Simultaneous analysis of angiotensin peptides by LC-MS and LC-MS/MS: metabolism by bovine adrenal endothelial cells

Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were developed to simultaneously determine the concentrations of angiotensin (Ang) II, Ang 1-7, Ang III, and Ang IV in biological samples. The samples were extracted with C18 solid-phase extraction cartridges and separated by a reverse-phase C18 column using acetonitrile in water with 0.1% formic acid as a mobile phase. Ang peptides were ionized by electrospray and detected by triple quadrupole MS in the positive ion mode. (M+3H)(3+) and (M+2H)(2+) ions were chosen as the detected ions in the single ion recording (SIR) mode for LC-MS. The limits of detection (signal/noise [S/N]=3) using SIR are 1 pg for Ang IV and 5 pg for Ang 1-7, Ang III, and Ang II. Multiple reaction monitoring (MRM) mode was used for LC-MS/MS. The limits of detection (S/N =3) using MRM are 20 pg for Ang IV and 25 pg for Ang 1-7, Ang III, and Ang II. These methods were applied to analyze Ang peptides in bovine adrenal microvascular endothelial cells. The results show that Ang II is metabolized by endothelial cells to Ang 1-7, Ang III, and Ang IV, with Ang 1-7 being the major metabolite.     

Synthesis and characterisation of sulfated amphiphilic alpha-, beta- and gamma-cyclodextrins: application to the complexation of acyclovir

The synthesis of sulfated amphiphilic alpha-, beta- and gamma-cyclodextrins was achieved according to the standard protection-deprotection procedure. The formation of inclusion complexes between the amphiphilic alpha-, beta- and gamma-cyclodextrins and an antiviral molecule, acyclovir (ACV) was investigated by UV-visible spectroscopy (UV-Vis) and electrospray ionisation mass spectrometry (ESIMS). UV-Vis spectroscopy allowed determination of the stoichiometry and stability constants of complexes, whereas ESIMS, a soft ionisation technique, allowed the detection of the inclusion complexes. The results showed that the non-sulfated amphiphilic cyclodextrins exhibit a 1:2 stoichiometry with acyclovir, while sulfated amphiphilic cyclodextrins, except gamma-cyclodextrin, exhibit a 1:1 stoichiometry indicating the loss of one interaction site. Non-covalent interactions between acyclovir and non-sulfated amphiphilic cyclodextrins appear to take place both in the cavity of the cyclodextrin and inside the hydrophobic zone generated by alkanoyl chains. In contrast, in the case of sulfated amphiphilic cyclodextrins, the interactions appear to involve only the hydrophobic region of the alkanoyl chains.     

Simultaneous analysis of prostaglandin glyceryl esters and prostaglandins by electrospray tandem mass spectrometry

A high-performance liquid chromatography (HPLC) positive-ion electrospray ionization tandem mass spectrometry method for the quantification of prostaglandin glyceryl esters (PG-Gs), a newly discovered class of eicosanoids, is described. All four PG-Gs (PGE(2)-G, PGD(2)-G, PGF(2alpha)-G, and 6-keto-PGF(1alpha)-G) and the prostaglandins (PGs) that are formed by their hydrolysis are simultaneously quantified. Analytes were purified via reverse-phase solid-phase extraction, separated by reverse-phase HPLC, and quantified on a triple-quadrupole mass spectrometer using selected reaction monitoring. Quantification was achieved by stable isotope dilution employing penta-deuterated (PG-Gs) or tetra-deuterated (PGs) analogs. Analyte recovery from cell culture medium was >43% for all analytes at four different concentration levels. The limit of quantification is in the range of 25fmol on-column for each analyte and the analytes exhibit a linear response over approximately a 500-fold range. This method allows simultaneous profiling of several PG-Gs and PGs without multistep sample purification or derivatization.     

