Unique guanidine-conjugated catechins from the leaves of Alchornea rugosa and their autophagy modulating activity.

Natural guanidines, molecules that contain the guanidine moiety, are structurally unique and often exhibit potent biological activities. A phytochemical investigation of the leaves of Alchornea rugosa (Lour.) Müll.Arg. by MS/MS-based molecular networking revealed eight undescribed guanidine-flavanol conjugates named rugonines A-H. The chemical structures of the isolated compounds were comprehensively elucidated by NMR spectroscopy, HRESIMS, and circular dichroism (CD) analysis. All isolated compounds were tested for autophagosome formation in HEK293 cells stably expressing GFP-LC3. The results revealed that compounds rugonines D-G showed potential autophagy inhibitory activity.

Cataract-causing G91del mutant destabilised ßA3 heteromers formation linking with structural stability and cellular viability.

BACKGROUND/AIMS: Congenital cataracts, which are genetically heterogeneous eye disorders, result in visual loss in childhood around the world. CRYBA1/BA3 serves as an abundant structural protein in the lens, and forms homomers and heteromers to maintain lens transparency. In previous study, we identified a common cataract-causing mutation, ßA3-glycine at codon 91 (G91del) (c.271-273delGAG), which deleted a highly conserved G91del and led to perinuclear zonular cataract. In this study, we aimed to explore the underlying pathogenic mechanism of G91del mutation. METHODS: Protein purification, size-exclusion chromatography, spectroscopy and molecular dynamics simulation assays were used to investigate the effects on the heteromers formation and the protein structural properties of ßA3-crystallin caused by G91del mutation. Intracellular ßA3-G91del overexpression, MTT (3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide) and cell apoptosis were used to investigate the cellular functions of ßA3-G91del. RESULTS: ßA3-crystallin and ßB2-crystallin could form heteromers, which have much more stable structures than ßA3 homomers. Interestingly, ßA3/ßB2 heteromers improved their resistance against the thermal stress and the guanidine hydrochloride treatment. However, the pathogenic mutation ßA3-G91del destroyed the interaction with ßB2, and thereby decreased its structural stability as well as the resistance of thermal or chemical stress. What's more, the ßA3-G91del mutation induced cell apoptosis and escaped from the protection of ßB2-crystallin. CONCLUSIONS: ßA3/ßB2 heteromers play an indispensable role in maintaining lens transparency, while the ßA3-G91del mutation destabilises heteromers formation with ßB2-crystallin, impairs cellular viability and induces cellular apoptosis. These all might contribute to cataract development.

Guanidine Biosensors Enable Comparison of Cellular Turn-on Kinetics of Riboswitch-Based Biosensor and Reporter.

Cell-based sensors are useful for many synthetic biology applications, including regulatory circuits, metabolic engineering, and diagnostics. While considerable research efforts have been made toward recognizing new target ligands and increasing sensitivity, the analysis and optimization of turn-on kinetics is often neglected. For example, to our knowledge there has been no systematic study that compared the performance of a riboswitch-based biosensor versus reporter for the same ligand. In this study, we show the development of RNA-based fluorescent (RBF) biosensors for guanidine, a common chaotropic agent that is a precursor to both fertilizer and explosive compounds. Guanidine is cell permeable and nontoxic to E. coli at millimolar concentrations, which in contrast to prior studies enabled direct activation of the riboswitch-based biosensor and corresponding reporter with ligand addition to cells. Our results reveal that the biosensors activate fluorescence in the cell within 4 min of guanidine treatment, which is at least 15 times faster than a reporter derived from the same riboswitch, and this rapid sensing activity is maintained for up to 1.6 weeks. Together, this study describes the design of two new biosensor topologies and showcases the advantages of RBF biosensors for monitoring dynamic processes in cell biology, biotechnology, and synthetic biology.

Fabrication of poly o-cresophthalein complexone film modified electrode and its application to simultaneous determination of purine bases in denatured DNA.

