RESUMEN
Incorporation of an (18)O atom into a peptide C-terminus by proteolytic cleavage in the presence of H2(18)O is one of the most effective ways of enhancing tandem mass spectrometry (MS/MS)-based de novo sequencing. Incorporation is usually accomplished by procedures including vacuum-assisted drying of tryptic peptides extracted from gels, their subsequent reconstitution in a H2(16)O/H2(18)O mixture and re-treatment with trypsin. In the present work, we propose a simplified procedure for (18)O incorporation into tryptic peptides by adding H2(18)O and trypsin to the original digest solution. In comparison to published methods, the proposed protocol for peptide de novo sequencing brings significant advantages in analysis and workflow with no deterioration in method performance. We show that labeling by this simplified method leads to a highlighting of the y-ion fragment series in the peptide matrix-assisted laser desorption/ionization (MALDI)- MS/MS data, which facilitates MS/MS data interpretation. We also prove that eliminating acid extraction of peptides from gels does not result in a decrease in sequence coverage or a qualitative loss of particular peptides detectable by MALDI-MS. The method was examined by MALDI-MS/MS on bovine serum albumin and recombinant histidine kinase CKI1 from Arabidopsis thaliana, and was verified by de novo sequencing of tryptic peptides originating from Apodemus sylvaticus salivary proteins.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/química , Isótopos de Oxígeno , Proteínas Quinasas/química , Análisis de Secuencia de Proteína/métodos , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Proteínas de Arabidopsis/análisis , Metilación , Datos de Secuencia Molecular , Proteínas Quinasas/análisis , Análisis de Secuencia de Proteína/instrumentación , Espectrometría de Masas en Tándem/instrumentación , Tripsina/químicaRESUMEN
Retention characteristics of selected synthetic 5'-terminal phosphate absent penta-nucleotides containing adenine, guanine, and thymine were studied in relation to their sequence by hydrophilic interaction chromatography and ion-interaction reversed-phase liquid chromatography. The organic solvent content, pH, and buffer concentration in mobile phases were evaluated as influential separation conditions. Data demonstrate that both compared chromatographic modes can be used to separate synthetic penta-nucleotides according to their nucleotide composition. Moreover, reversed-phase liquid chromatography allows separation according to their sequence. We have found a simple linear additive model to describe the retention order in both separation modes in regard to their sequence. In hydrophilic interaction chromatography, the retention behavior is controlled primarily by the hydrophilicity of involved nucleotides and minimally by their sequence position. For reversed-phase liquid chromatography, the nucleotide hydrophobicity plays an important role in their retention properties and the influence of their location in sequence on the retention increases toward the center and decreases toward the termini. Our results show that the penta-nucleotide sequence, and thus its spatial arrangement induced by the surrounding environment, is highly related to the retention properties, so it may be hypothetically used to read the sequence from the retention properties acquired under particular separation conditions.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Cromatografía Liquida/métodos , Oligonucleótidos/química , Cromatografía de Fase Inversa/métodos , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Oligonucleótidos/síntesis químicaRESUMEN
Quantitative protein extraction from biological samples, as well as contaminants removal before LC-MS/MS, is fundamental for the successful bottom-up proteomic analysis. Four sample preparation methods, including the filter-aided sample preparation (FASP), two single-pot solid-phase-enhanced sample preparations (SP3) on carboxylated or HILIC paramagnetic beads, and protein suspension trapping method (S-Trap) were evaluated for SDS removal and protein digestion from Arabidopsis thaliana (AT) lysate. Finally, the optimized carboxylated SP3 workflow was benchmarked closely against the routine FASP. Ultimately, LC-MS/MS analyses revealed that regarding the number of identifications, number of missed cleavages, proteome coverage, repeatability, reduction of handling time, and cost per assay, the SP3 on carboxylated magnetic particles proved to be the best alternative for SDS and other contaminants removal from plant sample lysate. A robust and efficient 2-h SP3 protocol for a wide range of protein input is presented, benefiting from no need to adjust the amount of beads, binding and rinsing conditions, or digestion parameters.
