RESUMO
We investigated the utility of an SPE-MS/MS platform in combination with a modified SISCAPA workflow for chromatography-free MRM analysis of proteotypic peptides in digested human plasma. This combination of SISCAPA and SPE-MS/MS technology allows sensitive, MRM-based quantification of peptides from plasma digests with a sample cycle time of â¼7 s, a 300-fold improvement over typical MRM analyses with analysis times of 30-40 min that use liquid chromatography upstream of MS. The optimized system includes capture and enrichment to near purity of target proteotypic peptides using rigorously selected, high affinity, antipeptide monoclonal antibodies and reduction of background peptides using a novel treatment of magnetic bead immunoadsorbents. Using this method, we have successfully quantitated LPS-binding protein and mesothelin (concentrations of â¼5000 ng/mL and â¼10 ng/mL, respectively) in human plasma. The method eliminates the need for upstream liquid-chromatography and can be multiplexed, thus facilitating quantitative analysis of proteins, including biomarkers, in large sample sets. The method is ideal for high-throughput biomarker validation after affinity enrichment and has the potential for applications in clinical laboratories.
Assuntos
Proteínas Sanguíneas/análise , Ensaios de Triagem em Larga Escala/métodos , Espectrometria de Massas/métodos , Peptídeos/sangue , Software , Proteínas de Fase Aguda/análise , Anticorpos Monoclonais/química , Afinidade de Anticorpos , Biomarcadores/sangue , Proteínas de Transporte/análise , Cromatografia Líquida , Proteínas Ligadas por GPI/sangue , Humanos , Glicoproteínas de Membrana/análise , Mesotelina , Proteômica/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fatores de TempoRESUMO
Oxidative stress converts lipids into DNA-damaging agents. The genomic lesions formed include 1,N(6)-ethenoadenine (epsilonA) and 3,N(4)-ethenocytosine (epsilonC), in which two carbons of the lipid alkyl chain form an exocyclic adduct with a DNA base. Here we show that the newly characterized enzyme AlkB repairs epsilonA and epsilonC. The potent toxicity and mutagenicity of epsilonA in Escherichia coli lacking AlkB was reversed in AlkB(+) cells; AlkB also mitigated the effects of epsilonC. In vitro, AlkB cleaved the lipid-derived alkyl chain from DNA, causing epsilonA and epsilonC to revert to adenine and cytosine, respectively. Biochemically, epsilonA is epoxidized at the etheno bond. The epoxide is putatively hydrolyzed to a glycol, and the glycol moiety is released as glyoxal. These reactions show a previously unrecognized chemical versatility of AlkB. In mammals, the corresponding AlkB homologs may defend against aging, cancer and oxidative stress.
Assuntos
Adenina/análogos & derivados , Citosina/análogos & derivados , Adutos de DNA/metabolismo , Reparo do DNA , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Oxigenases de Função Mista/metabolismo , Acetaldeído/análogos & derivados , Acetaldeído/toxicidade , Adenina/química , Adenina/metabolismo , Citosina/química , Citosina/metabolismo , Adutos de DNA/química , Dano ao DNA , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Peroxidação de Lipídeos , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , MutagêneseRESUMO
DinB, the E. coli translesion synthesis polymerase, has been shown to bypass several N2-alkylguanine adducts in vitro, including N2-furfurylguanine, the structural analog of the DNA adduct formed by the antibacterial agent nitrofurazone. Recently, it was demonstrated that the Fe(II)- and α-ketoglutarate-dependent dioxygenase AlkB, a DNA repair enzyme, can dealkylate in vitro a series of N2-alkyguanines, including N2-furfurylguanine. The present study explored, head to head, the in vivo relative contributions of these two DNA maintenance pathways (replicative bypass vs. repair) as they processed a series of structurally varied, biologically relevant N2-alkylguanine lesions: N2-furfurylguanine (FF), 2-tetrahydrofuran-2-yl-methylguanine (HF), 2-methylguanine, and 2-ethylguanine. Each lesion was chemically synthesized and incorporated site-specifically into an M13 bacteriophage genome, which was then replicated in E. coli cells deficient or proficient for DinB and AlkB (4 strains in total). Biochemical tools were employed to analyze the relative replication efficiencies of the phage (a measure of the bypass efficiency of each lesion) and the base composition at the lesion site after replication (a measure of the mutagenesis profile of each lesion). The main findings were: 1) Among the lesions studied, the bulky FF and HF lesions proved to be strong replication blocks when introduced site-specifically on a single-stranded vector in DinB deficient cells. This toxic effect disappeared in the strains expressing physiological levels of DinB. 2) AlkB is known to repair N2-alkylguanine lesions in vitro; however, the presence of AlkB showed no relief from the replication blocks induced by FF and HF in vivo. 3) The mutagenic properties of the entire series of N2-alkyguanines adducts were investigated in vivo for the first time. None of the adducts were mutagenic under the conditions evaluated, regardless of the DinB or AlkB cellular status. Taken together, the data indicated that the cellular pathway to combat bulky N2-alkylguanine DNA adducts was DinB-dependent lesion bypass.
