Liquid ultraviolet matrix-assisted laser desorption/ionization mass spectrometry for automated proteomic analysis

We have combined several key sample preparation steps for the use of a liquid matrix system to provide high analytical sensitivity in automated ultraviolet - matrix-assisted laser desorption/ ionisation - mass spectrometry (UV-MALDI-MS). This new sample preparation protocol employs a matrix-mixture which is based on the glycerol matrix-mixture described by Sze et al. U. Am. Soc. Mass Spectrom. 1998, 9, 166-174). The low-ferntomole sensitivity that is achievable with this new preparation protocol enables proteomic analysis of protein digests comparable to solid-state matrix systems. For automated data acquisition and analysis, the MALDI performance of this liquid matrix surpasses the conventional solid-state MALDI matrices. Besides the inherent general advantages of liquid samples for automated sample preparation and data acquisition the use of the presented liquid matrix significantly reduces the extent of unspecific ion signals in peptide mass fingerprints compared to typically used solid matrices, such as 2,5-dihydrox-ybenzoic acid (DHB) or alpha-cyano-hydroxycinnamic acid (CHCA). In particular, matrix and lowmass ion signals and ion signals resulting from cation adduct formation are dramatically reduced. Consequently, the confidence level of protein identification by peptide mass mapping of in-solution and in-gel digests is generally higher.

Investigation of liquid MALDI and optimization for instrument tuning and quantitative measurements

The success of Matrix-assisted laser desorption / ionisation (MALDI) in fields such as proteomics has partially but not exclusively been due to the development of improved data acquisition and sample preparation techniques. This has been required to overcome some of the short comings of the commonly used solid-state MALDI matrices such as - cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB). Solid state matrices form crystalline samples with highly inhomogeneous topography and morphology which results in large fluctuations in analyte signal intensity from spot to spot and positions within the spot. This means that efficient tuning of the mass spectrometer can be impeded and the use of MALDI MS for quantitative measurements is severely impeded. Recently new MALDI liquid matrices have been introduced which promise to be an effective alternative to crystalline matrices. Generally the liquid matrices comprise either ionic liquid matrices (ILMs) or a usually viscous liquid matrix which is doped with a UV lightabsorbing chromophore [1-3]. The advantages are that the droplet surface is smooth and relatively uniform with the analyte homogeneously distributed within. They have the ability to replenish a sampling position between shots negating the need to search for sample hot-spots. Also the liquid nature of the matrix allows for the use of additional additives to change the environment to which the analyte is added.

Sample preparation: a crucial factor for the analytical performance of rationally designed MALDI matrices

Evidence is presented that the performance of the rationally designed MALDI matrix 4-chloro-α-cyanocinnamic acid (ClCCA) in comparison to its well-established predecessor α-cyano-4-hydroxycinnamic acid (CHCA) is significantly dependent on the sample preparation, such as the choice of the target plate. In this context, it becomes clear that any rational designs of MALDI matrices and their successful employment have to consider a larger set of physicochemical parameters, including sample crystallization and morphology/topology, in addition to parameters of basic (solution and/or gas-phase) chemistry.

Coupling liquid MALDI MS to liquid chromatography

Matrix-assisted laser desorption/ionisation (MALDI) coupled with time-of-flight (TOF) mass spectrometry (MS) is a powerful tool for the analysis of biological samples, and nanoflow high-performance liquid chromatography (nanoHPLC) is a useful separation technique for the analysis of complex proteomics samples. The off-line combination of MALDI and nanoHPLC has been extensively investigated and straightforward techniques have been developed, focussing particularly on automated MALDI sample preparation that yields sensitive and reproducible spectra. Normally conventional solid MALDI matrices such as α-cyano-4-hydroxycinnamic acid (CHCA) are used for sample preparation. However, they have limited usefulness in quantitative measurements and automated data acquisition because of the formation of heterogeneous crystals, resulting in highly variable ion yields and desorption/ ionization characteristics. Glycerol-based liquid support matrices (LSM) have been proposed as an alternative to the traditional solid matrices as they provide increased shot-to-shot reproducibility, leading to prolonged and stable ion signals and therefore better results. This chapter focuses on the integration of the liquid LSM MALDI matrices into the LC-MALDI MS/MS approach in identifying complex and large proteomes. The interface between LC and MALDI consists of a robotic spotter, which fractionates the eluent from the LC column into nanoliter volumes, and co-spots simultaneously the liquid matrix with the eluent fractions onto a MALDI target plate via sheath flow. The efficiency of this method is demonstrated through the analysis of trypsin digests of both bovine serum albumin (BSA) and Lactobacillus plantarum WCFS1 proteins.

