RESUMO
Herein, we describe the development of a fully automatable technology that features online coupling of high-pH RP separation with conventional low-pH RP separation in a two-dimensional capillary liquid chromatography (2-D LC) system for shotgun proteomics analyses. The complete analysis comprises 13 separation cycles, each involving transfer of the eluate from the first-dimension, high-pH RP separation onto the second RP dimension for further separation. The solvent strength increases across the 13 fractions (cycles) to elute all peptides for further resolution on the second-dimension, low-pH RP separation, each under identical gradient-elution conditions. The total run time per analysis is 52 h. In triplicate analyses of a lysate of mouse embryonic fibroblasts, we used this technology to identify 2431 non-redundant proteins, of which 50% were observed in all three replicates. A comparison of RP-RP 2-D LC and strong cation exchange-RP 2-D LC analyses reveals that the two technologies identify primarily different peptides, thereby underscoring the differences in their separation chemistries.
Assuntos
Cromatografia de Fase Reversa/métodos , Fragmentos de Peptídeos/química , Mapeamento de Peptídeos/métodos , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Automação Laboratorial , Linhagem Celular , Embrião não Mamífero , Desenho de Equipamento , Fibroblastos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Fragmentos de Peptídeos/análise , Proteínas/análise , Proteínas/química , Proteômica/instrumentação , Proteínas de Peixe-Zebra/análise , Proteínas de Peixe-Zebra/químicaRESUMO
In this paper, we describe an online combination of reversed-phase/reversed-phase (RP-RP) and porous graphitic carbon (PGC) liquid chromatography (LC) for multicomponent analysis of proteomics and glycoproteomics samples. The online RP-RP portion of this system provides comprehensive 2-D peptide separation based on sequence hydrophobicity at pH 2 and 10. Hydrophilic components (e.g. glycans, glycopeptides) that are not retained by RP are automatically diverted downstream to a PGC column for further trapping and separation. Furthermore, the RP-RP/PGC system can provide simultaneous extension of the hydropathy range and peak capacity for analysis. Using an 11-protein mixture, we found that the system could efficiently separate native peptides and released N-glycans from a single sample. We evaluated the applicability of the system to the analysis of complex biological samples using 25 µg of the lysate of a human choriocarcinoma cell line (BeWo), confidently identifying a total of 1449 proteins from a single experiment and up to 1909 distinct proteins from technical triplicates. The PGC fraction increased the sequence coverage through the inclusion of additional hydrophilic sequences that accounted for up to 6.9% of the total identified peptides from the BeWo lysate, with apparent preference for the detection of hydrophilic motifs and proteins. In addition, RP-RP/PGC is applicable to the analysis of complex glycomics samples, as demonstrated by our analysis of a concanavalin A-extracted glycoproteome from human serum; in total, 134 potentially N-glycosylated serum proteins, 151 possible N-glycosylation sites, and more than 40 possible N-glycan structures recognized by concanavalin A were simultaneously detected.
Assuntos
Cromatografia de Fase Reversa/instrumentação , Cromatografia de Fase Reversa/métodos , Glicômica/métodos , Glicopeptídeos/análise , Grafite/química , Proteômica/métodos , Sequência de Aminoácidos , Animais , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/química , Proteínas Sanguíneas/isolamento & purificação , Configuração de Carboidratos , Linhagem Celular Tumoral , Concanavalina A/química , Desenho de Equipamento , Glicopeptídeos/química , Glicopeptídeos/isolamento & purificação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Dados de Sequência Molecular , Peptídeos/análise , Peptídeos/química , Peptídeos/isolamento & purificação , Polissacarídeos/análise , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Estatísticas não ParamétricasRESUMO
We have developed a novel system for coupling reverse-phase (RP) and hydrophilic interaction liquid chromatography (HILIC) online in a micro-flow scheme. In this approach, the inherent solvent incompatibility between RP and HILIC is overcome through the use of constant-pressure online solvent mixing, which allows our system to perform efficient separations of both hydrophilic and hydrophobic compounds for mass spectrometry-based proteomics applications. When analyzing the tryptic digests of bovine serum albumin, ribonuclease B, and horseradish peroxidase, we observed near-identical coverage of peptides and glycopeptides when using online RP-HILIC--with only a single sample injection event--as we did from two separate RP and HILIC analyses. The coupled system was also capable of concurrently characterizing the peptide and glycan portions of deglycosylated glycoproteins from one injection event, as confirmed, for example, through our detection of 23 novel glycans from turkey ovalbumin. Finally, we validated the applicability of using RP-HILIC for the analysis of highly complex biological samples (mouse chondrocyte lysate, deglycosylated human serum). The enhanced coverage and efficiency of online RP-HILIC makes it a viable technique for the comprehensive separation of components displaying dramatically different hydrophobicities, such as peptides, glycopeptides, and glycans.
