RESUMO
The bromodomain and PHD-finger containing transcription factor (BPTF) is part of the nucleosome remodeling factor (NURF) complex and has been implicated in multiple cancer types. Here, we report the discovery of a potent and selective chemical probe targeting the bromodomain of BPTF with an attractive pharmacokinetic profile enabling cellular and inâ vivo experiments in mice. Microarray-based transcriptomics in presence of the probe in two lung cancer cell lines revealed only minor effects on the transcriptome. Profiling against a panel of cancer cell lines revealed that the antiproliferative effect does not correlate with BPTF dependency score in depletion screens. Both observations and the multi-domain architecture of BPTF suggest that depleting the protein by proteolysis targeting chimeras (PROTACs) could be a promising strategy to target cancer cell proliferation. We envision that the presented chemical probe and the related negative control will enable the research community to further explore scientific hypotheses with respect to BPTF bromodomain inhibition.
Assuntos
Neoplasias Pulmonares , Fatores de Transcrição , Animais , Camundongos , Proliferação de Células , Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Targeted protein degradation offers an alternative modality to classical inhibition and holds the promise of addressing previously undruggable targets to provide novel therapeutic options for patients. Heterobifunctional molecules co-recruit a target protein and an E3 ligase, resulting in ubiquitylation and proteosome-dependent degradation of the target. In the clinic, the oral route of administration is the option of choice but has only been achieved so far by CRBN- recruiting bifunctional degrader molecules. We aimed to achieve orally bioavailable molecules that selectively degrade the BAF Chromatin Remodelling complex ATPase SMARCA2 over its closely related paralogue SMARCA4, to allow in vivo evaluation of the synthetic lethality concept of SMARCA2 dependency in SMARCA4-deficient cancers. Here we outline structure- and property-guided approaches that led to orally bioavailable VHL-recruiting degraders. Our tool compound, ACBI2, shows selective degradation of SMARCA2 over SMARCA4 in ex vivo human whole blood assays and in vivo efficacy in SMARCA4-deficient cancer models. This study demonstrates the feasibility for broadening the E3 ligase and physicochemical space that can be utilised for achieving oral efficacy with bifunctional molecules.
Assuntos
Adenosina Trifosfatases , Fatores de Transcrição , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteólise , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
Activating mutations in KRAS are the most frequent oncogenic alterations in cancer. The oncogenic hotspot position 12, located at the lip of the switch II pocket, offers a covalent attachment point for KRASG12C inhibitors. To date, KRASG12C inhibitors have been discovered by first covalently binding to the cysteine at position 12 and then optimizing pocket binding. We report on the discovery of the in vivo active KRASG12C inhibitor BI-0474 using a different approach, in which small molecules that bind reversibly to the switch II pocket were identified and then optimized for non-covalent binding using structure-based design. Finally, the Michael acceptor containing warhead was attached. Our approach offers not only an alternative approach to discovering KRASG12C inhibitors but also provides a starting point for the discovery of inhibitors against other oncogenic KRAS mutants.
Assuntos
Neoplasias , Proteínas Proto-Oncogênicas p21(ras) , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Genes ras , Mutação , Neoplasias/genética , CisteínaRESUMO
Phosphoglycerate dehydrogenase (PHGDH) is known to be the rate-limiting enzyme in the serine synthesis pathway in humans. It converts glycolysis-derived 3-phosphoglycerate to 3-phosphopyruvate in a co-factor-dependent oxidation reaction. Herein, we report the discovery of BI-4916, a prodrug of the co-factor nicotinamide adenine dinucleotide (NADH/NAD+)-competitive PHGDH inhibitor BI-4924, which has shown high selectivity against the majority of other dehydrogenase targets. Starting with a fragment-based screening, a subsequent hit optimization using structure-based drug design was conducted to deliver a single-digit nanomolar lead series and to improve potency by 6 orders of magnitude. To this end, an intracellular ester cleavage mechanism of the ester prodrug was utilized to achieve intracellular enrichment of the actual carboxylic acid based drug and thus overcome high cytosolic levels of the competitive cofactors NADH/NAD+.
