'Top down' protein characterization via tandem mass spectrometry

Technological and scientific advances over the past decade have enabled protein identification and characterization strategies to be developed that are based on subjecting intact protein ions and large protein fragments directly to tandem mass spectrometry. These approaches are referred to collectively as 'top down' to contrast them with 'bottom up' approaches whereby protein identification is based on mass spectrometric analysis of peptides derived from proteolytic digestion, usually with trypsin. A key step in enabling top down approaches has been the ability to assign tandem mass spectrometer product ion identities, which can be done either via high resolving power or through product ion charge state manipulation. The ability to determine product ion charge states has permitted studies of the reactions, including dissociation, ion-molecule reactions, ion-electron reactions and ion-ion reactions of high-mass, multiply charged protein ions. Electrospray ionization combined with high magnetic field strength Fourier transform ion cyclotron resonance has proven to be particularly powerful for detailed protein characterization owing to its high mass resolution and mass accuracy and its ability to effect electron capture-induced dissociation. Other types of tandem mass spectrometers are also beginning to find increasing use in top down protein identification/characterization studies. Charge state manipulation via ion-ion reactions in electrodynamic ion traps, for example, enables top down strategies to be considered using instruments with relatively modest mass resolution capabilities. Precursor ion charge state manipulation techniques have also recently been demonstrated to be capable of concentrating and charge-state purifying proteins in the gas phase. Advances in technologies applied to the structural analysis of whole protein ions and in understanding their reactions, such as those described here, are providing new options for the study of complex protein mixtures.     

Peptide and protein characterization by high-rate electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry

The analytical utility of the electron capture dissociation (ECD) technique, developed by McLafferty and co-workers, has substantially improved peptide and protein characterization using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). The limitations of the first ECD implementations on commercial instruments were eliminated by the employment of low-energy electron-injection systems based on indirectly heated dispenser cathodes. In particular, the ECD rate and reliability were greatly increased, enabling the combination of ECD/FTICR-MS with on-line liquid separation techniques. Further technique development allowed the combination of two rapid fragmentation techniques, high-rate ECD and infrared multiphoton dissociation (IRMPD), in a single experimental configuration. Simultaneous and consecutive irradiations of trapped ions with electrons and photons extended the possibilities for ion activation/dissociation and led to improved peptide and protein characterization. The application of high-rate ECD/FTICR-MS has demonstrated its power and unique capabilities in top-down sequencing of peptides and proteins, including characterization of post-translational modifications, improved sequencing of peptides with multiple disulfide bridges and secondary fragmentation (w-ion formation). Analysis of peptide mixtures has been accomplished using high-rate ECD in bottom-up mass spectrometry based on mixture separation by liquid chromatography and capillary electrophoresis. This paper summarizes the current impact of high-rate ECD/FTICR-MS for top-down and bottom-up mass spectrometry of peptides and proteins.     

De novo sequencing of antimicrobial peptides isolated from the venom glands of the wolf spider Lycosa singoriensis

Antimicrobial peptides (AMPs), named lycocitin 1, 2 and 3, and a peptide with a monoisotopic molecular mass of 3038.70 Da were detected in the venom glands of the wolf spider Lycosa singoriensis. Two of the peptides, lycocitin 1 and 2, are new AMPs whereas lycocitin 3 is highly homologous to lycotoxin II isolated from the venom of spider Lycosa carolinensis. In addition, two other peptides with monoisotopic masses of 2034.20 and 2340.28 Da showing the motif typical for antimicrobial peptides were also identified. These peptides and lycocitin 1, 2 and 3 were de novo sequenced using electron capture dissociation and low-energy collisional tandem mass spectrometry. The amino acid sequence of lycocitin 1 was determined as GKLQAFLAKMKEIAAQTL-NH(2). Lycocitin 2 differs from lycocitin 1 by a replacement of a lysine residue for an arginine residue at the second position. Lycocitin 3 differs from the known lycotoxin II consisting of 27 amino acid residues by a deletion of Gly-26. Both lycocitin 1 and 2 inhibit growth of Gram-positive (Staphylococcus aureus, Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria and fungi (Candida albicans, Pseudomonas aeruginosa) at micromolar concentrations.     