Assuntos
Cromatografia de Fase Reversa/instrumentação , Glicoproteínas/análise , Proteínas/análise , Proteômica/instrumentação , Sequência de Aminoácidos , Animais , Bovinos , Linhagem Celular , Cromatografia Líquida/instrumentação , Cromatografia Líquida/métodos , Cromatografia de Fase Reversa/métodos , Desenho de Equipamento , Glicoproteínas/isolamento & purificação , Humanos , Espectrometria de Massas/métodos , Camundongos , Dados de Sequência Molecular , Polissacarídeos/análise , Polissacarídeos/isolamento & purificação , Proteínas/isolamento & purificação , Proteômica/métodos , Soro/químicaRESUMO
Molecular radical cations of tripeptides of the form glycylglycyl(residue X) (GGX*+) are produced by the collision-induced, intramolecular one-electron transfer of [Cu(II)(L)GGX]*2+ complexes (L = triamine ligand). We demonstrate, for the first time, the formation of molecular radical cations of all of the aliphatic, basic, aromatic, acidic, and some heteroatom-bearing GGX tripeptides, albeit inefficiently in some cases, by altering the structure of the auxiliary polyamine ligand bound to the copper atom. The design of the ligand allows exquisite control over the nature of the dissociation pathway. Steric hindrance of bulky groups in the ligand affects the binding of the peptide to the copper ion; this interaction is an important factor in determining whether the electron transfer pathway predominates.
Assuntos
Cobre/análise , Cobre/química , Peptídeos/análise , Peptídeos/química , Piridinas/análise , Piridinas/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Cátions , Transporte de Elétrons , Ligantes , Ligação Proteica , EstereoisomerismoRESUMO
Previously, we described an online high-/low-pH RP-RP LC system exhibiting high-throughput, automatability, and performance comparable with that of SCX-RP. Herein, we report a variant of the RP-RP platform, RP-SCX-RP, featuring an additional SCX trap column between the two LC dimensions. The SCX column in combination with the second-dimension RP can be used as an SCX-RP biphasic column for trapping peptides in the eluent from the first RP column. We evaluated the performance of the new platform through proteomic analysis of Arabidopsis thaliana chloroplast samples and mouse embryonic mouse fibroblast STO cell lysate at low-microgram levels. In general, RP-SCX-RP enhanced protein identification by allowing the detection of a larger number of hydrophilic peptides. Furthermore, the platform was useful for the quantitative analyses of crude chloroplast samples for iTRAQ applications at low-microgram levels. In addition, it allowed the online removal of sodium dodecyl sulfate and other chemicals used in excess in iTRAQ reactions, avoiding the need for time-consuming offline SCX clean-up prior to RP-RP separation. Relative to the RP-RP system, our newly developed RP-SCX-RP platform allowed the detection of a larger number of differentially expressed proteins in a crude iTRAQ-labeled chloroplast protein sample.