A novel poly o-cresophthalein complexone film (POCF) modified electrode was fabricated and used as a sensor for the simultaneous detection of adenine and guanine. By comparing with the bare paraffin wax impregnated graphite electrode (PIGE), POCF modified electrode showed remarkable increase in the oxidation peak currents of adenine and guanine. The surface morphology of the POC polymer film and its nature on the PIGE were characterized by field emission scanning electron microscopy, X-ray photoelectron spectroscopy, Raman spectroscopy, cyclic voltammetry, electrochemical impedance spectroscopy and differential pulse voltammetry. The POCF modified electrode exhibited a linear range of 0.08 µM-200 µM with a low detection limit of 0.02 µM (S/N) for adenine and guanine, respectively. Further, the proposed POCF modified electrode was used for simultaneous detection of adenine and guanine in denatured CT-DNA and moth DNA with satisfactory results. The value of (G + C)/(A + T) for CT-DNA and moth DNA was calculated to be 0.79 and 0.78 respectively. The prepared POCF modified electrode showed high reproducibility and excellent stability.

Forensic Analysis and Differentiation of Black Powder and Black Powder Substitute Chemical Signatures by Infrared Thermal Desorption-DART-MS.

The trace detection and forensic analysis of black powders and black powder substitutes, directly from wipe-based sample collections, was demonstrated using infrared thermal desorption (IRTD) coupled with direct analysis in real time mass spectrometry (DART-MS). Discrete 15 s heating ramps were generated, creating a thermal desorption profile that desorbed more volatile species (e.g., organic and semivolatile inorganic compounds) at lower temperatures (250-400 °C) and nonvolatile inorganic oxidizers at high temperatures (450-550 °C). Common inorganic components of black powders (e.g., sulfur and potassium nitrate) as well as the alternative and additional organic and inorganic components of common black powder substitutes (e.g., dicyandiamide, ascorbic acid, sodium benzoate, guanidine nitrate, and potassium perchlorate) were detected from polytetrafluoroethylene-coated fiberglass collection wipes with no additional sample preparation. IRTD-DART-MS enabled the direct detection of intact inorganic salt species as nitrate adducts (e.g., [KClO4+NO3]-) and larger clusters. The larger ion distributions generated by these complex mixtures were differentiated using principal component analysis (PCA) of the mass spectra generated at two points during the thermal desorption profile (low and high temperatures), as well as at high in-source collision-induced dissociation. The PCA framework generated by the analysis of the two black powders and five black powder substitutes was used to classify samples collected from a commercial firecracker containing both flash powder and black powder. The coupling of IRTD-DART-MS and multivariate statistics demonstrated the powerful utility for detection and discrimination of trace fuel-oxidizer mixtures.

Enhancement effect of p-iodophenol on gold nanoparticle-catalyzed chemiluminescence and its applications in detection of thiols and guanidine.

In the present study, the effects of nine compounds on the luminol-O2 chemiluminescence (CL) reaction catalyzed by gold nanoparticles (Au NPs) were investigated. p-Iodophenol (PIP), known as a traditional enhancer in horseradish peroxidase (HRP)-catalyzed luminol-H2O2 CL reaction, exhibited the highest enhancement effect on the luminol-O2-Au NPs CL reaction. The addition of PIP at a final concentration of 1.25 mM to luminol-O2-Au NPs CL system enhanced the CL signal almost 30-fold. Interestingly, the enhanced CL reaction exhibited a slow and intense CL signal which is different from the CL profile of luminol-H2O2-Au NPs CL system reported in previously. The experimental conditions of the PIP enhanced luminol-O2-Au NPs CL system were investigated systematically in the present study. And the mechanism studies showed that superoxide anion (O2•-) and singlet oxygen (1O2) generated from dissolved oxygen played an important role during the CL reaction. Furthermore, the effects of some organic compounds on the enhanced CL system were investigated. The results showed that compounds containing -SH and guanidine group can inhibit the signal of the enhanced CL reaction indicating the applicability of the proposed CL reaction for the detection of such compounds.

Improved Gas Chromatographic Determination of Guanidino Compounds Using Isovaleroylacetone and Ethyl Chloroformate as Derivatizing Reagents.