Effective reversed-phase ion pair high-performance liquid chromatography method for the separation and characterization of intact low-molecular-weight heparins

A simple, selective, and efficient reversed-phase ion pair high-performance liquid chromatography (RPIP-HPLC) method was developed for the separation of various commercially available intact low-molecular-weight heparins (LMWHs). The developed method uses a C₁₈ column (150 × 4.6 mm) with diode array detection at 230 nm, flow rate at 1.0 ml/min, and a mobile phase containing acetonitrile/water (32:68%), tetrabutylammonium hydroxide (15 mM), and ammonium acetate (50 mM) at pH 7.0. The performance of this method was assessed in terms of selectivity, linearity, intra- and interday precision, and accuracy. The novel application of RPIP-HPLC with evaporative light scattering detection (ELSD) for the analysis of intact LMWHs was demonstrated. Intact LMWHs were analyzed with superior resolution and peak shape. Different chromatographic profiles were obtained for different LMWHs showing significant structural diversity. This method clearly showed chemical changes that occurred to LMWH under the stress condition. This method can be applied for the separation, identification, characterization, and pharmaceutical stability analysis of various LMWHs.     

High-performance liquid chromatography/electrospray ionization ion-trap mass spectrometry for analysis of oligosaccharides derivatized by reductive amination and N,N-dimethylation

Milk oligosaccharides derivatized by reductive amination with benzylamine followed by N,N-dimethylation (DMBA-oligosaccharides), were analyzed by high-performance liquid chromatography/electrospray ionization ion-trap mass spectrometry (HPLC/ESI-ITMS). Separation of DMBA-oligosaccharides was achieved on a graphitized carbon column eluted with aqueous acetonitrile and the DMBA-oligosaccharides were detected by positive-ion mode ESI-ITMS allowing sample amounts down to approximately 30fmol of single DMBA-oligosaccharides injected on the HPLC column. MS/MS operation of the mass spectrometer resulted in the detection of diagnostic fragments, mainly belonging to the Y-series, allowing differentiation between isomeric milk oligosaccharides. HPLC/ESI-ITMS/MS/MS experiments indicated the migration of fucose residues of the DMBA milk oligosaccharides to the modified reducing end glucose residue during analysis, a migration previously only observed for proton adduct ions.     

Structural analysis of the lipopolysaccharide derived core oligosaccharides of Actinobacillus pleuropneumoniae serotypes 1, 2, 5a and the genome strain 5b

The structures of the core oligosaccharides of the lipopolysaccharides (LPS) from Actinobacillus pleuropneumoniae serotypes 1, 2, 5a and 5b were elucidated. The LPS's were subjected to a variety of degradative procedures. The structures of the purified products were established by monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. The following structures for the core oligosaccharides were determined on the basis of the combined data from these experiments. [carbohydrate formula see text] For serotype 1: R is (1S)-GalaNAc-(1-->4,6)-alpha-Gal II-(1-->3)-beta-Gal I-(1-->, and R' is H For serotype 2: R is beta-Glc III-(1-->, and R' is D-alpha-D-Hep V-(1--> For serotypes 5a and 5b: R is H and R' is D-alpha-D-Hep V-(1--> All oligosaccharides elaborated a conserved inner core structure, as illustrated. All sugars were in the pyranose ring form apart from the open-chain N-acetylgalactosamine, the identification of which in the serotype 1 LPS was of interest.     

Hair analysis of histamine after fluorescence labeling by column-switching reversed-phase liquid chromatography with electrospray ionization mass spectrometry and application to human hair

Sensitive determination of histamine (HA) in hair was carried out by column-switching reversed-phase high-performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS). HA was labeled with excess amounts of 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) at 60 degrees C for 30 min in a mixture of 0.1 M borax (pH 9.3) and acetonitrile (CH(3)CN). The resulting DBD-HA derivative was roughly separated by a Mightysil RP-18 GP (100 x 2mm i.d., 3 microm) with an acidic mobile phase containing 0.1% trifluoroacetic acid. DBD-HA in the fraction flowing due to a position change in the six-port column-switching valve was then completely separated by a Wakopak Navi C30 (150 x 2mm i.d., 5 microm) with 20 mM AcONH(4)-CH(3)CN (8:2). The mass spectrometer was operated in the selected reaction monitoring (SRM) mode for the product ion (m/z 292) obtained from MS-MS measurement using the protonated molecular ion [M+H](+) (m/z 337) as the precursor ion. Good linearity was achieved from the calibration curve obtained by plotting peak area ratios of the internal standard (HA-d(4)) against the injected amounts of HA (1.66-16.6 pmol, r(2)=0.999). The coefficients of variation, at 1.66- and 16.6-pmol injections, were 5.6 and 3.7%, respectively (n=6). Furthermore, the detection limit was 0.167 pmol. The efficiency of the recommended procedure was identified from the determination in the rat hair root after intraperitoneal administration of HA. The proposed method was applied to HA determination in the hair shaft of Dark Agouti rats and healthy volunteers. The variations in the concentrations in 1mg of hair shaft were 0.80-1.84 pmol (mean+/-SD=1.33+/-0.33, n=12) in rats and 0.94-72.3 pmol (17.2+/-21.5, n=16) in humans. The determination of HA in the plasma of rats and humans was also performed successfully by this method. Because the proposed method provides good precision and trace detection of HA in hair, the analytical technique seems to be applicable for the determination of various biogenic amines in hair.     