RESUMEN
Trypsin (EC 3.4.21.4) is the protease of choice for proteome analysis using mass spectrometry of peptides in sample digests. In this work, trypsin from Streptomyces griseus (SGT) was purified to homogeneity from pronase. The enzyme was evaluated in in-gel digestion of protein standards followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) analyses of the digests. We recognized a remarkable cleavage performance of SGT. The number of produced and matching tryptic peptides was higher than in the case of commonly used bovine trypsin (BT) and allowed us to obtain higher identification scores in database searches. Interestingly, SGT was found to also generate nonspecific peptides whose sequencing by MALDI-TOF/TOF tandem mass spectrometry (MS/MS) revealed a partial F-X, Y-X, and W-X cleavage specificity. To suppress autolysis, either arginine or arginine plus lysine residues in SGT were modified by chemical reagents. In consequence, the autolytic pattern of SGT was reduced significantly, but specific activity dropped dramatically. As demonstrated by relative quantification of peptides at different times, SGT is more stable at 37 degrees C than is its bovine counterpart. We conclude that SGT represents a convenient alternative for proteomic applications involving protein digestion. Moreover, parallel digestions of sample aliquots by SGT and BT provide the possibility of combining partially different results (unique matching peptides) to improve protein identification.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteoma/análisis , Streptomyces griseus/enzimología , Tripsina/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Estabilidad de Enzimas , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Mapeo Peptídico , Proteoma/química , Proteoma/metabolismo , Proteómica/métodos , Reproducibilidad de los Resultados , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , TemperaturaRESUMEN
The anaphase-promoting complex (APC/C) is a large ubiquitin-protein ligase which controls progression through anaphase by triggering the degradation of cell cycle regulators such as securin and B-type cyclins. The APC/C is an unusually complex ligase containing at least 10 different, evolutionarily conserved components. In contrast to APC/C's role in cell cycle regulation little is known about the functions of individual subunits and how they might interact with each other. Here, we have analyzed Swm1/Apc13, a small subunit recently identified in the budding yeast complex. Database searches revealed proteins related to Swm1/Apc13 in various organisms including humans. Both the human and the fission yeast homologues are associated with APC/C subunits, and they complement the phenotype of an SWM1 deletion mutant of budding yeast. Swm1/Apc13 promotes the stable association with the APC/C of the essential subunits Cdc16 and Cdc27. Accordingly, Swm1/Apc13 is required for ubiquitin ligase activity in vitro and for the timely execution of APC/C-dependent cell cycle events in vivo.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Subunidades de Proteína/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Secuencia de Aminoácidos , Ciclosoma-Complejo Promotor de la Anafase , Animales , Subunidad Apc3 del Ciclosoma-Complejo Promotor de la Anafase , Ciclo Celular/fisiología , Cromátides/metabolismo , ADN Polimerasa III , Evolución Molecular , Prueba de Complementación Genética , Humanos , Meiosis/fisiología , Datos de Secuencia Molecular , Subunidades de Proteína/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe , Alineación de Secuencia , Complejos de Ubiquitina-Proteína Ligasa/química , Complejos de Ubiquitina-Proteína Ligasa/genética , Ubiquitina-Proteína LigasasRESUMEN
We studied sequence-dependent retention properties of synthetic 5'-terminal phosphate absent trinucleotides containing adenine, guanine and thymine through reversed-phase liquid chromatography (RPLC) and QSRR modelling. We investigated the influence of separation conditions, namely mobile phase composition (ion interaction agent content, pH and organic constituent content), on sequence-dependent separation by means of ion-interaction RPLC (II-RPLC) using two types of models: experimental design-artificial neural networks (ED-ANN), and linear regression based on molecular dynamics data. The aim was to determine those properties of the above-mentioned analytes responsible for the retention dependence of the sequence. Our results show that there is a deterministic relation between sequence and II-RPLC retention properties of the studied trinucleotides. Further, we can conclude that the higher the content of ion-interaction agent in the mobile phase, the more prominent these properties are. We also show that if we approximate the polar component of solvation energy in QSRR by the electrostatic work in transferring molecules from vacuum to water, and the non-polar component by the solvent accessible surface area, these parameters best describe the retention properties of trinucleotides. There are some exceptions to this finding, namely sequences 5'-NAN-3', 5'-ANN-3', 5'-TGN-3', 5'-NTA-3'and 5'-NGA-3' (N stands for generic nucleotide). Their role is still unknown, but since linear regression including these specific constellations showed a higher observable variance coverage than the model with only the basic descriptors, we may assume that solvent-analyte interactions are responsible for the exceptional behaviour of 5'-NAN-3' & 5'-ANN-3' trinucleotides and some intramolecular interactions of neighbouring nucleobases for 5'-TGN-3', 5'-NTA-3'and 5'-NGA-3' trinucleotides.