Assuntos
Proteínas de Escherichia coli/genética , Guanina/análogos & derivados , Oxigenases de Função Mista/genética , Bacteriófago M13/metabolismo , Adutos de DNA/química , Adutos de DNA/metabolismo , Dano ao DNA , Reparo do DNA , Escherichia coli/genética , Compostos Ferrosos/metabolismo , Genoma , Guanina/química , Mutagênese , Mutagênese Sítio-Dirigida , Oligonucleotídeos/genética , Resposta SOS em GenéticaRESUMO
Histone acetyltransferases (HATs) catalyze the transfer of an acetyl group from an acetyl-coenzyme A donor molecule to specific lysine residues within proteins. The acetylation state of proteins, particularly histones, is known to modulate their intermolecular binding properties and control various cellular processes, most notably transcriptional activation. In addition, deregulation of HAT activity has been linked to the development of a number of cancers; therefore, compounds that affect these enzymes have strong potential as therapeutic agents. The research presented here demonstrates three label-free HAT screening approaches, all based on the fast and direct measurement of one or more substrate-product pairs by high-throughput mass spectrometry techniques. The first approach involves monitoring all possible acetylation states of a peptide concurrently to measure HAT activity. The second approach measures acetylation reactions, on both peptides and whole protein substrates, via direct detection of the acetyl-coenzyme A cosubstrate and coenzyme A coproduct. Lastly, the authors demonstrate the ability to monitor directly the acetylation state of whole histone proteins in the same high-throughput manner using time-of-flight mass spectrometry. The generation of compound-mediated inhibition data using each of these techniques establishes mass spectrometry as a versatile, label-free, and biologically relevant screening approach to this challenging target class.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Histona Acetiltransferases/metabolismo , Acetilação/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Histonas/química , Espectrometria de Massas , Peptídeos/farmacologia , Proteína Supressora de Tumor p53/química , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
The sirtuin enzymes, a class of NAD(+)-dependent histone deacetylases, are a focal point of epigenetic research because of their roles in regulating gene expression and cellular differentiation by deacetylating histones and a host of transcription factors, including p53. Here, the authors present two label-free screening methodologies to study sirtuin activity using high-throughput mass spectrometry. The first method involves the detection of native peptides and provides a platform for more detailed mechanistic studies by enabling the concurrent and direct measurement of multiple modification states. The second method obviates the need for substrate-specific assay development by measuring the O-acetyl-ADP-ribose co-product formed by sirtuin-dependent deacetylation. Both methodologies were applied to investigating the deacetylation of multiple-peptide substrates by multiple-sirtuin enzymes. Kinetic data, including binding constants, inhibition, and, in some cases, activation, are demonstrated to correlate well, both between the methodologies and with previous literature precedent. In addition, the ability to monitor sirtuin activity via O-acetyl-ADP-ribose production permits experimentation on whole-protein substrates. The deacetylation of whole-histone proteins by SIRT3, and inhibition thereof, is presented and demonstrates the feasibility of screening sirtuins using more biologically relevant molecules.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Sirtuínas/análise , Sirtuínas/metabolismo , Acetilação/efeitos dos fármacos , Histonas/química , Humanos , Cinética , Espectrometria de Massas , O-Acetil-ADP-Ribose/análise , O-Acetil-ADP-Ribose/metabolismo , Peptídeos/farmacologia , Proteína Supressora de Tumor p53/químicaRESUMO
1,N(6)-ethanoadenine (EA) forms through the reaction of adenine in DNA with the antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea, a chemotherapeutic used to combat various brain, head, and neck tumors. Previous studies of the toxic and mutagenic properties of the DNA adduct EA have been limited to in vitro experiments using mammalian polymerases and have revealed the lesion to be both miscoding and genotoxic. This work explores lesion bypass and mutagenicity of EA replicated in vivo and demonstrates that EA is neither toxic nor mutagenic in wild-type Escherichia coli. Although the base excision repair glycosylase enzymes of both humans and E. coli possess a weak ability to act on the lesion in vitro, an in vivo repair pathway has not yet been demonstrated. Here we show that an enzyme mechanistically unrelated to DNA glycosylases, the adaptive response protein AlkB, is capable of acting on EA via its canonical mechanism of oxidative dealkylation. The reaction alleviates the unrepaired adduct's potent toxicity through metabolism at the C8 position (attached to N1 of adenine), producing a nontoxic and weakly mutagenic N(6) adduct. AlkB is shown here to be a geno-protective agent that reduces the toxicity of DNA damage by converting the primary adduct to a less toxic secondary product.