Formation and biliary excretion of glutathione conjugates of bile acids in the rat as shown by liquid chromatography/electrospray ionization-linear ion trap mass spectrometry

Acyl-adenylates and acyl-CoA thioesters of bile acids (BAs) are reactive acyl-linked metabolites that have been shown to undergo transacylation-type reactions with the thiol group of glutathione (GSH), leading to the formation of thioester-linked GSH conjugates. In the current study, we examined the transformation of cholyl-adenylate (CA-AMP) and cholyl-coenzyme A thioester (CA-CoA) into a cholyl-S-acyl GSH (CA-GSH) conjugate by rat hepatic glutathione S-transferase (GST). The reaction product was analyzed by liquid chromatography (LC)/electrospray ionization (ESI)-linear ion trap mass spectrometry (MS). The GST-catalyzed formation of CA-GSH occurred with both CA-AMP and CA-CoA. Ursodeoxycholic acid, lithocholic acid, and 2,2,4,4-²H₄-labeled lithocholic acid were administered orally to biliary fistula rats, and their corresponding GSH conjugates were identified in bile by LC/ESI-MS². These in vitro and in vivo studies confirm a new mode of BA conjugation in which BAs are transformed into their GSH conjugates via their acyl-linked intermediary metabolites by the catalytic action of GST in the liver, and the GSH conjugates are then excreted into the bile.     

Synthesis, crystal structure and characterization of alkali metal hydroxoantimonates

Several alkali metal hydroxoantimonates, K₂[Sb(O)(OH)₅], Na[Sb(OH)₆], Cs[Sb(OH)₆] and Cs₂[Sb₂(μ-O)₂(OH)₈] were isolated from aqueous solutions and characterized by single crystal and powder X-ray diffraction studies and by FTIR and thermal analysis. Crystal structures involving [Sb(O)(OH)₅]²⁻ were never anticipated before, and this is also the first disclosure of a dinuclear antimonate [Sb₂(μ-O)₂(OH)₈]²⁻. Aqueous antimonate solutions of different pH were studied by high resolution electrospray mass spectrometry showing pH indifferent spectra and predominance of the mono and dinuclear antimonate species at pH 4-10.     

Strategy for the investigation of O-linked oligosaccharides from mucins based on the separation into neutral, sialic acid- and sulfate-containing species

A method for the separation of O-linked oligosaccharides into neutral, sialic acid-containing and sulfated species was applied to oligosaccharides released by alkaline borohydride from mucin glycopeptides from porcine small intestine. The released mixture of reduced oligosaccharides was applied to an anion exchange column, and the neutral oligosaccharides were collected as the unretarded fraction. A mixture of dimethyl sulfoxide and iodomethane was passed through the column to convert the sialic acid-containing oligosaccharides into methyl esters that were eluted and converted to methyl amides by methyl amine. Finally the sulfated oligosaccharide fraction was eluted with salt. The neutral and the derivatized sialic acid-containing oligosaccharides were analysed by gas chromatography-mass spectrometry after permethylation and the sulfated oligosaccharide fraction was analysed by high performance anion exchange chromatography.Abbreviations GC gas chromatography - GC/MS gas chromatography-mass spectrometry - HPAEC-PAD high performance anion exchange chromatography-pulsed amperometric detection - Hex hexose - HexNAc N-acetyl hexosamine - HexNAcol N-acetyl hexosaminitol - Fuc Fucose - NeuAc N-acetyl neuraminic acid - NeuGc N-glycolyl neuraminic acid     

Structural and quantitative analysis of N-linked glycans by matrix-assisted laser desorption ionization and negative ion nanospray mass spectrometry