Asunto(s)
Oligonucleótidos/aislamiento & purificación , Adenina/análogos & derivados , Adenina/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Cromatografía de Fase Inversa , Guanina/análogos & derivados , Guanina/aislamiento & purificación , Simulación de Dinámica Molecular , Redes Neurales de la Computación , Relación Estructura-Actividad Cuantitativa , Solventes , Electricidad Estática , Timina/análogos & derivados , Timina/aislamiento & purificación , AguaRESUMEN
The study of experimental design conjunction with artificial neural networks for optimisation of isocratic ion-pair reverse phase HPLC separation of neuroprotective peptides is reported. Different types of experimental designs (full-factorial, fractional) were studied as suitable input and output data sources for ANN training and examined on mixtures of humanin derivatives. The independent input variables were: composition of mobile phase, including its pH, and column temperature. In case of a simple mixture of two peptides, the retention time of the most retentive component and resolution were used as the dependent variables (outputs). In case of a complex mixture with unknown number of components, number of peaks, sum of resolutions and retention time of ultimate peak were considered as output variables. Fractional factorial experimental design has been proved to produce sufficient input data for ANN approximation and thus further allowed decreasing the number of experiments necessary for optimisation. After the optimal separation conditions were found, fractions with peptides were collected and their analysis using off-line matrix assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF-MS) was performed.
Asunto(s)
Fraccionamiento Químico/métodos , Cromatografía Líquida de Alta Presión/métodos , Redes Neurales de la Computación , Sustitución de Aminoácidos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/aislamiento & purificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The extent of the DNA methylation of genomic DNA as well as the methylation pattern of many gene-regulatory areas are important aspects with regard to the state of genetic information, especially their expression. There is growing evidence that aberrant methylation is associated with many serious pathological consequences. As genetic research advances, many different approaches have been employed to determine the overall level of DNA methylation in a genome or to reveal the methylation state of particular nucleotide residues, starting from semiquantitative methods up to new and powerful techniques. In this paper, the currently employed techniques are reviewed both from the point of view of their relevance in genomic research and of their analytical application. The methods discussed include approaches based on chromatographic separation (thin-layer chromatography, high-performance liquid chromatography, affinity chromatography), separation in an electric field (capillary electrophoresis, gel electrophoresis in combination with methylation-sensitive restriction enzymes and/or specific sequencing protocols), and some other methodological procedures (mass spectrometry, methyl accepting capacity assay and immunoassays).
Asunto(s)
Biomarcadores , Citosina/análogos & derivados , Citosina/análisis , Metilación de ADN , 5-Metilcitosina , Cromatografía/métodos , Electroforesis/métodos , Espectrometría de Masas/métodosRESUMEN
The yeast phosphatidylinositol 4-kinase Pik1p is essential for proliferation, and it controls Golgi homeostasis and transport of newly synthesized proteins from this compartment. At the Golgi, phosphatidylinositol 4-phosphate recruits multiple cytosolic effectors involved in formation of post-Golgi transport vesicles. A second pool of catalytically active Pik1p localizes to the nucleus. The physiological significance and regulation of this dual localization of the lipid kinase remains unknown. Here, we show that Pik1p binds to the redundant 14-3-3 proteins Bmh1p and Bmh2p. We provide evidence that nucleocytoplasmic shuttling of Pik1p involves phosphorylation and that 14-3-3 proteins bind Pik1p in the cytoplasm. Nutrient deprivation results in relocation of Pik1p from the Golgi to the nucleus and increases the amount of Pik1p-14-3-3 complex, a process reversed upon restored nutrient supply. These data suggest a role of Pik1p nucleocytoplasmic shuttling in coordination of biosynthetic transport from the Golgi with nutrient signaling.
Asunto(s)
1-Fosfatidilinositol 4-Quinasa/metabolismo , Proteínas 14-3-3/metabolismo , Núcleo Celular/enzimología , Aparato de Golgi/enzimología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , 1-Fosfatidilinositol 4-Quinasa/química , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Proliferación Celular , Alimentos , Modelos Biológicos , Datos de Secuencia Molecular , Complejos Multiproteicos/metabolismo , Mutación/genética , Fosforilación , Fosfoserina/metabolismo , Unión Proteica , Proteínas de Saccharomyces cerevisiae/química , Red trans-Golgi/enzimologíaRESUMEN
Conjugating bovine trypsin with oligosaccharides maltotriose, raffinose and stachyose increased its thermostability and suppressed autolysis, without affecting its cleavage specificity. These conjugates accelerated the digestion of protein substrates both in solution and in gel, compared to commonly used unmodified and methylated trypsins.