De novo sequencing of a 21-kDa cytochrome c4 from Thiocapsa roseopersicina by nanoelectrospray ionization ion-trap and Fourier-transform ion-cyclotron resonance mass spectrometry

We have determined the primary structure of cytochrome c(4) from Thiocapsa roseopersicina by de novo protein sequencing using the 'bottom up' approach. Three different enzymes (trypsin, endoproteinase Lys-C, and endoproteinase Glu-C) were employed to prepare four different sets of proteolytic digests. The digestion strategy was designed to permit a gradual buildup of smaller peptides into larger ones that were overlapped to yield the complete protein sequence. In this way we countered the main problem: peptides larger than about 1500 Da were difficult to sequence fully by tandem mass spectrometry. Direct infusion and online liquid chromatography were used on a linear ion trap Fourier-transform ion-cyclotron resonance hybrid instrument. The high resolving power, high mass accuracy and the availability of electron capture dissociation and collision-induced dissociation were essential to achieve full sequence coverage. The software DeNovoX complemented by manual interpretation was used to generate sequence information from tandem mass spectra. The predominantly automated nature of data acquisition and handling allowed for a relatively straightforward and fast procedure, which could compete with the mainstream alternative of nucleotide sequence determination.     

Ion activation methods for tandem mass spectrometry

This tutorial presents the most common ion activation techniques employed in tandem mass spectrometry. In-source fragmentation and metastable ion decompositions, as well as the general theory of unimolecular dissociations of ions, are initially discussed. This is followed by tandem mass spectrometry, which implies that the activation of ions is distinct from the ionization step, and that the precursor and product ions are both characterized independently by their mass/charge ratios. In collision-induced dissociation (CID), activation of the selected ions occurs by collision(s) with neutral gas molecules in a collision cell. This experiment can be done at high (keV) collision energies, using tandem sector and time-of-flight instruments, or at low (eV range) energies, in tandem quadrupole and ion trapping instruments. It can be performed using either single or multiple collisions with a selected gas and each of these factors influences the distribution of internal energy that the activated ion will possess. While CID remains the most common ion activation technique employed in analytical laboratories today, several new methods have become increasingly useful for specific applications. More recent techniques are examined and their differences, advantages and disadvantages are described in comparison with CID. Collisional activation upon impact of precursor ions on solid surfaces, surface-induced dissociation (SID), is gaining importance as an alternative to gas targets and has been implemented in several different types of mass spectrometers. Furthermore, unique fragmentation mechanisms of multiply-charged species can be studied by electron-capture dissociation (ECD). The ECD technique has been recognized as an efficient means to study non-covalent interactions and to gain sequence information in proteomics applications. Trapping instruments, such as quadrupole ion traps and Fourier transform ion cyclotron resonance instruments, are particularly useful for the photoactivation of ions, specifically for fragmentation of precursor ions by infrared multiphoton dissociation (IRMPD). IRMPD is a non-selective activation method and usually yields rich fragmentation spectra. Lastly, blackbody infrared radiative dissociation is presented with a focus on determining activation energies and other important parameters for the characterization of fragmentation pathways. The individual methods are presented so as to facilitate the understanding of each mechanism of activation and their particular advantages and representative applications.     

Characterization of nucleic acid higher order structure by high-resolution tandem mass spectrometry

Mass spectrometry (MS) is extensively used for the identification and sequencing of nucleic acids but has so far seen limited use for characterization of their higher order structures. Here, we have applied a range of different tandem mass spectrometry techniques, including electron detachment dissociation (EDD), infrared multiphoton dissociation (IRMPD), activated ion (AI) EDD, and EDD/IRMPD MS³, in a Fourier transform ion cyclotron resonance mass spectrometer to the characterization of three isomeric 15mer DNAs with different sequences and predicted solution-phase structures. Our goal was to explore whether their structural differences could be directly probed with these techniques. We found that all three 15mers had higher order structures in the gas phase, although preferred structures were predicted for only two of them in solution. Nevertheless, EDD, AI EDD, and EDD/IRMPD MS³ experiments yielded different cleavage patterns with less backbone fragmentation for the more stable solution-phase structure than for the other two 15mers. By contrast, no major differences were observed in IRMPD, although the extent of backbone cleavage was higher with that technique for all three 15mers. Thus, experiments utilizing the radical ion chemistry of EDD can provide complementary structural information compared to traditional slow heating methods, such as IRMPD, for structured nucleic acids.     