Negative ion electrospray (ESI) fragmentation spectra derived from anion-adducted glycans were evaluated for structural determination of N-linked glycans and found to be among the most useful mass spectrometric techniques yet developed for this purpose. In contrast to the more commonly used positive ion spectra that contain isobaric ions formed by losses from different regions of the molecules and often lead to ambiguous deductions, the negative ion spectra contain ions that directly reflect structural features such as the branching pattern, location of fucose, and the presence of bisecting GlcNAc. These structural features are sometimes difficult to determine by traditional methods. Furthermore, the spectra give structural information from mixtures of isomers and from single compounds. The method was evaluated with well-characterized glycans from IgG and used to explore structures of N-linked glycans released from serum glycoproteins with the aim of identifying biomarkers for cancer. Quantities of glycans were measured by ESI and by matrix-assisted laser desorption ionization mass spectrometry; each technique produced virtually identical results for the neutral desialylated glycans.     

Synthesis, characterization and structure of metallocene dicyanamide complexes

The bis(cyclopentadienyl) complexes [Cp₂Ti(dca)]₂O and Cp₂V(dca)₂ (dca = dicyanamide) have been prepared by reaction of sodium dicyanamide with aqueous solution of titanocene dichloride and vanadocene dichloride, respectively. The X-ray structure analyses of both complexes confirmed monodentate coordination of dicyanamide ligand through the terminal nitrogen atom of cyano group.     

A simple cellulose column procedure for selective enrichment of glycopeptides and characterization by nano LC coupled with electron-transfer and high-energy collisional-dissociation tandem mass spectrometry

In this report we describe an on-column method for glycopeptide enrichment with cellulose as a solid-phase extraction material. The method was developed using tryptic digests of several standard glycoproteins and validated with more complex standard protein digest mixtures. Glycopeptides of different masses containing neutral and acidic glycoforms of both N- and O-linked sugars were obtained in good yield by this method. Upon isolation, glycopeptides may be subjected to further glycoproteomic and glycomic workflows for the purpose of identifying glycoproteins present in the sample and characterizing their glycosylation sites, as well as their global and site-specific glycosylation profiles at the glycopeptide level. Detailed structural analysis of glycoforms may then be performed at the glycan level upon chemical or enzymatic release of the oligosaccharides. Aiming at complementing other purification methods, this technique is extremely simple, cost-effective, and efficient. Glycopeptide enrichment was verified and validated by nano liquid chromatography-tandem mass spectrometry (LC-MS/MS) combining electron-transfer dissociation (ETD) and collision-activated dissociation (CAD) fragmentation techniques.     

n-Alkyl p-aminobenzoates as derivatizing agents in the isolation, separation, and characterization of submicrogram quantities of oligosaccharides by liquid secondary ion mass spectrometry

In this laboratory we are pursuing a comprehensive strategy for isolation and characterization of oligosaccharides from glycoproteins that are available only in limited quantities. To improve sensitivity in the analysis by liquid secondary ion mass spectrometry, we have investigated the relative behavior of a homologous series of n-alkyl esters of p-aminobenzoic acid as derivatizing agents. Ethyl p-aminobenzoate, the derivatizing agent used in many of our earlier studies, is one of these compounds. Our experiments using the hepatasaccharide maltoheptaose (M7) as a model oligosaccharide establish that by lengthening the alkyl chain from methyl to n-tetradecyl, a concomitant increase in the molecular ion abundance is obtained. The increase is a factor of 10 when 1 microgram of derivatized M7 is analyzed, and as much as 40 when 0.1 microgram of sample is examined. This series of derivatives of maltoheptaose form a suite of relatively abundant fragment ions in the negative ion mode as expected from our previous studies with the ethyl ester. Although very high mass spectral sensitivities were achieved with M7 n-tetradecyl and n-decyl p-aminobenzoates, the yields of derivative obtained were significantly lower than those obtained for M7 n-octyl, n-hexyl, n-butyl, ethyl, and methyl p-aminobenzoates, despite improvements made in the derivatization procedure. When analyzing biological samples, n-octyl and n-hexyl p-aminobenzoate were found to be optimal considering both yield of derivative and mass spectral sensitivity. This improved method of derivatization was incorporated into a simple but effective procedure for dealing with very small quantities of heterogeneous samples of oligosaccharides, such as those released from 250 micrograms (1 nmol) of nicotinic acetylcholine receptor from Torpedo californica and 90 micrograms (2 nmol) of human alpha 1 acid glycoprotein.     

