activation energy is well defined and is approximately given by the enthalpy of the chemical ionization reaction. Control over the fragmentation of peptide ions is demonstrated through reactions between desorbed peptide molecules with different reagent ion species. The fragmentation behavior of peptide ions with different internal energies is established by generation of a breakdown curve for the peptide under investigation. Breakdown curves are reported for the peptides Val-Pro, Val-Pro-Leu, Phe-Phe-Gly-Leu-Met NH₂, and Arg-Lys-Asp-Val-Tyr. The derived breakdown curve of Val-Pro has been fitted by using quasi-equilibrium Rice-Ramsperger-Kassel-Marcus theory to model the unimolecular dissociation of the protonated peptide to provide a better understanding of the mechanisms for the formation of fragment ions that originate from protonated peptides.     

CHCA-modified Au nanoparticles for laser desorption ionization mass spectrometric analysis of peptides

Gold nanoparticles (AuNPs) have been studied as a potential solid-state matrix for laser desorption/ionization mass spectrometry (LDI-MS) but the efficiency in ionization remains low. In this report, AuNPs are capped by a self-assembled monolayer of cysteamine and modified with α-cyano-4-hydroxycinnanic acid (CHCA) for effective MALDI measurements. CHCA-terminated AuNPs offer marked improvement on peptide ionization compared with citrate-capped or cysteamine-capped AuNPs. The coating also effectively suppresses formation of Au cluster ions and analyte fragment ions, leading to cleaner mass spectra. Addition of glycerol and citric acid to the peptide/AuNPs sample further improves the performance of these AuNPs for LDI-MS analysis. Glycerol appears to enhance the dispersion of AuNPs in sample spots, increasing the sample ionization and shot-to-shot reproducibility, while citric acid serves as an external proton donor, providing high production of protonated analyte ions and reducing fragmentation of peptides on the nanoparticle-based surface. Optimal ratios of citric acid, glycerol, and AuNPs in sample solution have been systematically studied. A more than 10-fold increase for desorption ionization of peptides can be achieved by combining 5% glycerol and 20 mM citric acid with the CHCA-terminated AuNPs. The applicability of the CHCA-AuNPs for LDI-MS analysis of protein digests has also been demonstrated. This work shows the potential of AuNPs for SALDI-MS analysis, and the improvement with chemical functionalization, controlled dispersion, and use of an effective proton donor.     

Substituent effects on the gas-phase fragmentation reactions of sulfonium ion containing peptides

The multistage mass spectrometric (MS/MS and MS3) gas-phase fragmentation reactions of methionine side-chain sulfonium ion containing peptides formed by reaction with a series of para-substituted phenacyl bromide (XBr where X=CH2COC6H4R, and R=--COOH, --COOCH3, --H, --CH3 and --CH2CH3) alkylating reagents have been examined in a linear quadrupole ion trap mass spectrometer. MS/MS of the singly (M+) and multiply ([M++nH](n+1)+) charged precursor ions results in exclusive dissociation at the fixed charge containing side chain, independently of the amino acid composition and precursor ion charge state (i.e., proton mobility). However, loss of the methylphenacyl sulfide side-chain fragment as a neutral versus charged (protonated) species was observed to be highly dependent on the proton mobility of the precursor ion, and the identity of the phenacyl group para-substituent. Molecular orbital calculations were performed at the B3LYP/6-31+G** level of theory to calculate the theoretical proton affinities of the neutral side-chain fragments. The log of the ratio of neutral versus protonated side-chain fragment losses from the derivatized side chain were found to exhibit a linear dependence on the proton affinity of the side-chain fragmentation product, as well as the proton affinities of the peptide product ions. Finally, MS3 dissociation of the nominally identical neutral and protonated loss product ions formed by MS/MS of the [M++H]2+ and [M++2H]3+ precursor ions, respectively, from the peptide GAILM(X)GAILK revealed significant differences in the abundances of the resultant product ions. These results suggest that the protonated peptide product ions formed by gas-phase fragmentation of sulfonium ion containing precursors in an ion trap mass spectrometer do not necessarily undergo intramolecular proton 'scrambling' prior to their further dissociation, in contrast to that previously demonstrated for peptide ions introduced by external ionization sources.     

