RESUMEN
Herein it is shown that a combination of direct analysis in real time (DART) with a corona discharge system consisting of only a needle electrode easily improves DART ionization efficiency. Positive and negative DC corona discharges led to a formation of abundant excited helium atoms as well as the reactant ions H3O(+)(H2O)n and O2Ë(-) in the DART analyte ionization area. These phenomena resulted in an increase in the absolute intensities of (de)protonated analytes by a factor of 2-20 over conventional DART. The other analyte ions detected in this corona-DART system (i.e., molecular ions, fragment ions, oxygenated (de)protonated analytes, dehydrogenated deprotonated analytes, and negative ion adducts) were quite similar to those obtained from DART alone. This indicates a lack of side reactions due to the corona discharge. The change in the relative intensities of individual analyte-related ions due to the combination of a corona discharge system with DART suggests that there is no effect of the abundant excited helium in the analyte ionization area on the fragmentation processes or enhancement of oxidation due to hydroxyl radicals HOË. Furthermore, it was found that the corona-DART combination can be applied to the highly sensitive analysis of n-alkanes, in which the alkanes are ionized as positive ions via hydride abstraction and oxidation, independent of the type of alkane or the mass spectrometer used.
RESUMEN
Highly protonated histone-derived peptides impede a sufficient mass spectrometry (MS)-based epigenetic analysis because their relatively low m/z, due to a high degree of proton addition to peptides, would make it difficult to analyze the resulting complex MS/MS spectra. To reduce the degree of protonations, we have developed a new interface, the Ionization Variable Unit (IVU), in which peptides are ionized under a vaporized organic solvent. It is demonstrated that the doubly charged histone tail H2B peptide, PEPAKSAPAPKKGSKKAVTKAQKK (m/z 1238.243, +2), which was not detectable before, can be detected by using the IVU interface and sequenced.
Asunto(s)
Epigénesis Genética , Histonas/química , Fragmentos de Péptidos/química , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Espectrometría de Masas en Tándem/instrumentaciónRESUMEN
The positive and negative ionization characteristics of 20 different α-amino acids were investigated using Direct Analysis in Real Time (DART) mass spectrometry. Almost all of the amino acids M were ionized to generate the (de)protonated analytes [M ± H](±)via proton transfer reactions with the typical background ions H3O(+)(H2O)n and O2Ë(-) and resonant electron capture by M. The application of DART to amino acids also resulted in molecular ion formation, fragmentation, oxidations involving oxygen attachment and hydrogen loss, and formation of adducts [M + R](-) with negative background ions R(-) (O2Ë(-), HCO2(-), NO2(-) and COO(-)(COOH)), depending on the physicochemical and/or structural properties of individual amino acids. The relationship between each amino acid and the ionization reactions observed suggested that fragmentation can be attributed to pyrolysis during analyte desorption as well as excess energy obtained via (de)protonation. Oxidation and [M + R](-) adduct formation, in contrast, most likely originate from reactions with active oxygen such as hydroxyl radicals HOË, indicating that the typical background neutral species involved in analyte ionization in DART mass spectrometry contain HOË.