Synthesis of a new gadolinium complex with a high affinity for human serum albumin and its manifold physicochemical characterization by proton relaxation rate analysis, NMR diffusometry and electrospray mass spectrometry

A novel gadolinium complex, derived from Gd-DTPA (DTPA: diethylenetriaminepentaacetic acid) and sulfaphenazole, intended to be a potential MRI contrast agent and to interact with human serum albumin (HSA), was synthesized and characterized. Its relaxometric properties were evaluated in water, and its binding to HSA was investigated by three techniques: proton relaxation rate analysis, NMR diffusometry, and electrospray mass spectrometry. The complex has a higher relaxivity than the parent compound (r(1)=7.8s(-1)mM(-1) at 310K and 0.47T and 7.7s(-1)mM(-1) at 310K and 1.41T), a fast water exchange, and a very good stability versus zinc(II) transmetallation. All techniques agree with a high affinity of the complex for HSA, and competition experiments indicate that this contrast agent competes with ibuprofen for HSA.     

Structural characterization of N-linked oligosaccharides on monoclonal antibody cetuximab by the combination of orthogonal matrix-assisted laser desorption/ionization hybrid quadrupole-quadrupole time-of-flight tandem mass spectrometry and sequential enzymatic digestion

Cetuximab is a novel therapeutic monoclonal antibody with two N-glycosylation sites: a conserved site in the CH2 domain and a second site within the framework 3 of the variable portion of the heavy chain. The detailed structures of these oligosaccharides were successfully characterized using orthogonal matrix-assisted laser desorption/ionization hybrid quadrupole-quadrupole time-of-flight mass spectrometry (oMALDI Qq-TOF MS) and tandem mass spectrometry (MS/MS) in combination with exoglycosidase digestion. The N-linked oligosaccharides were released by treatment with N-glycanase F, reductively aminated with anthranilic acid, and fractionated by normal phase high-performance liquid chromatography (NP-HPLC). The fluorescent-labeled oligosaccharide pool and fractions were analyzed by oMALDI Qq-TOF MS and MS/MS in negative ion mode. Each fraction was further digested with an array of exoglycosidase mixtures, and subsequent MALDI TOF MS analysis of the resulting products yielded information about structural features of the oligosaccharide. The combined data revealed the presence of 21 distinct oligosaccharide structures in cetuximab. These oligosaccharides differ mainly in degree of sialylation with N-glycolyl neuraminic acid and extent of galactosylation (zero-, mono-, di-, and alpha(1-3)-galactosidase). The individual oligosaccharides were further assigned to the specific sites in the Fab and Fc regions of the antibody. This study represents a unique approach in that MS/MS data were used to identify and confirm the oligosaccharide structures of a protein.     

Structural characterization of oligosaccharides by high-performance liquid chromatography, fast-atom bombardment-mass spectrometry, and exoglycosidase digestion

A method for structural characterization of oligosaccharides after preparing uv-absorbing derivatives is described. The derivatives can be rapidly analyzed and purified by high-performance liquid chromatography, with separation of various structures determined primarily by size and sugar composition. Derivatization requires as little as 0.5-1.0 nmol of oligosaccharide, and detection of down to 50 pmol of oligosaccharide is possible by monitoring absorbance at 229 nm. In addition, the carbohydrate portion of the derivative was found to retain its sensitivity to exoglycosidases, allowing sequential enzymatic digestions for determination of sugar sequence and anomerity to be performed. The derivatives also possessed a site of potential positive charge, making them amenable to analysis by fast-atom bombardment-mass spectrometry. Permethylation of the derivatives permitted their separation by capillary gas chromatography, thus allowing investigation of their structures by gas chromatography-mass spectrometry. The combination of these techniques will allow almost the complete structure of small amounts of oligosaccharides to be determined.     