Electrospray mass spectrometry of hydrophobic compounds using dimethyl sulfoxide and dimethylformamide as solvents.

The use of dimethyl sulfoxide (DMSO) and dimethylformamide (DMF) as solvents in electrospray ionization (ESI) is suggested for the analysis of hydrophobic compounds. Its use was shown to overcome solubility problems and resulted in good quality electrospray spectra of protected hydrophobic peptides, sugars and other hydrophobic compounds. Intense protonated and/or sodiated molecules were formed in positive ion mode while negative ion mode resulted in [M + 95](-) ions from DMSO and [M + Cl](-) ions from DMF in cases where no significant molecular ion related peaks could be observed applying commonly used protic solvents such as methanol or acetonitrile. Deuterium labeling (d6-DMSO), high resolution experiments and tandem mass spectrometric measurements showed that the methanesulfonic acid (MSA), present in DMSO as a common impurity, is responsible for the formation of protonated molecules in positive ion mode and for methane sulfonate anion adducts [M + 95](-) in negative ion mode.     

Tandem mass spectrometric analysis of distinct fragmentation patterns to [M+Na]/z and [M+H]/z of dendritic Al(III) and Zn(II) quinolates

The synthesis of two novel dendritic aluminum and zinc quinolates, which are soluble in common solvents, was monitored effectively by positive ion electrospray ionization mass spectrometry (ESI-MS). Through tandem mass spectrometric analysis of the complexes, distinct fragmentation pathways for sodium adduct molecular ion [M+Na]+ and protonated molecular ion [M+H]+ were observed.     

A binary matrix of 2,5-dihydroxybenzoic acid and glycerol produces homogenous sample preparations for matrix-assisted laser desorption/ionization mass spectrometry

We introduce a two-component matrix for ultraviolet matrix-assisted laser desorption/ionization mass spectrometry (UV-MALDI-MS) that consists of 2,5-dihydroxybenzoic acid (DHB) and glycerol. Upon slow evaporation of residual water/methanol solvents in a pre-vacuum chamber sample preparations are obtained that exhibit a homogeneous morphology with analyte-matrix crystals evenly distributed over the whole sample spot. At a molar DHB/glycerol ratio of approximately 1:5, the crystals range in length from approximately 100 to 300 microm and are about 15-30 microm wide. Mass spectra of peptides, proteins, and an oligosaccharide are presented and compared with those recorded from standard dried-droplet DHB matrix. The ion signals show a reproducibility of the order of 10-15% when scanning the surface of an individual sample or even different samples that contain the same amount of peptide, A close to linear relationship between peptide concentration and the corresponding peptide ion signal is found over three orders of magnitude of sample prepared. However, when a fixed position is irradiated with a large number of laser pulses, a monotonous decay of peptide ion signal with time is observed. Potentially, the binary matrix will be especially useful for the analysis of samples that are stabilized in buffered aqueous glycerol solution and preliminary results addressing this aspect are shown.     

Formation and reactions of clusters in the gas phase: small peptides and carboxylic acids

The cluster formation of seventeen small dipeptides with different primary structures and vanillic acid was investigated by means of a neutral laser desorption and supersonic beam expansion followed by multi photon ionization time of flight mass spectrometry. The structures of these clusters have been characterized by mass spectrometric methods as well as by DFT calculations. It is shown that the structure of the cluster from a dipeptide and vanillic acid is described by a hydrogen bond between the phenolic group of the vanillic acid and the N-terminal amino function of the dipeptide. The intensity of the cluster ion and the main fragmentation product, the protonated peptide ion, can be linked to the proton affinity of the peptide. Furthermore the fragmentation reactions of the protonated peptide are accompanied by extensive hydrogen rearrangements yielding both a and y fragments. The intensities of these fragments follow the proton affinity of the dipeptide.     