Assuntos
Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Fosfoglicerato Desidrogenase/antagonistas & inibidores , Serina/antagonistas & inibidores , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Indóis/síntese química , Indóis/química , Modelos Moleculares , Estrutura Molecular , Fosfoglicerato Desidrogenase/metabolismo , Serina/biossíntese , Relação Estrutura-AtividadeRESUMO
Here, we report the fragment-based discovery of BI-9321, a potent, selective and cellular active antagonist of the NSD3-PWWP1 domain. The human NSD3 protein is encoded by the WHSC1L1 gene located in the 8p11-p12 amplicon, frequently amplified in breast and squamous lung cancer. Recently, it was demonstrated that the PWWP1 domain of NSD3 is required for the viability of acute myeloid leukemia cells. To further elucidate the relevance of NSD3 in cancer biology, we developed a chemical probe, BI-9321, targeting the methyl-lysine binding site of the PWWP1 domain with sub-micromolar in vitro activity and cellular target engagement at 1 µM. As a single agent, BI-9321 downregulates Myc messenger RNA expression and reduces proliferation in MOLM-13 cells. This first-in-class chemical probe BI-9321, together with the negative control BI-9466, will greatly facilitate the elucidation of the underexplored biological function of PWWP domains.
Assuntos
Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , Sistemas CRISPR-Cas , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Domínios Proteicos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
The transcription factor BCL6 is a known driver of oncogenesis in lymphoid malignancies, including diffuse large B cell lymphoma (DLBCL). Disruption of its interaction with transcriptional repressors interferes with the oncogenic effects of BCL6. We used a structure-based drug design to develop highly potent compounds that block this interaction. A subset of these inhibitors also causes rapid ubiquitylation and degradation of BCL6 in cells. These compounds display significantly stronger induction of expression of BCL6-repressed genes and anti-proliferative effects than compounds that merely inhibit co-repressor interactions. This work establishes the BTB domain as a highly druggable structure, paving the way for the use of other members of this protein family as drug targets. The magnitude of effects elicited by this class of BCL6-degrading compounds exceeds that of our equipotent non-degrading inhibitors, suggesting opportunities for the development of BCL6-based lymphoma therapeutics.
Assuntos
Proteólise , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Concentração Inibidora 50 , Cinética , Modelos Moleculares , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-6/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-6/química , Pirimidinas/farmacologia , Relação Estrutura-Atividade , Ubiquitinação/efeitos dos fármacosRESUMO
Embryonic stem cell (ESC) cultures display a heterogeneous gene expression profile, ranging from a pristine naïve pluripotent state to a primed epiblast state. Addition of inhibitors of GSK3ß and MEK (so-called 2i conditions) pushes ESC cultures toward a more homogeneous naïve pluripotent state, but the molecular underpinnings of this naïve transition are not completely understood. Here, we demonstrate that DAZL, an RNA-binding protein known to play a key role in germ-cell development, marks a subpopulation of ESCs that is actively transitioning toward naïve pluripotency. Moreover, DAZL plays an essential role in the active reprogramming of cytosine methylation. We demonstrate that DAZL associates with mRNA of Tet1, a catalyst of 5-hydroxylation of methyl-cytosine, and enhances Tet1 mRNA translation. Overexpression of DAZL in heterogeneous ESC cultures results in elevated TET1 protein levels as well as increased global hydroxymethylation. Conversely, null mutation of Dazl severely stunts 2i-mediated TET1 induction and hydroxymethylation. Our results provide insight into the regulation of the acquisition of naïve pluripotency and demonstrate that DAZL enhances TET1-mediated cytosine hydroxymethylation in ESCs that are actively reprogramming to a pluripotent ground state.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Murinas/fisiologia , Células-Tronco Pluripotentes/fisiologia , Proteínas Proto-Oncogênicas/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Diferenciação Celular , Reprogramação Celular , Citosina/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Camadas Germinativas/fisiologia , Camundongos , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , TranscriptomaRESUMO
Embryonic stem (ES) cells are pluripotent and characterized by open chromatin and high transcription levels, achieved through auto-regulatory and feed-forward transcription factor loops. ES-cell identity is maintained by a core of factors including Oct4 (also known as Pou5f1), Sox2, Klf4, c-Myc (OSKM) and Nanog, and forced expression of the OSKM factors can reprogram somatic cells into induced pluripotent stem cells (iPSCs) resembling ES cells. These gene-specific factors for RNA-polymerase-II-mediated transcription recruit transcriptional cofactors and chromatin regulators that control access to and activity of the basal transcription machinery on gene promoters. How the basal transcription machinery is involved in setting and maintaining the pluripotent state is unclear. Here we show that knockdown of the transcription factor IID (TFIID) complex affects the pluripotent circuitry in mouse ES cells and inhibits reprogramming of fibroblasts. TFIID subunits and the OSKM factors form a feed-forward loop to induce and maintain a stable transcription state. Notably, transient expression of TFIID subunits greatly enhanced reprogramming. These results show that TFIID is critical for transcription-factor-mediated reprogramming. We anticipate that, by creating plasticity in gene expression programs, transcription complexes such as TFIID assist reprogramming into different cellular states.
