Alpha-cyano 4-hydroxycinnamic acid MALDI matrix

The MALDI-TOF technique was first developed for the analysis of large biomolecules (Karas and others 1987). This technique presents some interesting characteristics. Of these, the high speed of analysis and the sensitivity of the technique have been pointed out as important advantages compared with other methods. In MALDI the samples are cocrystallized with a matrix that is usually composed of organic compounds, such as 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid), 2, 4, 6 -trihydroxyacetophenone, a-cyano-4-hydroxycinnamic acid (alpha-cyano or alpha-matrix), and 2,5-dihydroxybenzoic acid (DHB). After the cocrystallization, the laser is fired and the matrix absorbs energy and allows a soft ionization of the samples. Afterward the ions are analyzed by a TOF mass spectrometer. 

Peptide extracts prepared as described above were analyzed on a MALDI-Tof mass spectrometer (Micromass TofSpec E). A small portion of the extract was mixed with an equal volume of a-cyano-4-hydroxycinnamic acid (alpha CHC) matrix in methanol and loaded onto a MALDI plate. Calibration of the flight tube was performed with the internal standards angiotensin I and adrenocorticotrophic hormone (both from Sigma). 

For non-quantitative LC-MALDI applications, derivatization chemistry is not restricted to compounds which allow a convenient incorporation of heavy isotopes. For example for improved MS/MS detection sensitivity, tryptic peptides were labeled with sulfonated coumarin-tags at the N-termini after guanidylation of lysines (Pashkova et al. 2005). Despite reduced MS sensitivity for arginine-terminated peptides (in alpha cyano-4-hydroxycinnamic acid matrix), formation of y-ions was enhanced in MS/MS by the second mobile proton provided from the sulfonic acid group. For a SCX fraction from a E.coli hydrolysate 50% more peptides and 30% more proteins could be identified by multiplexed LC-MALDI MS and MS/MS after derivatization. 

The samples were analyzed on the VG TofSpec-SE MALDI TOP mass spectrometer in the reflectron mode with positive ion detection. The samples were spotted on the sample plate in acetonitrile water (60 40) or chloroform methanohTFA (1 1 0.1) mixture plus ammonium suliate on alpha-C (a-cyano-4-hydroxycinnamic acid) matrix. The ionization of the samples was carried out with Nd YAG laser at 355 nm or nitrogen laser at 337 nm. Some of the fractions were analyzed by SIMS on a Kratos 890 mass spectrometer equipped with a Phrasor Scientific SIMS source. The mass spectral data were analyzed by the MSFIT program at the University of California, San Francisco. 

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... 

Beavis, R. C. Chaudary, T. Chait, B. T. Alpha-cyano-4-hydroxycinnamic acid as a matrix for MALDI. Org. Mass Spectrom. 1992, 27, 156-158. 

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