An improved GC method in terms of sensitivity and decrease in the analysis time has been developed for the analysis of eight guanidino compounds: guanidine (G), methylguanidine (MG), creatinine (CTN), guanidinoacetic acid (GAA), guanidinobutyric acid (GBA), guanidinopropionic acid (GPA), argenine (Arg), and guanidinosuccinic acid (GSA), using isovaleroylacetone (IVA) and ethyl chloroformate (ECF) as derivatizing reagents. The separation was obtained from column HP-5 (30 m × 0.32 mm i.d.) with film thickness of 0.25 µm within 11 min. The linear calibrations were obtained with 0.5 to 50 µg/mL with coefficient of determination (R(2)) within 0.9969 - 0.9998. Limits of detections (LODs) were within 5 - 140 ng/mL. The derivatization, separation and determination was repeatable (n = 6) with relative standard deviation (RSD) within 1.2 - 3.1%. The guanidino compounds were determined in deproteinized serum of healthy volunteers and uremic patients within below LOD to 8.8 µg/mL and below LOD to 43.99 µg/mL with RSD within 1.4 - 3.6%. The recovery of guanidino compounds calculated by standard addition from serum was within 96.1 - 98.9%, with RSD 1.4 - 3.6%.

A new approach for trace analysis of guanidine compounds in surface water with resorcinarene-based ion chromatography columns.

Trace levels of pharmaceuticals have been detected in surface water and may pose a health risk to humans and other organisms. New chromatographic materials will help identify and quantify these contaminants. We introduce a new ion chromatographic (IC) material designed to separate cationic pharmaceuticals and report its ability to separate a group of guanidine compounds. Guanidine moieties are strongly basic and protonated under acid conditions, and therefore can potentially be separated on the newly designed stationary phase and detected by ion exchange chromatography. The new column packing material is based on glutamic acids bonded to resorcinarene moieties that in turn are bound to divinylbenzene macroporous resin. Detection limits in the range of 5-30 µg L(-1) were achieved using integrated pulsed amperometric detection (IPAD) for guanidine (G), methylguanidine (MG), 1,1-dimethylbiguanide (DMG), agmatine (AGM), guanidinobenzoic acid (GBA) and cimetidine (CIM). Suppressed conductivity (CD) and UV-vis detection resulted in limits of detection similar to IPAD, in the range of 2-66 µg L(-1), but were not able to detect all of the analytes. Three water sources, river, lake, and marsh, were analyzed and despite matrix effects, sensitivity for guanidine compounds was in the 100 µg L(-1) range and apparent recoveries were 80-96%. The peak area precision was 0.01-2.89% for IPAD, CD and UV-vis detection.

Trace analysis of biocidal oligoguanidines in environmental water samples.

This paper demonstrates the determination of residues of biocidal oligoguanidines manufactured by polycondensation of guanidine hydrochloride and 2,2'-(ethylenedioxy)bis(ethylamine) in environmental water samples. The analytes were preconcentrated from samples adjusted to pH 4 by solid-phase extraction using a mixed-mode sorbent with weak cation exchange functionalities. Elution from the sorbent was achieved by 2M hydrochloric acid in methanol. After evaporation and reconstitution in water, the extract was analyzed by reversed-phase high performance liquid chromatography combined with quadrupole/time-of-flight or triple quadrupole mass spectrometry employing electrospray ionization. A preconcentration factor of 10,000 could be achieved and detection limits were in the sub-µgL(-1) range. This method looks promising to monitor the fate of these biocides released into the aquatic environment during different applications.

Guanidination of tryptic peptides without desalting for matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry analysis.

Derivatizations that enhance mass spectral quality often require desalting, which presents as a bottleneck in matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS)-proteomics. Guanidination, which converts lysine to homoarginine, an arginine analogue, can increase detection of those peptides 5-15-fold. Our aim was to improve guanidination by using a novel reagent, O-methylisourea-freebase. In a simple reaction, interfering salts were removed prior to guanidination. Freebase preparation took about 30 min and could be applied to samples all at once as opposed to desalting samples one-by-one for 5 min each. For freebase guanidinated BSA tryptic peptides, more than 6-times the peptides were observed relative to tryptic peptides or those guanidinated with the conventional reagent, O-methylisourea hemisulfate. Peptide signals increased more than 10-fold relative to those from guanidination with the conventional reagent and were equivalent to those from conventional guanidination with desalting. In addition, freebase guanidination allowed for a lower limit of detection when combined with another derivatization, N-terminal sulfonation, as evidenced by tandem mass spectrometry (MS/MS) fragmentation analysis of in-gel digests of cytochrome c. Freebase guanidination of rat lung proteins after 2-D gel electrophoresis allowed for identification of all tested protein spots regardless of protein characteristics (MW or pI) or abundance. Co-derivatization with N-terminal sulfonation confirmed the identity of low-abundance proteins in 2-D gel spots that contained more than one protein. The freebase guanidination reagent is simple to prepare and to implement. Desalting is not needed prior to MALDI-TOF MS. Freebase guanidination effectively increases the dynamic range of detection of lysine-containing peptides while decreasing the work needed for sample preparation.