Assuntos
Células-Tronco Pluripotentes/metabolismo , Fator de Transcrição TFIID/metabolismo , Transcrição Gênica , Animais , Reprogramação Celular/genética , Cromatina/genética , Cromatina/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Masculino , Camundongos , Células-Tronco Pluripotentes/citologia , Regiões Promotoras Genéticas/genética , RNA Polimerase II/metabolismo , Fatores Associados à Proteína de Ligação a TATA/deficiência , Fatores Associados à Proteína de Ligação a TATA/genética , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Proteína de Ligação a TATA-Box/metabolismo , Fator de Transcrição TFIID/deficiência , Fator de Transcrição TFIID/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The TATA binding protein (TBP) plays a pivotal role in RNA polymerase II (Pol II) transcription through incorporation into the TFIID and B-TFIID complexes. The role of mammalian B-TFIID composed of TBP and B-TAF1 is poorly understood. Using a complementation system in genetically modified mouse cells where endogenous TBP can be conditionally inactivated and replaced by exogenous mutant TBP coupled to tandem affinity purification and mass spectrometry, we identify two TBP mutations, R188E and K243E, that disrupt the TBP-BTAF1 interaction and B-TFIID complex formation. Transcriptome and ChIP-seq analyses show that loss of B-TFIID does not generally alter gene expression or genomic distribution of TBP, but positively or negatively affects TBP and/or Pol II recruitment to a subset of promoters. We identify promoters where wild-type TBP assembles a partial inactive preinitiation complex comprising B-TFIID, TFIIB and Mediator complex, but lacking TFIID, TFIIE and Pol II. Exchange of B-TFIID in wild-type cells for TFIID in R188E and K243E mutant cells at these primed promoters completes preinitiation complex formation and recruits Pol II to activate their expression. We propose a novel regulatory mechanism involving formation of a partial preinitiation complex comprising B-TFIID that primes the promoter for productive preinitiation complex formation in mammalian cells.
Assuntos
Fatores Associados à Proteína de Ligação a TATA/metabolismo , Proteína de Ligação a TATA-Box/metabolismo , Transcrição Gênica , Animais , Regulação da Expressão Gênica , Teste de Complementação Genética , Genoma , Camundongos , Mutação , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Fatores Associados à Proteína de Ligação a TATA/química , Proteína de Ligação a TATA-Box/química , Proteína de Ligação a TATA-Box/genética , Fator de Transcrição TFIID/metabolismo , TranscriptomaRESUMO
Protein modifications are biologically important events that may be studied by mass spectrometry-based high-throughput proteome analyses. In recent years, several new technologies have emerged that have widened and deepened the targeted analysis of one important, albeit functionally ill-defined modification, namely protein acetylation. This modification can take place both co- and post-translationally by the transfer of acetyl groups under the catalysis of acetyltransferases. The acetyl group can modify either the α-amino group at the N-terminus, so-called N-terminal acetylation, or the ε-amino group on the side chain of lysine residues. Here, we review several emerging targeted technologies to chart both N-terminal acetylation as well as acetylation at the lysine side chain, on a proteome-wide scale, highlighting in particular studies that have expanded the biological knowledge on the appearance and function of these common but functionally still less investigated co- and post-translational modifications.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Proteínas/química , Proteômica/métodos , Acetilação , Animais , Cromatografia Líquida , Humanos , Técnicas de Sonda Molecular , Processamento de Proteína Pós-Traducional , Proteínas/análise , Proteínas/metabolismoRESUMO
Recent emergence of new mass spectrometry techniques (e.g. electron transfer dissociation, ETD) and improved availability of additional proteases (e.g. Lys-N) for protein digestion in high-throughput experiments raised the challenge of designing new algorithms for interpreting the resulting new types of tandem mass (MS/MS) spectra. Traditional MS/MS database search algorithms such as SEQUEST and Mascot were originally designed for collision induced dissociation (CID) of tryptic peptides and are largely based on expert knowledge about fragmentation of tryptic peptides (rather than machine learning techniques) to design CID-specific scoring functions. As a result, the performance of these algorithms is suboptimal for new mass spectrometry technologies or nontryptic peptides. We recently proposed the generating function approach (MS-GF) for CID spectra of tryptic peptides. In this study, we extend MS-GF to automatically derive scoring parameters from a set of annotated MS/MS spectra of any type (e.g. CID, ETD, etc.), and present a new database search tool MS-GFDB based on MS-GF. We show that MS-GFDB outperforms Mascot for ETD spectra or peptides digested with Lys-N. For example, in the case of ETD spectra, the number of tryptic and Lys-N peptides identified by MS-GFDB increased by a factor of 2.7 and 2.6 as compared with Mascot. Moreover, even following a decade of Mascot developments for analyzing CID spectra of tryptic peptides, MS-GFDB (that is not particularly tailored for CID spectra or tryptic peptides) resulted in 28% increase over Mascot in the number of peptide identifications. Finally, we propose a statistical framework for analyzing multiple spectra from the same precursor (e.g. CID/ETD spectral pairs) and assigning p values to peptide-spectrum-spectrum matches.