Chemistry of aqueous silica nanoparticle surfaces and the mechanism of selective peptide adsorption

Control over selective recognition of biomolecules on inorganic nanoparticles is a major challenge for the synthesis of new catalysts, functional carriers for therapeutics, and assembly of renewable biobased materials. We found low sequence similarity among sequences of peptides strongly attracted to amorphous silica nanoparticles of various size (15-450 nm) using combinatorial phage display methods. Characterization of the surface by acid base titrations and zeta potential measurements revealed that the acidity of the silica particles increased with larger particle size, corresponding to between 5% and 20% ionization of silanol groups at pH 7. The wide range of surface ionization results in the attraction of increasingly basic peptides to increasingly acidic nanoparticles, along with major changes in the aqueous interfacial layer as seen in molecular dynamics simulation. We identified the mechanism of peptide adsorption using binding assays, zeta potential measurements, IR spectra, and molecular simulations of the purified peptides (without phage) in contact with uniformly sized silica particles. Positively charged peptides are strongly attracted to anionic silica surfaces by ion pairing of protonated N-termini, Lys side chains, and Arg side chains with negatively charged siloxide groups. Further, attraction of the peptides to the surface involves hydrogen bonds between polar groups in the peptide with silanol and siloxide groups on the silica surface, as well as ion-dipole, dipole-dipole, and van-der-Waals interactions. Electrostatic attraction between peptides and particle surfaces is supported by neutralization of zeta potentials, an inverse correlation between the required peptide concentration for measurable adsorption and the peptide pI, and proximity of cationic groups to the surface in the computation. The importance of hydrogen bonds and polar interactions is supported by adsorption of noncationic peptides containing Ser, His, and Asp residues, including the formation of multilayers. We also demonstrate tuning of interfacial interactions using mutant peptides with an excellent correlation between adsorption measurements, zeta potentials, computed adsorption energies, and the proposed binding mechanism. Follow-on questions about the relation between peptide adsorption on silica nanoparticles and mineralization of silica from peptide-stabilized precursors are raised.     

Gas phase hydrogen/deuterium exchange of arginine and arginine dipeptides complexed with alkali metals

The hydrogen/deuterium (H/D) exchange of protonated and alkali-metal cationized Arg-Gly and Gly-Arg peptides with D(2)O in the gas phase was studied using electrospray ionization quadropole ion trap mass spectrometry. The Arg-Gly and Gly-Arg alkali metal complexes exchange significantly more hydrogens than protonated Arg-Gly and Gly-Arg. We propose a mechanism where the peptide shifts between a zwitterionic salt bridge and nonzwitterionic charge solvated conformations. The increased rate of H/D exchange of the alkali metal complexes is attributed to the peptide metal complexes' small energy difference between the salt-bridge conformation and the nonzwitterionic charge-solvated conformation. Implications for the applicability of this mechanism to other zwitterionic systems are discussed.     

Effects of the position of internal histidine residues on the collision-induced fragmentation of triply protonated tryptic peptides

The collision-induced dissociation spectra of a series of synthetic, tryptic peptides that differed by the position of an internal histidine residue were studied. Electrospray ionization of these peptides produced both doubly and triply protonated molecular ions. Collision-induced fragmentation of the triply protonated peptide ions had better efficiency than that of the doubly protonated ions, producing a higher abundance of product ions at lower collision energies. The product ion spectra of these triply protonated ions were dominated by a series of doubly charged y-ions and the amount of sequence information was dependent on the position of the histidine residue. In the peptides where the histidine was located towards the C-terminus of the peptide, a more extensive series of sequence specific product ions was observed. As the position of the histidine residue was moved towards the N-terminus of the peptide, systematically less sequence information was observed. The peptides were subsequently modified with diethylpyrocarbonate to manipulate the product ion spectra. Addition of the ethoxyformyl group to the N-terminus and histidine residue shifted the predominant charge state of the modified peptide to the doubly protonated form. These peptide ions fragmented efficiently, producing product ion spectra that contained more sequence information than could be obtained from the corresponding unmodified peptide.     

Is there a ‘matrix suppression effect’ under fast‐atom bombardment liquid secondary ion mass spectrometry of ionic surfactants in glycerol?