A competitive binding study of chemokine, sulfated receptor, and glycosaminoglycan interactions by nano-electrospray ionization mass spectrometry

Chemokines are secreted proteins that play roles in inducing chemotaxis, extravasation, and activation of leukocytes associated with inflammatory or homeostatic processes. Tyrosine sulfation of the chemokine receptor has been shown to be important for binding and signaling. We have applied a mass spectrometry method to measure the contribution of this posttranslational modification to binding of its ligand chemokine. Using nano-electrospray time-of-flight mass spectrometry (nano-ESI TOF MS), we determined the association constants of C-C motif chemokine 7 (CCL7) with C-C chemokine receptor type 2 (CCR2), monosulfated CCR2, and disulfated CCR2 peptides to be 1.1 × 10⁴ M⁻¹, 3.9 × 10⁴ M⁻¹, and 4.0 × 10⁵ M⁻¹, respectively. To our knowledge, this is the first reported association constant measurement between a protein and sulfated peptide using MS. Furthermore, nano-ESI MS was used to characterize noncovalent binding interactions among CCL7, Arixtra (a pentasaccharide glycosaminoglycan [GAG] analog), and disulfated CCR2 peptide. A lack of observable ternary complex formation prompted investigation of competitive binding. Results of this study suggest that CCR2 competes partially with GAG for CCL7 binding and that disulfated CCR2 peptide has a higher binding affinity than Arixtra, which correlates with data from association constant measurements for CCL7-disulfated CCR2 and CCL7-Arixtra.     

Quantitative determination of the antitumor alkyl ether phospholipid edelfosine by reversed-phase liquid chromatography-electrospray mass spectrometry: application to cell uptake studies and characterization of drug delivery systems

Edelfosine is a synthetic alkyl ether phospholipid that represents a promising class of antitumor agents. However, analytical methods to measure these type compounds are scarce. The lack of a reliable methodology to quantify edelfosine is a major problem in ongoing and scheduled preclinical and clinical trials with this drug. We evaluated the applicability of high-performance liquid chromatography-mass spectrometry to determine edelfosine in biological samples and polymeric delivery systems. Sample pre-treatment involved polymer precipitation or cell lysis with methanol. HPLC separation was performed on an Alltima RPC(18) narrow-bore column and edelfosine quantification was done by electrospray ionization mass spectrometry (ESI-MS) using positive ion mode and selected ion monitoring. Assays were linear in the tested range of 0.3-10 microg/ml. The limit of quantification was 0.3 ng/sample in both matrices, namely biological samples and polymeric delivery systems. The interassay precision ranging from 0.79 to 1.49%, with relative errors of -6.7 and 12.8%. Mean extraction recovery was 95.6%. HPLC-ESI-MS is a reliable system for edelfosine analysis and quantification in samples from different sources, combining advantages of full automation (rapidity, ease of use, no need of extensive extraction procedures) with high analytical performance and throughput.     

Estimation of pseudopeptide metal ion complexation tendencies by special MS methods, HPCE and circular dichroism

To obtain more insight into catalytic mechanisms of metallo enzymes and specific metal complexation by proteins we use linear and cyclic pseudopeptides as mimetics. Knowledge about tendencies of complex formation of different ligands with selected transition metal ions is an indispensable prerequisite for the development of homo- and hetero-dinuclear metallo enzyme mimetics. Three pseudotripeptide ligands were investigated with respect to formation tendency and properties of complexes with the transition metal ions Cu²⁺, Zn²⁺, Ni²⁺, Co²⁺ and Mn²⁺. To study complexation tendencies we applied different methods. One of the important prerequisites for the application in a screening of series of peptide ligands is the necessity for a minimal amount of substance. We used and compared certain masspectrometric methods for the estimation of a rank order of complexation of certain transition metal ions. We also applied spectrophotometric titration, circular dichroism measurements, capillary electrophoresis and pH-rate profile of catalytic activity in the attempt to evaluate complex formation tendencies. Except for the spectrophotometric pH-titration and the pH-profile of catalytic activity all methods were applicable, but each method has its advantages and disadvantages depending on the separation effect of the ligand from the metal complex, and depending on the spectroscopic properties of ligand and complex. The results regarding complex formation are compared to each other. Comparison of pairs by MALDI-TOF- and ESI-MS allows an estimation of the rank order of complexation tendency of one ligand with different metal ions and requires the least amount of substance. The other investigated methods provided additional information on structural properties of the formed complexes; however either they required too much pseudopeptide ligand or were not applicable for all transition metal ions used in this study.