Assuntos
Bases de Dados de Proteínas , Espectrometria de Massas em Tandem/métodos , Algoritmos , Linhagem Celular , Humanos , Mapeamento de Peptídeos , Tripsina/químicaRESUMO
Isobaric stable isotope labeling of peptides using iTRAQ is an important method for MS based quantitative proteomics. Traditionally, quantitative analysis of iTRAQ labeled peptides has been confined to beam-type instruments because of the weak detection capabilities of ion traps for low mass ions. Recent technical advances in fragmentation techniques on linear ion traps and the hybrid linear ion trap-orbitrap allow circumventing this limitation. Namely, PQD and HCD facilitate iTRAQ analysis on these instrument types. Here we report a method for iTRAQ-based relative quantification on the ETD enabled LTQ Orbitrap XL, which is based on parallel peptide quantification and peptide identification. iTRAQ reporter ion generation is performed by HCD, while CID and ETD provide peptide identification data in parallel in the LTQ ion trap. This approach circumvents problems accompanying iTRAQ reporter ion generation with ETD and allows quantitative, decision tree-based CID/ETD experiments. Furthermore, the use of HCD solely for iTRAQ reporter ion read out significantly reduces the number of ions needed to obtain informative spectra, which significantly reduces the analysis time. Finally, we show that integration of this method, both with existing CID and ETD methods as well as with existing iTRAQ data analysis workflows, is simple to realize. By applying our approach to the analysis of the synapse proteome from human brain biopsies, we demonstrate that it outperforms a latest generation MALDI TOF/TOF instrument, with improvements in both peptide and protein identification and quantification. Conclusively, our work shows how HCD, CID and ETD can be beneficially combined to enable iTRAQ-based quantification on an ETD-enabled LTQ Orbitrap XL.
Assuntos
Peptídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Cromatografia de Fase Reversa , Humanos , Marcação por Isótopo , Peptídeos/químicaRESUMO
The Saccharomyces cerevisiae Spt-Ada-Gcn5 acetyltransferase (SAGA) protein complex is a coactivator for transcription by RNA polymerase II and has various activities, including acetylation and deubuiqitination of histones and recruitment of TATA-binding protein to promoters. The Spt7p subunit is subject to proteolytic cleavage at its C terminus resulting in removal of the Spt8p-binding domain and generation of the SAGA-related SALSA/SAGA-like (SLIK) protein complex. Here, we report identification of the protease responsible for this cleavage. Screening of a protease knock-out collection revealed PEP4 to be required for cleavage of Spt7p within SAGA in vitro. Endogenous formation of truncated Spt7p was abolished in cells lacking PEP4. Purified Pep4p but not catalytic dead mutant Pep4p or unrelated Prc1p protease specifically cleaved Spt7p within SAGA into SLIK-related Spt7p. Interestingly, SAGA lacking Spt8p was more sensitive to Pep4p-mediated truncation of Spt7p, suggesting that Spt8p counteracted its own release from SAGA. Strains mimicking constitutive SLIK formation showed increased resistance to rapamycin treatment, suggesting a role for SLIK in regulating cellular responses to nutrient stress.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Transativadores/metabolismo , Ácido Aspártico Endopeptidases/deficiência , Ácido Aspártico Endopeptidases/genética , Farmacorresistência Fúngica , Técnicas de Inativação de Genes , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Sirolimo/farmacologia , Transativadores/químicaRESUMO
In large scale mass spectrometry-based phosphoproteomics, a current bottleneck is the unambiguous assignment of the phosphorylation site within the peptide. An additional problem is that it has been reported that under conditions wherein peptide ions are collisionally activated the phosphate group may migrate to a nearby phosphate group acceptor, thus causing ambiguity in site assignment. Here, we generated and analyzed a statistically significant number of phosphopeptides. Starting with a human cell lysate, we obtained via strong cation exchange fractionation nearly pure phosphopeptide pools from trypsin and Lys-N digestions. These pools were subjected to nano-LC-MS using an Orbitrap mass spectrometer that is equipped with both CID and electron transfer dissociation with supplemental activation (ETcaD) functionality. We configured a method to obtain sequentially both ETcaD and CID spectra for each peptide ion. We exploited the resistant nature of ETcaD toward rearrangement of phosphate groups to evaluate whether there is potentially phosphate group relocation occurring during CID. We evaluated a number of peptide and spectral annotation properties and found that for â¼75% of the sequenced phosphopeptides the assigned phosphosite was unmistakably identical for both the ETcaD and CID spectra. For the remaining 25% of the sequenced phosphopeptides, we also did not observe evident signs of relocation, but these peptides exhibited signs of ambiguity in site localization, predominantly induced by factors such as poor fragmentation, sequences causing inefficient fragmentation, and generally poor spectrum quality. Our data let us derive the conclusion that both for trypsin- and Lys-N-generated peptides there is little relocation of phosphate groups occurring during CID.