Assuntos
Cromatografia por Troca Iônica/métodos , Cromatografia de Fase Reversa/métodos , Mapeamento de Peptídeos/métodos , Proteômica/métodos , Animais , Proteínas de Arabidopsis/química , Cátions , Cloroplastos/química , Fibroblastos/química , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Marcação por Isótopo , Espectrometria de Massas , Camundongos , Fragmentos de Peptídeos/análiseRESUMO
Extensive front-end separation is usually required for complex samples in bottom-up proteomics to alleviate the problem of peptide undersampling. Isobaric Tags for Relative and Absolute Quantification (iTRAQ)-based experiments have particularly higher demands, in terms of the number of duty cycles and the sensitivity, to confidently quantify protein abundance. Strong cation exchange (SCX)/reverse phase (RP) liquid chromatography (LC) is currently used routinely to separate iTRAQ-labeled peptides because of its ability to simultaneously clean up the iTRAQ reagents and byproducts and provide first-dimension separation; nevertheless, the low resolution of SCX means that peptides can be redundantly sampled across fractions, leading to loss of usable duty cycles. In this study, we explored the combinatorial application of offline SCX fractionation with online RP-RP applied to iTRAQ-labeled chloroplast proteins to evaluate the effect of three-dimensional LC separation on the overall performance of the quantitative proteomics experiment. We found that the higher resolution of RP-RP can be harnessed to complement SCX-RP and increase the quality of protein identification and quantification, without significantly impacting instrument time and reproducibility.
Assuntos
Cromatografia por Troca Iônica/métodos , Cromatografia Líquida/métodos , Proteoma/análise , Proteômica/métodos , Sequência de Aminoácidos , Proteínas de Arabidopsis/análise , Cátions , Cloroplastos/metabolismo , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Peptídeos/análise , Reprodutibilidade dos TestesRESUMO
Gas phase fragmentations of two isomeric radical cationic tripeptides of glycylglycyltryptophan-[G(*)GW](+) and [GGW](*+)-with well-defined initial radical sites at the alpha-carbon atom and the 3-methylindole ring, respectively, have been studied using collision-induced dissociation (CID), density functional theory (DFT), and Rice-Ramsperger-Kassel-Marcus (RRKM) theory. Substantially different low-energy CID spectra were obtained for these two isomeric GGW structures, suggesting that they did not interconvert on the time scale of these experiments. DFT and RRKM calculations were used to investigate the influence of the kinetics, stabilities, and locations of the radicals on the competition between the isomerization and dissociation channels. The calculated isomerization barrier between the GGW radical cations (>35.4 kcal/mol) was slightly higher than the barrier for competitive dissociation of these species (<30.5 kcal/mol); the corresponding microcanonical rate constants for isomerization obtained from RRKM calculations were all considerably lower than the dissociation rates at all internal energies. Thus, interconversion between the GGW isomers examined in this study cannot compete with their fragmentations.
Assuntos
Carbono/química , Cátions/química , Oligopeptídeos/química , Sequência de Aminoácidos , Isomerismo , Espectrometria de Massas , TermodinâmicaRESUMO
Although reverse-phase liquid chromatography (RP-LC) is a common technique for peptide separation in shotgun proteomics and glycoproteomics, it often provides unsatisfactory results for the analysis of glycopeptides and glycans. This bias against glycopeptides makes it difficult to study glycoproteins. By coupling mass spectrometry (MS) with a combination of RP-LC and normal-phase (NP)-LC as an integrated front-end separation system, we demonstrate that effective identification and characterization of both peptides and glycopeptides mixtures, and their constituent glycan structures, can be achieved from a single sample injection event.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Glicoproteínas/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Bovinos , Glicopeptídeos/química , Peroxidase do Rábano Silvestre/química , Espectrometria de Massas/métodos , Polissacarídeos/química , Proteoma , Proteômica/métodos , Ribonucleases/química , Albumina Sérica/química , Solventes/química , Tripsina/químicaRESUMO
Molecular radical cations have proven to be difficult to generate from aliphatic peptides under electrospray ionization mass spectrometry (ESI-MS) conditions. For a family of small aliphatic peptides GGX, where X = G, A, P, I, L and V, these cations have been generated by electrospraying a mixture of Cu.2+, 12-crown-4 and GGX in methanol/water. GGX.+ is readily formed from the collision-induced dissociation (CID) of [CuII(12-crown-4)(GGX)].2+. The formation of these aliphatic peptide radical ions from these complexes, in cases where it is not possible from the corresponding complexes involving a series of amine ligands instead of 12-crown-4, is likely due to the second ionization energy of the [CuI(12-crown-4)(GGX)]+ complex being higher than that of the corresponding [CuI(amine)(GGX)]+ complex. Using these 12-crown-4 complexes, GGI can be differentiated from the isomeric GGL by comparing the CID spectra of their [a3 + H].+ ions.