Manipulating internal energy of protonated biomolecules in electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

The internal energy of protonated leucine enkephalin has been manipulated in electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry with two newly designed pump-probe experiments. Blackbody infrared radiation was applied to pump an ion population into a well-defined internal energy distribution below the dissociation threshold. Following this pumping stage, the internal energy distribution was probed using on-resonance collisional activation to dissociate the ions. These pump-probe experiments were carried out in two different ways: (a) using on-resonance collisional activation with variable kinetic energies to dissociate the ions at a constant initial ion temperature (determining the precursor ion survival percentage as a function of kinetic energy) and (b) using on-resonance collisional activation with a constant kinetic energy to dissociate the ions at variable initial ion temperatures (to investigate the ion survival yield-initial ion temperature dependence). Using this approach, a detailed study of the effects of the initial ion temperature, the probing kinetic energy and the internal energy loss rate on the effective conversion efficiency of (laboratory-frame) kinetic energy to internal energy was conducted. This conversion efficiency was found to be dependent on the initial ion temperature. Depending on the experimental conditions the conversion efficiency (for collisions with argon) was estimated to be about 4.0 +/- 1.7%, which agrees with that obtained from a theoretical modeling. Finally, the reconstructed curves of the ion survival yield versus the mode of the (final) total internal energy distribution of the activated ion population (after pump and probe events) at different pump-probe conditions reveal the internal energy content of the activated ions.     

Analysis of MALDI FT-ICR mass spectrometry data: A time series approach

Matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry is a technique for high mass-resolution analysis of substances that is rapidly gaining popularity as an analytic tool. Extracting signal from the background noise, however, poses significant challenges. In this article, we model the noise part of a spectrum as an autoregressive, moving average (ARMA) time series with innovations given by a generalized gamma distribution with varying scale parameter but constant shape parameter and exponent. This enables us to classify peaks found in actual spectra as either noise or signal using a reasonable criterion that outperforms a standard threshold criterion.     

Comparison of sustained off-resonance irradiation collisionally activated dissociation and multipole storage-assisted dissociation for top-down protein analysis

Tandem mass spectrometric data acquired for small (8-18 kDa) intact proteins by sustained off-resonance irradiation collisionally activated dissociation (SORI-CAD) and multipole storage-assisted dissociation (MSAD) were compared, and the results indicate that the two activation methods do not always provide the same fragmentation patterns. In MSAD experiments, the charge state distribution made available by the ionization conditions may dictate the range of fragment ions that can be generated. In addition, conditions of high space charge within the hexapole impair transmission and/or trapping of high m/z species, which can result in loss of important precursor and product ions. Finally, the non-resonant nature of activation in MSAD can provide access to secondary dissociation processes that are not available by SORI. Because of these considerations, MSAD is less reliable than SORI for generating sequence tag data. However, it appears that MSAD samples 'preferred' cleavage processes (i.e. those occurring at D and P residues) just as well as SORI, which implies that MSAD data may be somewhat more compatible with search algorithms that utilize unprocessed fragment ion masses.     

Fourier transform ion cyclotron resonance mass spectrometry of covalent adducts of proteins and 4-hydroxy-2-nonenal, a reactive end-product of lipid peroxidation

Covalent adduction of the model protein apomyoglobin by 4-hydroxy-2-nonenal, a reactive end-product of lipid peroxidation, was characterized by nanoelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (FTICR). The high mass resolving power and mass measurement accuracy of the instrument facilitated a detailed compositional analysis of the complex reaction product without the need for deconvolution and transformation to clearly show the pattern of adduction and component molecular weights. Our study has also demonstrated the value of electron capture dissociation over collision-induced dissociation for the tandem mass spectrometric determination of site modification for the 4-hydroxy-2-nonenal adduct of oxidized insulin B chain as an example. Figure FTICR allowed characterization of 4-hydroxy-2-nonenal (HNE)-modified apomyoglobin (an expanded spectrum of the +15 charge state is shown)     

Analysis of lipids with desorption atmospheric pressure photoionization‐mass spectrometry (DAPPI‐MS) and desorption electrospray ionization‐mass spectrometry (DESI‐MS)