Some features of a ‘matrix suppression effect’ caused by ionic surface‐active compounds under fast‐atom bombardment (FAB) liquid secondary ion mass spectrometry (LSIMS) are being revised. It is shown that abundant transfer of the glycerol matrix molecules to the gas phase does occur under FAB‐LSIMS of ionic surfactants, contrary to popular belief. This process can be obscure because of the dependence of the charge state of the glycerol‐containing cluster ions on the type of ionic surfactant. It is revealed that, while glycerol matrix signals are really completely suppressed in the positive ion mass spectra of cationic surfactants (decamethoxinum, aethonium), abundant deprotonated glycerol and glycerol‐anion clusters are recorded in the negative ion mode. In the case of an anionic surfactant (sodium dodecyl sulfate), on the contrary, glycerol is completely suppressed in the negative ion mode, but is present in the protonated and cationized forms in the positive ion mass spectra. It is suggested that such patterns of positive and negative ion FAB‐LSIMS spectra of ionic surfactants solutions reflect the structure and composition of the electric double layer formed at the vacuum‐liquid interface by organic cations or anions and their counterions. Processes leading to the formation of the glycerol‐containing ions preferentially of positive or negative charge are discussed. The most obvious of them is efficient binding of glycerol to inorganic counterions of the salts Cl^? or Na⁺, which is confirmed by data from quantum chemical calculations. The high content of the counterions and relatively small content of glycerol in the sputtered zone may be responsible for the charge‐selective suppression of neat glycerol clusters of opposite charge to the counterions. In the case of a mixture of cationic and anionic surfactants the substitution of inorganic counterions by organic ones was observed. The dependence of the exchange rate in the surface layer is not a linear function of the bulk solution concentration, and an effect of abrupt recharging of the surface can be registered. No both positively or negatively charged pure glycerol and glycerol‐inorganic counterion clusters are recorded for the mixture. Correlations between the mass spectrometric observations and some phenomena of surface and colloid chemistry and physics are discussed. Copyright © 2007 John Wiley & Sons, Ltd.     

Negative ion production from peptides and proteins by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Negative ion production from peptides and proteins was investigated by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry. Although most research on peptide and protein identification with ionization by MALDI has involved the detection of positive ions, for some acidic peptides protonated molecules are not easily formed because the side chains of acidic residues are more likely to lose a proton and form a deprotonated species. After investigating more than 30 peptides and proteins in both positive and negative ion modes, [M–H]⁻ ions were detected in the negative ion mode for all peptides and proteins although the matrix used was 2,5‐dihydroxybenzoic acid (DHB), which is a good proton donor and favors the positive ion mode production of [M+H]⁺ ions. Even for highly basic peptides without an acidic site, such as myosin kinase inhibiting peptide and substance P, good negative ion signals were observed. Conversely, gastrin I (1‐14), a peptide without a highly basic site, will form positive ions. In addition, spectra obtained in the negative ion mode are usually cleaner due to absence of alkali metal adducts. This can be useful during precursor ion isolation for MS/MS studies. Copyright © 2008 John Wiley & Sons, Ltd.     

Evaluation of a novel approach for peptide sequencing: laser-induced acoustic desorption combined with P(OCH(3))(2)(+) chemical ionization and collision-activated dissociation in a Fourier transform ion cyclotron resonance mass spectrometer

A novel mass spectrometric method has been developed for obtaining sequence information on small peptides. The peptides are desorbed as intact neutral molecules into a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR) by means of laser-induced acoustic desorption (LIAD). Reactions of the neutral peptides with the dimethoxyphosphenium ion, P(OCH(3))(2)(+), occur predominantly by addition of the peptide to P(OCH(3))(2)(+) followed by the loss of two methanol molecules, thus yielding product ions with the composition (peptide + P - 2H)(+). Upon sustained off-resonance irradiation for collision-activated dissociation (SORI-CAD), the (peptide + P - 2H)(+) ions undergo successive losses of CO and NHCHR or H(2)O, CO, and NHCHR to yield sequence-related fragment ions in addition to the regular a(n)- and b(n)-type ions. Under the same conditions, SORI-CAD of the analogous protonated peptides predominantly yields the regular a(n)- and b(n)-type ions. The mechanisms of the reactions of peptides with P(OCH(3))(2)(+) and the dissociation of the (peptide + P - 2H)(+) ions were examined by using model peptides and molecular orbital calculations.     