Rapid detection of economic adulterants in fresh milk by liquid chromatography-tandem mass spectrometry.

A method to aid in the detection of the economically driven adulteration of fresh milk with a range of small, nitrogen containing compounds, including melamine, ammeline, ammelide, cyanuric acid, allantoin, thiourea, urea, biuret, triuret, semicarbazide, aminotriazine, 3- and 4-aminotriazole, cyanamide, dicyandiamide, guanidine, choline, hydroxyproline, nitrate, and a range of amino acids, has been developed. (15)N2-Urea is used as an internal standard. The adulteration of milk with exogenous urea has previously been difficult to detect because of the variation in the naturally occurring levels of urea in milk. However, by monitoring the contaminants biuret and triuret, which comprise up to 1% of synthetic urea, the adulteration of milk with urea-based fertilizer can be detected. We estimate that to be economically viable, adulteration of the order of 90-4000ppm of the above adulterants would need to be added to fresh milk. For most of the compounds, an arbitrary detection threshold of 2ppm is therefore more than sufficient. For biuret, a lower detection threshold, better than 0.5ppm, is desirable and the sensitivity for biuret and triuret can be improved by the post-column addition of lithium to create lithium adducts under electrospray ionisation. Sample handling involves a two-step solvent precipitation method that is deployed in a 96-well plate format, and the hydrophilic interaction liquid chromatography uses a rapid gradient (1.2min). Three separate injections, to detect the positively charged compounds, the negatively charged compounds and amino acids and finally the lithium adducts, are used. This rapid and qualitative survey method may be deployed as a second tier screening method to quickly reduce sample numbers indicated as irregular by an FTIR based screening system, and to direct analysis to appropriate quantification methods.

Guanidinium chloride-induced spectral perturbations of 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid confound interpretation of data on molten globule states.

We describe limitations in the use of 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) to examine unfolding intermediates associated with guanidinium chloride (GuHCl)-induced protein denaturation. Several studies have used alterations in fluorescence emission of bis-ANS to quantify the population of "molten globule" states. Our findings indicate that the observed changes in bis-ANS spectroscopic properties could originate from the interactions of bis-ANS and GuHCl and the aggregation of the dye at higher GuHCl concentrations. We posit that in the absence of additional complementary structural or spectroscopic measurements, the use of bis-ANS emission alone to monitor protein conformations can be misleading.

Characterization of specific noncovalent complexes between guanidinium derivatives and single-stranded DNA by MALDI.

Noncovalently bound complexes between highly basic sites of 12 guanidinium compounds and single-stranded DNA were studied using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. 6-Aza-2-thiothymine (ATT) was used as the matrix in the presence of ammonium citrate, and spectra were recorded in the positive ion mode. Detailed control experiments confirmed unambiguously the high selectivity and specificity of the guanidinium moiety for phosphate groups of DNA. The results verify the binding stoichiometry and show preferential binding of hydrophobic binders (pyrene and anthracene guanidinium derivatives) to all sequences examined. In addition, we demonstrate that electrostatic noncovalent interactions are strengthened with phosphorothioate analogs of DNA. These results clearly highlight the structure-directing role of the self-assembling organic species and strongly emphasize the significance of concentration, hydrophobicity, hydrogen-bonding, and pi-pi interactions of the artificial receptor in the formation of these noncovalent complexes. Because of the ability of DNA-binding compounds to influence gene expression, and therefore cell proliferation and differentiation, the interactions described above could be important in providing a better understanding of the mechanism of action of these noncovalent genetic regulators.

Effects of energy restriction and fish oil supplementation on renal guanidino levels and antioxidant defences in aged lupus-prone B/W mice.