Asunto(s)
Tripsina/química , Animales , Bovinos , Citocromos c/química , Estabilidad de Enzimas , Fructosa-Bifosfato Aldolasa/química , Geles , Calefacción , Mioglobina/química , Oligosacáridos/química , Proteómica , Rafinosa/química , Albúmina Sérica Bovina/química , Soluciones , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Trisacáridos/químicaRESUMEN
In-gel digestion of proteins isolated by gel electrophoresis is a cornerstone of mass spectrometry (MS)-driven proteomics. The 10-year-old recipe by Shevchenko et al. has been optimized to increase the speed and sensitivity of analysis. The protocol is for the in-gel digestion of both silver and Coomassie-stained protein spots or bands and can be followed by MALDI-MS or LC-MS/MS analysis to identify proteins at sensitivities better than a few femtomoles of protein starting material.
Asunto(s)
Proteínas/metabolismo , Proteómica/métodos , Electroforesis , Espectrometría de MasasRESUMEN
Syntheses of conjugates of garden pea (Pisum sativum) and grass pea (Lathyrus sativus) amine oxidases (PSAO and GPAO respectively) with BCD (beta-cyclodextrin), performed to improve the thermostability of the enzymes, are described in the present study. Periodate-oxidized BCD reacted with the enzyme proteins via free primary amino groups in a buffered solution containing cyanoborohydride as a reductant. Although the specific activities of PSAO and GPAO partially decreased after modification, Km values determined for the best diamine substrates remained almost unchanged. Both the BCD conjugates could be incubated at 65 degrees C for 30 min without considerable inactivation, and the residual activity remained detectable even after incubation at 75 degrees C. The conjugates contained approx. 30% of neutral sugars. Molecular masses of BCD-PSAO and BCD-GPAO (180 kDa), as estimated by gel-permeation chromatography, were higher compared with the value of 145 kDa for the native enzymes. This was in good correlation with the number of modified lysine residues determined by a spectrophotometric method. Peptide mass fingerprints of tryptic digests of BCD-PSAO and BCD-GPAO were less specific than those of the native enzymes when compared with the database sequence of PSAO. As a consequence of the modification, many unidentified peaks were observed in the digests of the studied conjugates that were not seen in the digests of native PSAO and GPAO. Only some of these peaks overlapped between BCD-PSAO and BCD-GPAO. The BCD conjugates described in the present study represent suitable candidates for biotechnological applications, e.g. in analyses using biosensors, which might benefit from increased storage stability and amine oxidation at high temperatures.
Asunto(s)
Lathyrus/enzimología , Pisum sativum/enzimología , beta-Ciclodextrinas/química , Activación Enzimática , Estabilidad de Enzimas , Isoenzimas/química , Relación Estructura-Actividad , Especificidad por Sustrato , TemperaturaRESUMEN
Cohesion established between sister chromatids during pre-meiotic DNA replication mediates two rounds of chromosome segregation. The first division is preceded by an extended prophase wherein homologous chromosomes undergo recombination. The persistence of cohesion during prophase is essential for recombination and both meiotic divisions. Here we show that Mnd2, a subunit of the anaphase-promoting complex (APC/C) from budding yeast, is essential to prevent premature destruction of cohesion in meiosis. During S- and prophase, Mnd2 prevents activation of the APC/C by a meiosis-specific activator called Ama1. In cells lacking Mnd2 the APC/C-Ama1 enzyme triggers degradation of Pds1, which causes premature sister chromatid separation due to unrestrained separase activity. In vitro, Mnd2 inhibits ubiquitination of Pds1 by APC/C-Ama1 but not by other APC/C holo-enzymes. We conclude that chromosome segregation in meiosis depends on the selective inhibition of a meiosis-specific form of the APC/C.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Segregación Cromosómica/fisiología , Meiosis/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Anafase/genética , Anafase/fisiología , Ciclosoma-Complejo Promotor de la Anafase , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/genética , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/metabolismo , Proteínas Cdc20 , Proteínas de Ciclo Celular/genética , Cromátides/genética , Cromátides/metabolismo , Segregación Cromosómica/genética , Endopeptidasas/metabolismo , Meiosis/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Desnaturalización Proteica/fisiología , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Securina , Separasa , Complejos de Ubiquitina-Proteína Ligasa/genéticaRESUMEN
A combination of nanoelectrospray tandem mass spectrometry and (18)O-labeled peptide internal standards was applied for the absolute quantification of proteins from their in-solution and in-gel tryptic digests. Although absolute quantification from in-solution digests was accurate, we observed that in-gel digestion compromised the quantification accuracy by affecting the recovery of individual peptides and, therefore, the provided estimates might be strongly influenced by the selection of reference peptides. Under optimized experimental conditions, it was possible to provide a semiquantitative estimate of the absolute amount of gel separated proteins within better than 50% error margin.