Chemical ionization mass spectrometric characteristics of morphine alkaloids

The ion-molecular reaction behavior of ten morphine alkaloids with several commonly used reagent gases are studied under chemical ionization mass spectrometry conditions. These studies emphasize the correlation of the structural characteristics of the 10 alkaloids with the following four mass spectrometric parameters: (i) mass shifts of the protonated ion as a result of replacing ammonia with deuterated ammonia as the reagent gas, (ii) relative tendencies of the adduct ion and the protonated ion to lose molecules of water, (iii) relative intensity ratio of the adduct ion and the protonated ion and (iv) tendency of a compound to undergo a reduction reaction.     

Cooperative effect of factors governing molecular ion yields in desorption/ionization mass spectrometry

Factors governing the molecular ion yields of amino acids and peptides have been studied using fast atom bombardment (FAB) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) in positive-ion mode. The ion yields of protonated amino acids under FAB conditions are dependent on proton affinity (PA), hydrophobicity, and aromaticity of amino acids. Both PA and hydrophobicity contribute to an increase in the ion yields, while aromaticity contributes to a decrease. In MALDI, the ion yields increase linearly with the increase of PA of amino acids with the exception of lysine. In both FAB and MALDI experiments with peptides, the presence of arginine residues is essential for producing abundant protonated peptides. In FAB, the presence of aliphatic and hydrophobic amino acids (leucine and isoleucine) increases the ion yields of protonated peptides, while some hydrophilic amino acids (aspartic acid and asparagines) decrease the ion yields. The presence of two or more arginine residues does not give higher ion yields in FAB. In MALDI, the presence of aromatic amino acids (phenylalanine and tyrosine) enhances the signals for protonated peptides. Thus, physicochemical factors of individual amino acids cooperatively affect the ion yields of protonated amino acids and peptides. These factors governing the ion yields in FAB and MALDI affect two processes, desorption and ionization, that can be considered independently.     

Ammonia chemical ionization mass spectra of esters and amides of oxyacids of phosphorus

Chemical ionization mass spectrometry using ammonia as the reagent gas has been carried out with esters and amides of a variety of oxyacids of phosphorus (phosphates, phosphonates, phosphites and phosphoramidates). In all cases, the protonated molecular ion is a major species in the spectrum and the percentage of the total ion current carried by these protonated molecular ions is always considerably greater than that carried by the molecular ions in the corresponding electron impact mass spectra. In the chemical ionization mass spectra only limited fragmentation of the protonated molecular ion occurs from which useful information on the structure of phosphorus derivatives may be inferred.     

Degree of Ionization in MALDI of Peptides: Thermal Explanation for the Gas-Phase Ion Formation

Degree of ionization (DI) in matrix-assisted laser desorption ionization (MALDI) was measured for five peptides using α-cyano-4-hydroxycinnanmic acid (CHCA) as the matrix. DIs were low 10(-4) for peptides and 10(-7) for CHCA. Total number of ions (i.e., peptide plus matrix) was the same regardless of peptides and their concentration, setting the number of gas-phase ions generated from a pure matrix as the upper limit to that of peptide ions. Positively charged cluster ions were too weak to support the ion formation via such ions. The total number of gas-phase ions generated by MALDI, and that from pure CHCA, was unaffected by the laser pulse energy, invalidating laser-induced ionization of matrix molecules as the mechanism for the primary ion formation. Instead, the excitation of matrix by laser is simply a way of supplying thermal energy to the sample. Accepting strong Coulomb attraction felt by cations in a solid sample, we propose three hypotheses for gas-phase peptide ion formation. In Hypothesis 1, they originate from the dielectrically screened peptide ions in the sample. In Hypothesis 2, the preformed peptide ions are released as part of neutral ion pairs, which generate gas-phase peptide ions via reaction with matrix-derived cations. In Hypothesis 3, neutral peptides released by ablation get protonated via reaction with matrix-derived cations.     