Energy restriction (ER) and dietary fish oil (FO) are known to reduce the severity of glomerulonephritis and increase the lifespan of lupus-prone (NZB x NZW) F1 (B/W) mice. In the present study, mice were fed either ad libitum or energy-restricted (a 40 % lower energy intake than the diet ad libitum), semi-purified diets containing 5 % maize oil or 5 % fish oil supplementation. To estimate the renal damage associated with oxidative stress, the total amounts of reactive oxygen species (ROS), cyclooxygenase-derived ROS and levels of guanidino compounds were measured. Additionally, we assessed the putative action of ER and FO on several key antioxidant enzymes measured in the kidney post-mitochondrial fraction. Results showed that the age-related increase in creatinine level was significantly reduced by ER and FO in old mice. In contrast, arginine and guanidino acetic acid levels showed a decrease with age but were increased by ER and FO. The GSH:GSSG ratio showed a significant decrease with age, whereas ER and FO feeding prevented the decrease. The age-related decrease in antioxidant scavenging superoxide dismutase, catalase and glutathione peroxidase activities were all reversed by ER and FO. The moderately decreased glutathione reductase and glutathione-S-transferase activities with age were significantly increased by ER and FO. Furthermore, the increased total ROS and cyclooxygenase-derived ROS levels were effectively reduced by ER and FO. In conclusion, our data strongly indicate that ER and FO maintain antioxidant status and GSH:GSSG ratio, thereby protecting against renal deterioration from oxidative insults during ageing.

Analysis of guanidine in high salt and protein matrices by cation-exchange chromatography and UV detection.

A simple and sensitive HPLC method for quantitative determination of guanidine in high salt and protein matrices was developed. The HPLC system consisted of an Agilent 1100 pump with an online degasser, a UV detector, an autosampler, and Dionex CS 14 cation-exchange guard (4 mm x 50 mm) and analytical (4 mm x 250 mm) columns. The mobile phase was 3.75 mM methanesulfonic acid (MSA) with a flow rate of 1 mL/min. The other analysis parameters were: 50 microL injection volume, 195 nm UV detection, and 21 min runtime. The limit of quantitation (LOQ) for guanidine HCl was determined to be 0.25 mg/L and the standard curve ranged from 0.25 mg/L to 10 mg/L. Sample preparation was required for the samples containing high protein concentrations. Proteins were removed by centrifuging a sample in a 30 K NanoSep centrifugal filter at 15,300 x g for 20 min. The method could determine guanidine accurately in sample matrices containing up to 200 mM sodium ion or up to 50 mM potassium ion. The method can be used for clearance testing of guanidine in biopharmaceutical products.

Improved high-performance liquid chromatographic determination of guanidino compounds by precolumn dervatization with ninhydrin and fluorescence detection.

Ninhydrin has been investigated as a pre-column derivatization reagent for guanidino compounds. The reaction takes place under strongly alkaline conditions, followed by a second step at low pH and elevated temperature. This procedure yields derivatives with favourable fluorescence properties (excitation at 390 nm, emission at 470 nm). Amino acids do not react with ninhydrin under these conditions so that the method can easily be used for biological samples. Reversed-phase HPLC separations of the derivatives of several representative guanidino compounds in human blood have been achieved with gradients consisting of aqueous formic acid and methanol. Fluorescence detection yields quantification limits of about 20 microg L(-1). Hyphenation with electrospray mass spectrometry has been used to confirm the identity of the derivatives.

Thermodynamic analysis of receptors based on guanidinium/boronic acid groups for the complexation of carboxylates, alpha-hydroxycarboxylates, and diols: driving force for binding and cooperativity.

The thermodynamics of guanidinium and boronic acid interactions with carboxylates, alpha-hydroxycarboxylates, and diols were studied by determination of the binding constants of a variety of different guests to four different hosts (7-10). Each host contains a different combination of guanidinium groups and boronic acids. The guests included molecules with carboxylate and/or diol moieties, such as citrate, tartrate, and fructose, among others. The Gibbs free energies of binding were determined by UV/Vis absorption spectroscopy, by use of indicator displacement assays. The receptor based on three guanidinium groups (7) was selective for the tricarboxylate guest. The receptors that incorporated boronic acids (8-10) had higher affinities for guests that included alpha-hydroxycarboxylate and catechol moieties over guests containing only carboxylates or alkanediols. Isothermal titration calorimetry revealed the enthalpic and entropic contributions to the Gibbs free energies of binding. The binding of citrate and tartrate was investigated with hosts 7-10, for which all the binding events were exothermic, with positive entropy. Because of the selectivity of hosts 8-10, a simple boronic acid (14) was also investigated and determined to be selective for alpha-hydroxycarboxylates and catechols over amino acids and alkanediols. Further, the cooperativity of 8 and 9 in binding tartrate was also investigated, revealing little or no cooperativity with 8, but negative cooperativity with 9. A linear entropy/enthalpy compensation relationship for all the hosts 7-10, 14, and the carboxylate-/diol-containing guests was also obtained. This relationship indicates that increasing enthalpy of binding is offset by similar losses in entropy for molecular recognition involving guanidinium and boronic acid groups.