Asunto(s)
Aminoácidos/química , Espectrometría de Masas/métodos , Oxidación-ReducciónRESUMEN
For high throughput proteome analysis of highly complex protein mixtures, we have constructed a fully automated online system for multi-dimensional protein profiling, which utilizes a combination of two-dimensional liquid chromatography and tandem mass spectrometry (2D-LC-MS-MS), based on our well-established offline system described previously [K. Fujii, T. Nakano, T. Kawamura, F. Usui, Y. Bando, R. Wang, T. Nishimura, J. Proteome Res. 3 (2004) 712]. A two-valve switching system on a programmable auto sample injector is utilized for online two-dimensional chromatography with strong cation-exchange (SCX) and reversed-phase (RP) separations. The SCX separation is carried out during the equilibration of RP chromatography and the entire sequence of analysis was performed under fully automated conditions within 4 h, based on six SCX fractionations, and 40 min running time for the two-dimensional RP chromatography. In order to evaluate its performance in the detection and identification of proteins, digests of six standard proteins and yeast 20S proteasome have been analyzed and their results were compared to those obtained by the one-dimensional reversed-phase chromatography system (ID-LC-MS-MS). The 2D-LC-MS-MS system demonstrated that both the number of peptide fragments detected and the protein coverage had more than doubled. Furthermore, this multi-dimensional protein profiling system was also applied to the human 26S proteasome, which is one of the highly complex protein mixtures. Consequently, 723 peptide fragments were identified as 31 proteasome components, together with other coexisting proteins in the sample. The identification could be comprehensively performed with a 63% sequence coverage on an average, and additionally, with modifications at the N-terminus. These results indicated that the online 2D-LC-MS-MS system being described here is capable of analyzing highly complex protein mixtures in a high throughput manner, and that it would be applicable to dynamic proteomics.
Asunto(s)
Proteínas/química , Automatización , Resinas de Intercambio de Catión , Cromatografía por Intercambio Iónico/métodos , HumanosRESUMEN
Smoking is a risk factor for lung diseases, including chronic obstructive pulmonary disease and lung cancer. However, the molecular mechanisms mediating the progression of these diseases remain unclear. Therefore, we sought to identify signaling pathways activated by tobacco-smoke exposure, by analyzing nuclear phosphoprotein expression using phosphoproteomic analysis of lung tissue from mice exposed to tobacco smoke. Sixteen mice were exposed to tobacco smoke for 1 or 7 days, and the expression of phosphorylated peptides was analyzed by mass spectrometry. A total of 253 phosphoproteins were identified, including FACT complex subunit SPT16 in the 1-day exposure group, keratin type 1 cytoskeletal 18 (K18), and adipocyte fatty acid-binding protein, in the 7-day exposure group, and peroxiredoxin-1 (OSF3) and spectrin ß chain brain 1 (SPTBN1), in both groups. Semi-quantitative analysis of the identified phosphoproteins revealed that 33 proteins were significantly differentially expressed between the control and exposed groups. The identified phosphoproteins were classified according to their biological functions. We found that the identified proteins were related to inflammation, regeneration, repair, proliferation, differentiation, morphogenesis, and response to stress and nicotine. In conclusion, we identified proteins, including OSF3 and SPTBN1, as candidate tobacco smoke-exposure markers; our results provide insights into the mechanisms of tobacco smoke-induced diseases.
RESUMEN
In clinical and diagnostic proteomics, it is essential to develop a comprehensive and robust system for proteome analysis. Although multidimensional liquid chromatography/tandem mass spectrometry (LC/MS/MS) systems have been recently developed as powerful tools especially for identification of protein complexes, these systems still some drawbacks in their application to clinical research that requires an analysis of a large number of human samples. Therefore, in this study, we have constructed a technically simple and high throughput protein profiling system comprising a two-dimensional (2D)-LC/MS/MS system which integrates both a strong cation exchange (SCX) chromatography and a microLC/MS/MS system with micro-flowing reversed-phase chromatography. Using the microLC/MS/MS system as the second dimensional chromatography, SCX separation has been optimized as an off-line first dimensional peptide fractionation. To evaluate the performance of the constructed 2D-LC/MS/MS system, the results of detection and identification of proteins were compared using digests mixtures of 6 authentic proteins with those obtained using one-dimensional microLC/MS/MS system. The number of peptide fragments detected and the coverage of protein sequence were found to be more than double through the use of our newly built 2D-LC/MS/MS system. Furthermore, this multidimensional protein profiling system has been applied to plasma proteome in order to examine its feasibility for clinical proteomics. The experimental results revealed the identification of 174 proteins from one serum sample depleted HSA and IgG which corresponds to only 1 microL of plasma, and the total analysis run time was less than half a day, indicating a fairly high possibility of practicing clinical proteomics in a high throughput manner.