A-cyano-4-hydroxycinnamic acid

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. 

J. Gobom, M. Schuerenberg, M. Mueller, D. Theiss, H. Lehrach, and E. Nordhoff. a-Cyano-4-hydroxycinnamic Acid Affinity Sample Preparation. A Protocol for MALDI-MS Peptide Analysis in Proteomics. Anal. Chem., 73(2001) 434-438. 

Note It is commonplace to use acronyms rather than compound names for matrices. However, these are not always consistently used, e.g., a-CHC, 4-HCCA, CHCA, and CCA all refer to a-cyano-4-hydroxycinnamic acid. Others may be easily confused, e.g., nicotinic acid (NA) and 9-nitroanthracene (9-NA). 

Beavis, R.C. Chaudhary, T. Chait, B.T. a-Cyano-4-Hydroxycinnamic Acid As a Matrix for Matrix-Assisted Laser Desorp-tion-MS. Org. Mass Spectrom. 1992, 27, 156-158. 

In the last years, ILs have been applied as matrices for matrix-assisted laser desorption/ionization (MALDI) MS [42], thus expanding the use of MALDI. In Ref. 38 the suitability of alkylammonium- and alkylimidazolium salts of a-cyano-4-hydroxycinnamic acid was investigated as a MALDI matrix and at the same time as the additive of BGE. The alkylammonium salt produced better separation of phenolic compounds than the alkylimidazolium salt. The investigation suggests that it is possible to synthesize ILs suitable for electrophoretic analysis as well as for online MALDI-MS analysis. 

Most commonly used matrix systems are derivatives of benzoic acid (e.g., 2,5-dihydroxybenzoic acid (DHB), derivatives of cinnamic acid (e.g., a-cyano-4-hydroxycinnamic acid (CHCA) or sinapinic acid (SA) (Figure 14.6) as well as heteroaromatic compounds containing nitrogen but numberless other substances and substance classes have been applied as matrices [36]. 

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). 

CFTR cystic fibrosis transmembrane conductance regulator CGE capillary gel electrophoresis CHCA a-cyano-4-hydroxycinnamic acid CHO Chinese hamster ovary cells CL chemiluminescence... 

A matrix adapted for <10 kDa peptides such as a-cyano-4-hydroxycinnamic acid and the adapted solvents (4HCCA, 40 mg/mL in acetone) for preparing the different solutions for sample preparation acetone, TFA, acetonitrile, and water (all HPLC grade). To select the best methodologies for sample preparation, some tricks are discussed in (26). 

For MALDI-TOF MS several sample preparations are available with different matrices (26). The choice of the matrix and of the sample preparation should be adapted to the molecular mass of the compounds and to the complexity of the samples to analyze. a-Cyano-4-hydroxycinnamic acid (4HCCA) is preferred for peptides between 1 and 15 kDa, and the sandwich sample preparation can be universally used for molecular mass determination of pure peptides and enzymatic fragments or complex mixtures (e.g., crude hemolymph, enzymatic digests). The procedures reported below are the ones used for the discovery of the Drosophila immune-induced peptides (19,27,30). 

After purification, lyophilize the peptide constructs and check their purity on analytical HPLC and MALDI-TOF mass spectroscopy. The conventional matrix for peptide mass spectroscopy, a-cyano-4-hydroxycinnamic acid, appears to work properly for the vaccine constructs. While it is less frequently used in standard peptide chemistry (and thus not detailed here), the size of these multiple peptide antigens allows quality control by SDS-PAGE as shown in Fig. 2. For this purpose we recommend the use of 16.5% precast peptide gels. These gels tend to break easily, so be careful during gel handling. 

MALDI-MS employs a matrix and the use of a matrix with the samples has several purposes extraction of analyte from the cocrystallization surface, formation of analyte-dropped crystals, and absorption of the laser energy for soft-ionization of analyte molecules into MS analyzer. The commonly used MALDI matrices include a-cyano-4-hydroxycinnamic acid (CHCA), 2,5-Dihydroxybenzoic acid (DHB), Sinapinic acid (SA), et al. [61], A typical matrix solution would comprise the matrix at a concentration of 10-20 mg/mL in a solvent that is compatible with the... 

Peptides/proteins a-Cyano-4-hydroxycinnamic acid CHCA... 

The matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra of 49a-d and macrocycle 50, using a-cyano-4-hydroxycinnamic acid (CCA) as a matrix, exhibited, besides [M+Na]+ as the main peak, [M+K]+ adducts and ions corresponding to protonated molecules [M+H]+ <1999RCM2359>. 

Mueller, M., Theiss, D., Lehragh, H., Nordhoff, E. (2001). a-Cyano-4-hydroxycinnamic acid affinity sample preparation. A protocol for MALDI-MS peptide analysis in proteomics. Anal. Chem. 73, 434 38. 

Approximately 0.5 nl of each fractions was analyzed on a Kratos Kompact in TOP instrument. Samples were prepared using a-cyano-4-hydroxycinnamic acid as matrix, liie sample wells were prespotted with matrix dissolved in acetone, dried, and respotted with a 1 1 solution of peptide and matrix dissolved in 30% acetonitrileA)<01% TFA. 

---