Small amounts of urea and guanidine hydrochloride can be detected by a far-UV spectrophotometric method in dialysed protein solutions.

The quantization of small amounts of chemical denaturants as urea or guanidine hydrochloride in protein solutions after dialysis is a difficult task in the molecular biology laboratory practice. Refractometric methods are useful to quantify a denaturant in the molar range but this methodology is not helpful when the denaturant is present in small amounts. The method herein described is a new comparative method that requires, a priori, the quantification of the stock solutions of urea (8 M) and guanidine hydrochloride (6 M) by refractometry to prepare by sequential dilution the standards used for comparison in the spectropolarimeter. The method is based on the observation that the wavelengths, at which the absorbance of polarized light increases in the far-UV region, as observed by spectropolarimetry, is related to the concentration of the chemical denaturant present in the protein solution. In the quantitation method herein reported, the urea and guanidine hydrochloride detection limits range from 1.2 x 10(-4) to 6 x 10(-6) M depending on the protein dialysis buffer used for a standard cell path length of 1 cm. The sensibility of this method results to be comprised in a range 4-5 orders of magnitude higher than that measured by refractometry. The determinations in both the sample and the control preparations are virtually completed within approximately 10 min.

Determination of DNA guanine sites forming photo-adducts with Ru(II)-labeled oligonucleotides; DNA polymerase inhibition by the resulting photo-crosslinking.

The influence of the distance between the anchoring site of the tethered [Ru(TAP)(2)dip](2+) complex (TAP=1,4,5,8-tetraazaphenanthrene; dip=4,7-diphenyl-1,10-phenanthroline) on a probe sequence and the guanines of the complementary target strand was studied by (1) the luminescence quenching of the complex (by electron transfer) and (2) the oligodeoxyribonucleotide adduct (ODN adduct) formation which results in photo-crosslinking of the two strands. Moving the guanine moieties away from the complex induces an important decrease of the efficiency of both processes, but clearly affects the ODN adduct formation more specifically than the quenching process. From these results, we determined the positions of the guanine bases in the duplex ODN that are able to form a photo-adduct with the tethered complex. We also examined the possible competition between a long-range hole migration in the duplex ODN and the formation of a photo-adduct by using a sequence labeled with the complex at the 5'-phosphate end. Such a hole migration appears to be inefficient as compared to the ODN adduct formation. Finally, we studied the influence of the photo-crosslinking on the function of two different DNA polymerases. A 17-mer Ru(II)-labeled ODN was hybridized to its complementary sequence located on the 5'-side of a 40-mer matrix. After illumination, the elongation of a 13-mer DNA primer hybridized to the 3'-extremity of the same matrix was stopped at a position corresponding to the formation of the ODN adduct.

Probing of arginine residues in peptides and proteins using selective tagging and electrospray ionization mass spectrometry.

A general labelling method is presented which allows the determination of the number of guanidine groups (related to arginine and homoarginine in peptides and proteins) by means of mass spectrometry. It implies a guanidine-selective derivatization step with 2,3-butanedione and an arylboronic acid under aqueous, alkaline conditions (pH 8-10). The reaction mixture is then directly analysed by electrospray ionization mass spectrometry without further sample pretreatment. Other amino acids are not affected by this reaction although it is demonstrated that lysine side-chains may be unambiguously identified when they are converted to homoarginine prior to derivatization. Guanidine functionalities, as e.g. in the amino acid arginine, are easily identified by the characteristic mass shift between underivatized and derivatized analyte. The tagging procedure is straightforward and selective for guanidine groups. The influence of several experimental parameters, especially the pH of the solution and the choice of reagents, is examined and the method is applied to various arginine-containing peptides and to lysozyme as a representative protein. Possible applications of this technique and its limitations are discussed.