Alpha-cyano 4-hydroxycinnamic acid

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. 

MALDI-TOF Mass Spectrometry. Peptide/detergent mixtures as well as purified peptides were analyzed using a Vestec LaserTec BenchTop II system (PerSeptive Biosystems). Peptides were mixed with alpha-cyano-4-hydroxycinnamic acid as previously described (4). 

Figure 3. MALDI-TOF mass spectrometry of peaks 1 (A-C) and 2 (D-F) purified after digestion of PVDF-bound transferrin using 1% RTX-100 (A, D), octylglucopyranoside (B, E), and decylglucopyranoside (C, F) as shown in Figure 2. Approximately 0.5% of the purified peptide (-150 fmole) was mixed with alpha-cyano-4-hydroxycinnamic acid and analyzed by MALDI-TOF mass spectrometry as described in Materials and Methods. Ninety percent of the peptide was amino terminally sequenced (Table II).

Matrix alpha-cyano-4-hydroxycinnamic acid (HCCA, 1 mg)... 

Gobom, J., Schuerenberg, M., Mueller, M., Theiss, D., Lehrach, H., and Nordhoff, E., Alpha-cyano-4-hydroxycinnamic acid affinity sample preparation. A protocol for MALDI-MS peptide analysis in proteomics. Analytical Chemistry, 73, 434-438, 2001. 

Fourier transform ion cyclotron resonance Full-width at half-maximum, the standard way of deter- mining peak width. It is used to calculate resolving power (see below). alpha-Cyano-4-hydroxycinnamic acid Ion cyclotron resonance Infrared... 

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... 

Calibrations of all instrument devices are performed with the MSCAL4 kit. Mass calibration of the Orbitrap detector in particular is done with the peptides therein and two main signals from alpha-cyano 4-hydroxycinnamic acid (HCCA). Mass calibration of the Orbitrap detector is typically done weekly and can be performed from another MALDI sample plate or the calibration sample can be placed next to a tissne imaging sample on the plate. In the herein described experiments, a separate calibration plate was used for mass calibration and the Orbitrap mass calibra-... 

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. 

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). 

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. 

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