Asunto(s)
Electroforesis en Gel de Poliacrilamida/métodos , Proteínas/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Proteínas/química , SolucionesRESUMEN
We have developed a dried-droplet probe preparation method for peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), which uses AnchorChip targets and alpha-cyano-4-hydroxycinnamic acid (CHCA) as a matrix. Upon drying of a matrix and analyte mixture on the AnchorChip, salts and low molecular weight contaminants were pooled at the hydrophilic metal anchor, whereas 10-50 microm matrix/peptide crystals firmly adhered at the surface of a hydrophobic polymer and the entire target could be subsequently washed by submerging it in 5% formic acid for 2-3 min. Epifluorescence microscopy suggested that peptides were completely co-localized with CHCA crystals at the AnchorChip surface. Fluorescent images of the probes were of good contrast and were background-free, compared with images taken by a video camera built into the ion source. CHCA/peptide crystals were easy to recognize at the surface and peptide mass maps were acquired from them without further adjustment of the position of the laser beam. These crystals were remarkably stable towards the laser depletion and almost no matrix-related ions were typically observed in the low m/z region of peptide mass maps. The sensitivity of the peptide mass mapping was at the low-femtomole level.
Asunto(s)
Mapeo Peptídico/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Bovinos , Cristalización , Fluorescencia , Microscopía Fluorescente/métodos , Microscopía por Video , Mapeo Peptídico/instrumentación , Sensibilidad y Especificidad , Albúmina Sérica Bovina/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/instrumentaciónRESUMEN
Kinetics of in-gel digestion of proteins by modified and native trypsins was studied by MALDI TOF mass spectrometry using 18O-labeled peptides as internal standards. The effect of the temperature, enzyme concentration, digestion time, and surface area of gel pieces on the yield of digestion products was characterized. Based on the kinetic data, we developed a protocol that enabled the identification of gel-separated proteins with 30-min digestion time without compromising the peptide yield and the sensitivity compared to conventional protocols that typically rely upon overnight enzymatic cleavage. The accelerated digestion protocol was tested in identification of more than 120 proteins from budding and fission yeasts at the subpicomole level.
Asunto(s)
Proteínas/análisis , Proteínas/metabolismo , Proteómica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Electroforesis en Gel de Poliacrilamida , Proteínas Fúngicas/análisis , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Cinética , Proteínas/química , Saccharomycetales/química , Schizosaccharomyces/química , Factores de Tiempo , Tripsina/metabolismoRESUMEN
1,5-diamino-2-pentyne (DAPY) was found to be a weak substrate of grass pea (Lathyrus sativus, GPAO) and sainfoin (Onobrychis viciifolia, OVAO) amine oxidases. Prolonged incubations, however, resulted in irreversible inhibition of both enzymes. For GPAO and OVAO, rates of inactivation of 0.1-0.3 min(-1) were determined, the apparent KI values (half-maximal inactivation) were of the order of 10(-5) m. DAPY was found to be a mechanism-based inhibitor of the enzymes because the substrate cadaverine significantly prevented irreversible inhibition. The N1-methyl and N5-methyl analogs of DAPY were tested with GPAO and were weaker inactivators (especially the N5-methyl) than DAPY. Prolonged incubations of GPAO or OVAO with DAPY resulted in the appearance of a yellow-brown chromophore (lambda(max) = 310-325 nm depending on the working buffer). Excitation at 310 nm was associated with emitted fluorescence with a maximum at 445 nm, suggestive of extended conjugation. After dialysis, the color intensity was substantially decreased, indicating the formation of a low molecular mass secondary product of turnover. The compound provided positive reactions with ninhydrin, 2-aminobenzaldehyde and Kovacs' reagents, suggesting the presence of an amino group and a nitrogen-containing heterocyclic structure. The secondary product was separated chromatographically and was found not to irreversibly inhibit GPAO. MS indicated an exact molecular mass (177.14 Da) and molecular formula (C10H15N3). Electrospray ionization- and MALDI-MS/MS analyses yielded fragment mass patterns consistent with the structure of a dihydropyridine derivative of DAPY. Finally, N-(2,3-dihydropyridinyl)-1,5-diamino-2-pentyne was identified by means of 1H- and 13C-NMR experiments. This structure suggests a lysine modification chemistry that could be responsible for the observed inactivation.