Phenolic Composition of the Edible Parts (Flesh and Skin) of Bordo Grape (Vitis labrusca) Using HPLC-DAD-ESI-MS/MS

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Phenolic Composition of the Brazilian Seedless Table Grape Varieties BRS Clara and BRS Morena

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Chromatic characteristics and color-related phenolic composition of Brazilian young red wines made from the hybrid grape cultivar BRS Violeta (BRS Rúbea×IAC 1398-21)

The color characteristics and the phenolic composition related to color of young red wines elaborated with the hybrid grape cultivar BRS Violeta, developed for its adaptation to sub-tropical climates in Brazil, have been studied. These wines are characterized with a deep red-purplish color, reaching color intensity averaging 24 units. In spite of being young red wines elaborated with short maceration time, their content in total polyphenols was very high (around 3692. mg/L, as gallic acid equivalents), especially when compared to similar Vitis vinifera young red wines. Within polyphenols, anthocyanins predominated (around 2037. mg/L, as malvidin 3,5-diglucoside equivalents) and they were almost exclusively anthocyanidin 3,5-glucosides (ca. 97%), mainly built from B-ring tri-substituted anthocyanidins (delphinidin. >. petunidin. ≈. malvidin) and having high proportion of p-coumaroylated derivatives (ca. 28%) that confer higher stability. The content of hydroxycinnamic acid derivatives was also remarkable (ca. 95. mg/L, as caffeic acid equivalents) and unknown glucose derivatives of p-coumaric acid accounted for ca. 42% of total HCAD. Finally, these found flavonols were mainly based on myricetin whereas kaempferol derivatives were missing, their total content being within the ranges usually found for V. vinifera wines, but reaching their top values (ca. 91. mg/L, as quercetin 3-glucoside equivalents). All the aforementioned data suggest that Violeta wine could be considered an important dietary source of healthy polyphenols with a moderate alcoholic content (ca. 11.6%). © 2013 Elsevier Ltd.

Phenolic composition of the berry parts of hybrid grape cultivar BRS Violeta (BRS Rubea×IAC 1398-21) using HPLC-DAD-ESI-MS/MS

The grape is considered a major source of phenolic compounds when compared to other fruits and vegetables, however, there are many cultivars with distinct characteristics directly linked to phenolic profile. Thus, the present study aimed to identify and quantify, for the first time and in detail, the phenolic compounds present in the skin, flesh and seeds of BRS Violeta grape berry using combination of SPE methodologies and analytical HPLC-DAD-ESI-MS/MS. The study was extended to the different berry parts and the most important grape and wine phenolic families, and has revealed interesting features. Violeta grape has a very thick skin (46% of grape weight) that accumulated the most of grape phenolic compounds: great amount of anthocyanins (3930. mg/kg, as malvidin 3,5-diglucoside), together with also important amounts of flavonols (150. mg/kg, as quercetin 3-glucoside), hydroxycinnamic acid derivatives (HCAD; 120. mg/kg, as caftaric acid), and proanthocyanidins (670. mg/kg, as (+)-catechin); in contrast, it seems to be a low resveratrol producer. Violeta grape seeds accounted for similar proportions of low molecular weight flavan-3-ols (mainly monomers; 345. mg/kg, as (+)-catechin) and proanthocyanidins (480. mg/kg, as (+)-catechin). Violeta grape is a teinturier cultivar, but it only contained traces of anthocyanins and low amounts of all the other phenolic types in its red-colored flesh. The anthocyanin composition of Violeta grape was dominated by anthocyanidin 3,5-diglucosides (90%). Within flavonols, myricetin-type predominated and kaempferol-type was missing. In addition to expected hydroxycinnamoyl-tartaric acids, several isomeric esters of caffeic and p-coumaric acids with hexoses were tentatively identified, accounting for relevant proportions within the pool of HCAD. Although pending of further confirmation over successive vintages, the aforementioned results suggest that BRS Violeta grape cultivar could be considered an interesting candidate for the elaboration of highly colored and antioxidant-rich grape juices and wines. © 2013 Elsevier Ltd.

Estudos bioquímicos, físico-químicos e tecnológicos de uvas paulistas

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Desenvolvimento de Plutella xylostella (Linnaeus, 1758) (Lepidoptera: Plutellidae) em Brassicaceae ao longo de gerações

Pós-graduação em Agronomia (Entomologia Agrícola) - FCAV

Peptídeos cíclicos hepatotóxicos: MALDI-TOF como uma ferramenta analítica

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Produção de uva passa de BRS Morena: pré-tratamento, caracterização físico-química e composição fenólica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Composição fenólica das partes comestíveis das uvas BRS Carmem e BRS Magna

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Secagem por atomização do suco de uva: microencapsulação das antocianinas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Supercritical CO2 extraction of phenolic compounds from Baccharis dracunculifolia

Extracts from Baccharis dracunculifolia leaves were obtained using the following solvents: supercritical carbon dioxide (SC-CO2), ethanol and methanol. Supercritical extraction was carried out at temperatures of 40, 50 and 60 degrees C and pressures of 20, 30 and 40 MPa. Four phenolic compounds were analysed in the extracts by high-performance liquid chromatography: 3,5-diprenyl-4-hydroxycinnamic acid (DHCA or artepillin C); 3-prenyl-4-hydroxycinnamic acid (PHCA); 4-hydroxycinnamic acid (p-coumaric acid) and 4-methoxy-3,5,7-trihydroxyflavone (kaempferide). The global extraction yields (X-0) obtained by the conventional methods with ethanol and methanol were higher than those obtained by SC-CO2. However on analysing the components of interest extracted at 60 degrees C and 40 MPa, the extraction yields of kaempferide, DHCA and PHCA were 156%, 98% and 64% higher, respectively, than in the ethanolic extracts. Only the p-coumaric acid extraction yield was better when extracted using the conventional method. (C) 2008 Elsevier B.V. All rights reserved.