In this article, the effect of spray solvent on the analysis of selected lipids including fatty acids, fat‐soluble vitamins, triacylglycerols, steroids, phospholipids, and sphingolipids has been studied by two different ambient mass spectrometry (MS) methods, desorption electrospray ionization‐MS (DESI‐MS) and desorption atmospheric pressure photoionization‐MS (DAPPI‐MS). The ionization of the lipids with DESI and DAPPI was strongly dependent on the spray solvent. In most cases, the lipids were detected as protonated or deprotonated molecules; however, other ions were also formed, such as adduct ions (in DESI), [M‐H]⁺ ions (in DESI and DAPPI), radical ions (in DAPPI), and abundant oxidation products (in DESI and DAPPI). DAPPI provided efficient desorption and ionization for neutral and less polar as well as for ionic lipids but caused extensive fragmentation for larger and more labile compounds because of a thermal desorption process. DESI was more suitable for the analysis of the large and labile lipids, but the ionization efficiency for less polar lipids was poor. Both methods were successfully applied to the direct analysis of lipids from pharmaceutical and food products. Although DESI and DAPPI provide efficient analysis of lipids, the multiple and largely unpredictable ionization reactions may set challenges for routine lipid analysis with these methods. Copyright © 2012 John Wiley & Sons, Ltd.     

Ionization energies of multiply protonated polypeptides obtained by tandem ionization in Fourier transform mass spectrometers

Ionization energies (IE) of [M + zH](z+) (z+) electrospray-produced polypeptides were determined by electron ionization in a Penning cell of 4.7 and 9.4 T Fourier transform mass spectrometers. For z = 1+ and substance P, the found IE value of 11.0 +/- 0.4 eV is in agreement with that obtained earlier for ions generated with matrix-assisted laser desorption/ionization. For higher z, the following values were found: 11.7 +/- 0.3 eV for 2+ of [Arg-8]-vasopressin, 11.1 +/- 0.6 eV for 2+ of substance P, 12.2 +/- 0.7 eV for 2+ of renin substrate, 13.3 +/- 0.4 eV for 3+ of B-chain of insulin and 14.6 +/- 0.6 eV for 4+ and 15.1 +/- 0.4 eV for 5+ of melittin. It was found that 90% of existing IE data on polypeptides in the 1.0-3.5 kDa mass range are described with 相似文献   

Characterization of barley leaf tissue using direct and indirect desorption electrospray ionization imaging mass spectrometry

Chemical profiling of barley (Hordeum vulgare) leaves was demonstrated using direct and indirect desorption electrospray ionization (DESI) imaging mass spectrometry. Direct DESI analysis of the untreated leaves was not possible despite a significant content of hydroxynitrile glucosides known to reside in the epidermis of the leaves. Instead, the epidermis was stripped off the leaves, thus allowing direct DESI imaging to be performed on the back of the epidermis. Furthermore, indirect DESI imaging was performed by making imprints in porous Teflon of the intact leaves as well as of the stripped epidermis. The DESI images reveal accumulation of hydroxynitrile glucosides in the leaf epidermis, homogeneously distributed throughout the surface. The indirect DESI approach enables relative quantitation, confirming variations of hydroxynitrile glucosides content in primary leaves of three different cultivars of barley seedlings. The study presents an example of how to overcome the morphological barriers from the plant surface and perform rapid and repeatable DESI imaging. In addition, a comparison is made of direct and indirect DESI imaging, contributing to the characterization of the recently developed method of indirect DESI imaging of plant material via porous Teflon imprints.     

Determination of bond dissociation energies using mass spectrometry

Fourier transform ion cyclotron resonance mass spectrometry (FT‐ICR/MS) offers the opportunity for gas phase cluster formation reactions at very low pressures and at temperatures that are different from room temperature. Reactions take place with single positive‐charge metal ions that are normally +2, +3, +4, etc., charged in solution. The ions formed are detected by measuring the current induced by their cyclotron rotation, but they cannot be physically separated and collected. Collision‐induced dissociation (CID) is widely used for ion‐structure determination via the fragmentation of the excited ions. CID study aims to determine the relationship between the V_pp [peak‐to‐peak voltage of the radiofrequency (rf) pulse] and the mass‐to‐charge (m/z) ratio, which will be used for the calculation of the center‐of‐mass translational kinetic energy (Ek_cm) of the excited ion under investigation. CID studies are restricted to stable ions with relatively high abundance. Nevertheless, with the evolution of computational chemistry, such problems can be overcome whereby CID calculations will be used to provide the substantial parameters for computer software, such as the Gaussian 03 program, for the structure determination of the less stable Ni_xS anions. The latter constitutes the core for our current research. © 2006 Wiley Periodicals, Inc. Int J Quantum Chem, 2007     

