Sinapinic protein analysis

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. ... 

Fig. 15, which shows a characteristic MALDI-TOF spectrum of a two-component protein mixture using sinapinic acid (SA) as a matrix. A broad, tailing peak is observed in the lower mass region of the spectrum (<2000 Da) that is associated with ions from the matrix. This matrix interference is not disturbing in this particular case of protein analysis but could be a disadvantage when smaller molecules are studied. One way to eliminate the matrix interference is the application of the so-called desorption/ionization on silicon (DIOS) technique [36]. 

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). 

Matrix Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS) Mass spectra of native and denatured antibodies were obtained with a PerSeptive Biosystems (Farmingham, MA.) Voyager Elite mass spectrometer operated in the linear mode with a Laser Sciences Inc., 337 nm nitrogen laser. hAB-1 was denatured by boiling the sample in 1.0 M guanidine-HCl, 50 mM Tris pH 7.5 buffer. Native and denatured samples were diluted with 20 mM Tris, 10 mM octylglucoside (Tris/OG) pH 6.8 buffer prior to MALDl-MS analysis. Proteins were spotted on the sample plate as a sandwich between two layers of the matrix. The bottom layer consisted of 100 mM sinapinic acid in acetonitrile and the top layer consisted of 50 mM sinapinic acid in 30% acetonitrile / 70% H2O / 0.07% TFA. The m/z scale of the instrument was calibrated using a Hewlett-Packard protein standard mixture. 

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... 

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