Tandem mass spectrometry of kahalalides: identification of two new cyclic depsipeptides, kahalalide R and S from Elysia grandifolia

Spectra obtained using electrospray ionization mass spectrometry (ESI-MS) of the mollusk Elysia grandifolia showed a cluster of molecular ion peaks centered at a molecular mass of 1478 Da (kahalalide F, an anticancer agent). Two new molecules, kahalalide R (m/z 1464) and S (m/z 1492) were characterized using tandem mass spectrometry. The mass differences of 14 Da suggest that they are homologous molecules. In addition, previously identified kahalalide D and kahalalide G are also reported. However, the ESI-MS of the mollusk's algal diet Bryopsis plumosa showed the presence of only kahalalide F. The amino acid sequences of kahalalide R and S are proposed using collision-induced dissociation (CID) experiments of singly and doubly charged molecular ions and by comparison with the amino acid sequence of kahalalide F. The pathway is presented for the loss of amino acid residues in kahalalide F. It is observed that there is sequential loss of amino acids in the linear peptide chain, but in the cyclic part the ring opens at the amide bond rather than at the lactone linkage, and the loss of amino acid residues is not sequential. The CID experiment of the alkali-metal-cationized molecular ions shows that the sodium and potassium ions coordinate to the amide nitrogen/oxygen in the linear peptide chain of the molecule and not to the lactone oxygen of the lactone. In the case of kahalalide D, CID of the protonated peptide opens the depsipeptide ring to form a linear peptide with acylium ion, and fragment ion signals indicate losses of amino acids in sequential order. In this study, tandem mass spectrometry has provided the detailed information required to fully characterize the new peptides.     

Practical considerations in analysing neuropeptides, calcitonin gene-related peptide and vasoactive intestinal peptide, by nano-electrospray ionisation and quadrupole time-of-flight mass spectrometry: monitoring multiple protonations

We investigated the pre-electrospray ionisation (pre-ESI) factors; analyte concentration (1-2500 ng/mL), concentration of formic acid (FA) in the mobile phase (0.01, 0.1 and 1%), concentration of the organic modifier (acetonitrile 50-90%) and flow rate (<10 μL/min) on the number of multiple protonations and ESI response for two neuropeptides (of ~3.3 kDa molecular mass); calcitonin gene-related peptide (CGRP) and vasoactive intestinal peptide (VIP). A pH of 3.23 (0.1% FA), nano-flow rate range of 350-750 nL/min and acetonitrile concentration of 50% were optimum for both neuropeptides where the highest intensities were observed. An inverse relationship between decreasing flow rate and ESI response for both peptides was also observed. The quadruply charged ([M+4H](4+)) ion was dominant for CGRP at all analyte concentrations, and also for VIP, but only at the higher analyte concentrations (250-2500 ng/mL); none of the [M+4H](4+), [M+5H](5+) or [M+6H](6+) ions were dominant at the lower concentrations. Linear correlations were obtained for the protonated states and ESI response at analyte concentrations (1-750 ng/mL). Acetonitrile concentration was critical; severe ion suppression was observed for VIP when the concentration of acetonitrile was ≥60%. Ion suppression was also observed for both peptides in an equimolar mixture, with the extent of ion suppression more severe for VIP. Our study concludes that it is important to monitor several protonated species when a single protonated state does not dominate, especially during label-free peptide quantitations.     

Sequence-specific fragmentation of deprotonated peptides containing H or alkyl side chains

The [M - H]- ions of a variety of di- to pentapeptides containing H or alkyl side chains have been prepared by electrospray ionization and low-energy collision-induced dissociation (CID) of the deprotonated species carried out in the interface region between the atmospheric pressure source and the quadrupole mass analyzer. Using the nomenclature applied to the fragmentation of protonated peptides, deprotonated dipeptides fragment to give a2 ions (CO2 loss) and y1 ions, where the y1 ion has two fewer hydrogens than the y"1 ions formed from protonated peptides. Deprotonated tri- and tetrapeptides fragment to give primarily y1, c1, and "b2 ions, where the "b2 ion has two fewer hydrogens than the b2 ion observed for protonated peptides. More minor yields of y2, c2, and a2 ions also are observed. The a ion formed by loss of CO2 from the [M - H]- ion shows loss of the N-terminal residue for tripeptides and sequential loss of two amino acid residues from the N-terminus for tetrapeptides. The formation of c(n) ions and the sequential loss of N-terminus residues from the [M - H - CO2]- ion serves to sequence the peptide from the N-terminus, whereas the formation of y(n) ions serves to sequence the peptide from the C-terminus. It is concluded that low-energy CID of deprotonated peptides provides as much (or more) sequence information as does CID of protonated peptides, at least for those peptides containing H or alkyl side chains. Mechanistic aspects of the fragmentation reactions observed are discussed.