The application of electrospray ionization coupled to ultrahigh resolution mass spectrometry for the molecular characterization of natural organic matter

Mass spectrometry has recently played a key role in the understanding of natural organic matter (NOM) by providing molecular-level details about its composition. NOM, a complex assemblage of organic molecules present in natural waters and soils/sediments, has the ability to bind and transport anthropogenic materials. An improved understanding of its composition is crucial in order to understand how pollutants interact with NOM and how NOM cycles through global carbon cycles. In the past, low-resolution (>3000) mass analyzers have offered some insights into the structure of NOM, but emerging ultrahigh resolution (>200,000) techniques such as electrospray ionization (ESI) coupled to Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) have significantly advanced our knowledge of NOM chemistry. Here, a review of the recent literature on the advancements of NOM characterization and the applications of mass spectrometry to this central task is presented. Various methods for the analysis and display of the extremely complex mass spectra, such as the van Krevelen diagram and Kendrick mass defect analysis, are discussed. We also review tandem mass spectrometry techniques employed to gain structural information about NOM components. Finally, we show how ESI-FT-ICR-MS has been applied to examine specific issues that are important to the NOM scientific community, such as NOM reactivity, transport and fate, degradation, and existence of components, which are indicators of NOM origin. In general, ultrahigh resolution provided by FT-ICR-MS is essential for the complete separation of the thousands of peaks present in the complex NOM mixture and will clearly lead to additional future advancements in the areas of aquatic, soil, and analytical chemistry.     

Negative-ion mass spectra of some N-mesyl derivatives of o-(cycloalk-2-enyl)-, o-(cycloalk-1-enyl)-, o-(pent-2-enyl)-, o-dibromopentylanilines and (α-bromoalkyl)indolines

The formation and fragmentation of negative ions of some N-(methylsulfonyl)anilines upon resonance electron capture have been studied. The formation of long-living molecular ions is due to the presence of the mesyl groups in the molecules. The difference in negative-ion mass spectra of isomeric N-(methylsulfonyl)anilines has been revealed.     

Fragmentation behavior of Amadori‐peptides obtained by non‐enzymatic glycosylation of lysine residues with ADP‐ribose in tandem mass spectrometry

Mono‐ and poly‐adenosine diphosphate (ADP)‐ribosylation are common post‐translational modifications incorporated by sequence‐specific enzymes at, predominantly, arginine, asparagine, glutamic acid or aspartic acid residues, whereas non‐enzymatic ADP‐ribosylation (glycation) modifies lysine and cysteine residues. These glycated proteins and peptides (Amadori‐compounds) are commonly found in organisms, but have so far not been investigated to any great degree. In this study, we have analyzed their fragmentation characteristics using different mass spectrometry (MS) techniques. In matrix‐assisted laser desorption/ionization (MALDI)‐MS, the ADP‐ribosyl group was cleaved, almost completely, at the pyrophosphate bond by in‐source decay. In contrast, this cleavage was very weak in electrospray ionization (ESI)‐MS. The same fragmentation site also dominated the MALDI‐PSD (post‐source decay) and ESI‐CID (collision‐induced dissociation) mass spectra. The remaining phospho‐ribosyl group (formed by the loss of adenosine monophosphate) was stable, providing a direct and reliable identification of the modification site via the b‐ and y‐ion series. Cleavage of the ADP‐ribose pyrophosphate bond under CID conditions gives access to both neutral loss (347.10 u) and precursor‐ion scans (m/z 348.08), and thereby permits the identification of ADP‐ribosylated peptides in complex mixtures with high sensitivity and specificity. With electron transfer dissociation (ETD), the ADP‐ribosyl group was stable, providing ADP‐ribosylated c‐ and z‐ions, and thus allowing reliable sequence analyses. Copyright © 2010 John Wiley & Sons, Ltd.     

