Dihydroxybenzoic protein analysis

As a practical example, when a mouse brain section to which 2,5-dihydroxybenzoic acid (DHB) has been applied as a matrix is subjected directly to MS in positive ion-detection conditions, strong peaks which are mainly derived from phospholipids were observed in mass region of 700 < m/z < 900 [8] on the other hand, signals derived from proteins, meanwhile, are scarcely detected at m/z > 3000 [9].This is because phospholipids ionize much more efficiently than proteins, and they, on the other hand, suppress protein/peptide ionization. Therefore, for detecting/imaging proteins and peptides, removal of such lipids improves the sensitivity for proteins analysis. To this end, tissue sections should be rinsed with organic solvent, to remove lipids from tissue samples [9-11]. 

In a typical MALDI experiment (see Fig. 7.3), e.g., for peptide or protein analysis, 0.3-1 pi of an aqueous analyte solution is mixed with 0.5-1 pi of a 5 mM so-Intion of an appropriate matrix, e.g., 2, 5-dihydroxybenzoic add (DHB), sinapinic acid, or a-cyano-4-hydroxycinnaniic acid (CHCA), in 50% aqueous acetonitrile containing 0.1% trifluoroacetic add, and then deposited onto a metal taiget. Other... 

MALDI matrix is typically a small organic acid that co-crystallizes with analytes extracted from the tissne section to allow absorption of energy of the pulsed laser. Matrix and analytes are ablated and ionized from the tissne snrfece with subsequent MS analysis. Common matrixes used for the analysis of peptides, lipids, and small drng molecules are 2,5-dihydroxybenzoic acid (DHB) and a-cyano-4-hydroxycinnamic acid (CHCA) while sinapinic acid (S A) is most often used for protein analysis. 1.23,26,27) An organic solvent snch as acetonitrile or methanol can be used for extraction of small molecules from tissne sections prior to matrix co-crystallization. ... 

MALDl-MS owes much of its popularity to its usefulness as an analytical tool for the characterization of peptides and proteins. While an extremely large number of matrix candidates have been investigated for their applicability in peptide and protein analysis, a relatively small group has proven reliable over the past 15 + years. The members of this small group include 2,5-dihydroxybenzoic acid (2,5-DHB), a-cyanohydroxycinnamic acid (CHCA or HCCA), and sinapinic acid (SA). 

In the aligned primary structures of class I decarboxylases, the conserved amino acid residues are scattered over their primary structures. There have been few reports to identify the amino acid residues essential for catalytic activity or substrate binding. Huang et al. reported the E-X-P motif in the alignment analysis for 4-hydroxybenzoate decarboxylase of C. hydroxybenzoicum and its homologous unidentified proteins. The E-X-P motif is also conserved in pyrrole-2-carboxylate decarboxylase and indole-3-carboxylate decarboxylase (unpublished data). However, the corresponding motif sequence is not observed in the primary structures of 3,4-dihydroxybenzoate decarboxylase of E. cloacae P241. ... 

Matrix-assisted iaser desorption/ionization (MALDI). This is another ionization method for the analysis of large molecules such as peptides, proteins, and nucleic acids, as well as some synthetic polymers. In MALDI, the analyte is first cocrystallized with an excess of a matrix, e.g., sinapinic acid or dihydroxybenzoic acid, that has a constituent aromatic component able to absorb photons from a UV laser beam. When the dried analyte matrix mixture is exposed (inside the vacuum chamber) to a sudden input of energy from a laser pulse the matrix evaporates, essentially instantaneously, carrying with it the analyte molecules. The matrix forms reagent ions that protonate the analytes. The selection of the matrix is critical as different compound classes exhibit substantial, matrix-dependent differences in ionization efficiency. The MALDI matrix should not be confused with the alternative use of the term matrix that is used to denote the medium in which biological and/or environmental components are presented, e.g., blood plasma, urine, sediment. 

Traditional methods to generate peptide maps involve fractionation of complex mixtures of peptides in a protein digest either with one-dimensional SDS-PAGE or RP-HPLC [28,29]. The mass spectrometry peptide-mapping protocol, in principle, is similar to these techniques, but it provides an added dimension of structure-specific data (i.e., the molecular mass). MALDI-MS [30,31], ESl-MS [32], LC/ESI-MS [33], and CE/ESI-MS [34] have currently replaced the traditional biochemical approaches. MALDI allows the direct analysis of unfractionated protein digests. The commonly used matrices are sinapinic acid, a-cyano-4-hydroxy cinnamic acid (a-CHCA), and 2,5-dihydroxybenzoic acid (DHB). 

For the MALDI-MS analysis of intact proteins, FI CCA (alpha-cyano-4-hydroxycinnamic acid), SA (sinapinic acid) or DHB (2,5-dihydroxybenzoic acid) matrices and the dried-droplet deposition method for sample preparation are typically used [13, 14] (Table 3.1). Depending on the properties of the protein, it is often necessary to test a series of solvents and matrices to optimize the outcome of the MALDI-MS experiment. Peptides and small proteins below molecular weight 20000 Da are often amenable to analysis using HCCA matrix and reflector TOF-MS mode, whereas larger proteins may produce better results with SA or DHB matrix in the linear TOF-MS mode. Hydrophobic proteins can be analyzed using the HCCA matrix dissolved in high concentrations of formic acid (up to 30%) [15]. When using cirmamic acid matrices, SA and HCCA, and the... 

A major difference between ESI and MALDI is that ESI requires liquid samples whereas MALDI, so far, works best with crystalHne samples. Another important difference is that MALDI requires a special matrix compound for the analysis of biological macromolecules, e.g., a-cyano-4-hydroxycinnamic acid (CHCA) for small and medium-size peptides, sinapic acid (SA) for large peptides and proteins, 2,5-dihydroxybenzoic acid (DHB) for glycosylated peptides and proteins, and 3-hydroxypicolinic acid for nucleic acids. It is noteworthy that all these compounds have been found solely empirically and that only a very few out of many hundred candidates turned out to be successful. 

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