Assuntos
Fosfatos/química , Fosfopeptídeos/metabolismo , Transporte de Elétrons , Espectrometria de Massas , Fosfopeptídeos/química , FosforilaçãoRESUMO
The basal transcription factor TFIID and the chromatin-modifying complex SAGA, which have several subunits in common, are crucial for transcription regulation. Here, we describe an in-depth profiling of post-translational modifications (PTMs) on both TFIID and SAGA from yeast. We took a multipronged approach using high-resolution mass spectrometry (LC-MS) in combination with the proteases Trypsin, Chymotrypsin and Glu-C. The cumulative peptide identification data, at a false discovery rate <1%, allowed us to cover most TFIID and SAGA subunit sequences to near completion. Additionally, for TFIID/SAGA subunits, we identified 118/102 unique phosphorylated and 54/61 unique lysine acetylated sites. Especially, several lysine residues on the SAGA subunits Spt7p and Sgf73p were found to be acetylated. Using a spectral counting approach, we found that the shared subunit TAF5p is phosphorylated to a significant greater extent in SAGA than in TFIID. Finally, we were able to map for the first time the cleavage site in Spt7p that is related to formation of the SAGA-like complex SLIK/SALSA. In general, our combination of tandem affinity enrichment, digestion with different proteases, extensive prefractionation and high-resolution LC-MS identifies a large number of PTMs of TFIID and SAGA/SLIK that might aid in future functional studies on these transcription factors.
Assuntos
Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae , Transativadores , Fator de Transcrição TFIID/química , Fator de Transcrição TFIID/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia Líquida/métodos , Regulação Fúngica da Expressão Gênica , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Humanos , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transativadores/química , Transativadores/metabolismo , Fator de Transcrição TFIID/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
In proteomics, proteolytic peptides are often chemically modified to improve MS analysis, peptide identification, and/or to enable protein/peptide quantification. It is known that such chemical modifications can alter peptide fragmentation in collision induced dissociation MS/MS. Here, we investigated the fragmentation behavior of such chemically modified peptides in MS/MS using the relatively new activation method electron transfer dissociation (ETD). We generated proteolytic peptides using the proteases Lys-N and trypsin and compared the fragmentation behavior of the unlabeled peptides with that of their chemically modified cognates. We investigated the effect of several commonly used modification reactions, namely, guanidination, dimethylation, imidazolinylation, and nicotinylation (ICPL). Of these guanidination and imidazolinylation specifically target the epsilon-amino groups of lysine residues in the peptides, whereas dimethylation and nicotinylation modify both N-termini and epsilon-amino groups of lysine residues. Dimethylation, guanidination, and particularly imidazolinylation of doubly charged Lys-N peptides resulted in a significant increase in peptide sequence coverage, resulting in more reliable peptide identification using ETD. This may be rationalized by the increased basicity and resulting positive charge at the N-termini of these peptides. Nicotinylation of the peptides, on the other hand, severely suppressed backbone fragmentation, hampering the use of this label in ETD based analysis. Doubly charged C-terminal lysine containing tryptic peptides also resulted in an enhanced observation of a single type of fragment ion series when guanidinated or imidazolinylated. These labels would thus facilitate the use of de novo sequencing strategies based on ETD for both arginine and lysine containing tryptic peptides. Since isotopic analogues of the labeling reagents applied in this work are commercially available, one can combine quantitation with improved ETD based peptide sequencing for both Lys-N and trypsin digested samples.