Collision-induced fragmentation pathways including odd-electron ion formation from desorption electrospray ionisation generated protonated and deprotonated drugs derived from tandem accurate mass spectrometry

The rapid desorption electrospray ionisation (DESI) of some small molecules and their fragmentation using a triple-quadrupole and a hybrid quadrupole time-of-flight mass spectrometer (Q-ToF) have been investigated. Various scanning modes have been employed using the triple-quadrupole instrument to elucidate fragmentation pathways for the product ions observed in the collision-induced dissociation (CID) spectra. Together with accurate mass tandem mass spectrometry (MS/MS) measurements performed on the hybrid Q-ToF mass spectrometer, unequivocal product ion identification and fragmentation pathways were determined for deprotonated metoclopramide and protonated aspirin, caffeine and nicotine. Ion structures and fragmentation pathway mechanisms have been proposed and compared with previously published data. The necessity for elevated resolution for the differentiation of isobaric ions are discussed.     

Comprehensive characterization of natural organic matter by MALDI- and ESI-Fourier transform ion cyclotron resonance mass spectrometry

Natural organic matter (NOM) is a complex and non-uniform mixture of organic compounds which plays an important role in environmental processes. Due to the complexity, it is challenging to obtain fully detailed structural information about NOM. Although Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) has been demonstrated to be a powerful tool for providing molecular information about NOM, multiple ionization methods are needed for comprehensive characterization of NOM at the molecular level considering the ionizing selectivity of different ionization methods. This paper reports the first use of matrix assisted laser desorption/ionization (MALDI) method coupled with FT-ICR-MS for molecular characterization of NOM within a mass range of 200–800 Da. The mass spectral data obtained by MALDI were systematically compared with data generated by electrospray ionization (ESI). It showed that complementary molecular information about NOM which could not be detected by ESI, were provided by MALDI. More unsaturated and aromatic constituents of NOM with lower O/C ratio (O/C ratio < 0.5) were preferentially ionized in MALDI negative mode, whereas more polar constituents of NOM with higher O/C ratio were preferentially ionized in ESI negative mode. Molecular anions of NOM appearing at even m/z in MALDI negative ion mode were detected. The results show that NOM molecules with aromatic structures, moderate O/C ratio (0.7 > O/C ratio > 0.25) and lower H/C ratio were liable to form molecular anions at even m/z, whereas those with higher H/C ratio are more likely to form deprotonated ions at odd m/z. It is speculated that almost half of the NOM molecules identified by MALDI may be aromatic or condensed aromatic compounds with special groups which are liable to absorb electron from other molecules to generate free radical anions during MALDI ionization.     

Characterization of a phosphorylated peptide and peptoid and peptoid-peptide hybrids by mass spectrometry

Nano-electrospray tandem mass spectrometry (nano-ES-MS/MS) was used to record collision-induced dissociation (CID) spectra of a set of peptoid-peptide hybrids and the complete peptoid derived from the phosphopeptide Ac-pTyr-Glu-Thr-Leu-NH(2) (1). The presence of B and Y'-type fragment ions in the tandem mass spectra of the protonated molecular ions [M + H](+) allowed confirmation of sequence similar to mass spectrometric sequence analysis in peptides. In the isomeric peptoid compounds studied, one or several amino acid residues were replaced by peptoid residues (N-substituted glycine residues), which resulted in characteristic tandem mass spectra with differently increased relative abundances of Y'-and B-type fragment ions. The increment of a particular Y'-ion was directly correlated to the position of a peptoid residue present. In addition to these increased peak intensities, other characteristic peaks were also observed compared with the spectrum of reference peptide 1. When a peptoid phosphotyrosine was incorporated, the presence of this residue was apparent from the occurrence of a relatively intense peak at m/z 187 representing the positively charged side-chain of phosphotyrosine, which was almost absent in the spectrum of the reference peptide 1. Since the threonine side-chain had to be translated into the homo peptoid analog this substitution was apparent from the presence of [M + H](+) and fragment ions 14 mass units higher than observed in the spectrum of the reference phosphopeptide 1. The presence of an NLeu peptoid residue could be confirmed by the specific fragmentation of the immonium ion showing an intense peak in its tandem mass spectrum at m/z 57, which results from the loss of an neutral imine molecule leading to a positively charged [C(4)H(9)](+) ion. By means of these mass spectrometric characteristics, all isomeric peptoid compounds could be distinguished from each other and characterized. The methods used appear to be very useful in future studies of peptoids and peptoid-peptide hybrids.