A-Cyano-3-hydroxycinnamate

To each of six tubes containing buffer was added 50 ph of a suspension of rat liver mitochondria that contained 20mgmL 1 of protein. Radioactively labeled pyruvate (0.07 mCi mmol-1) was added to each tube. a-Cyano-3-hydroxycinnamate (an inhibitor of transport) was added to each tube in turn at intervals of 5 s. Each solution was filtered immediately after the addition of inhibitor, and the radioactivities of the filters (which retained the mitochondria) were found to be 0.17, 0.35, 0,51, 0.66, 0.82, and 0.96 nCi for the filters obtained at 5, 10, 15, 20, 25, and 30 s, respectively. What is the rate of uptake of pyruvate in nmolmin-1 mg-1 of protein ... 

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. 

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). 

ESMS was perfonned with a Fisons VG Quattro outfitted with a Hsons Electrospray Source. Samples were dissolved in 1.0 mL of 50% methanol-1% acetic acid, then diluted 1 10 with 50% acetonitrile-1.0 mM anmumium acetate to give 25 pmol/pL. A 10 pL aliquot of each sample was injected into a 10 pL/min stream of 50% acetonitrile-1.0 mM ammonium acetate. Data was processed using Fisons MassLynx Software. MALDI-MS was performed with a Vestec Benchtop lit linear dme-of-flight mass spectrometer, opmted in the linear mode with an N2 laser (337 nm). Samples were dissolved in 1.0 mL of 25% acetonitrile-0.1% TFA, then diluted 3 100 to give 5-10 pmol/pL. A 0.5 pL aliquot of each sample solution was added to 0.5 pL of matrix [a-cyano-4-hydroxycinnamic aci saturated solution in 50% acetonitrile-2% TFA]. Samples were dried at ambient temperature and pressure. Each spectrum was the sum of ion intensity from 10-50 larer pulses. Tlie mass axis was calibrated externally. 

Hydroxypicolinic acid (HPA) 3-Aminopicolinic acid (APA) Dihydroxybenzoic acid (DHB) a-Cyano-4-hydroxycinnamic acid (qCHCA) Sinapinic acid (SA)... 

FIGURE 3.4 An example of the severe ion suppression effect on the tissue surface. A peptide solution (0.5 pL of 100 nM ACTH) was spotted on both ITO glass slide smface and brain-homogenate section. After the spraying of matrix solution (10 mg/mL a-cyano-4-hydroxycinnamic add [a-CHCA] 50% acetonitrile, 0.1% TFA), the spotted peptide was visuaUzed by IMS. 

A general challenge is the analyte-to-matrix molar ratio as many matrices caused interfering mass peaks at low m/z values. High matrix amount decreases selectivity, while a low matrix amount reduces the mass signal intensity and thus detectability. A suitable matrix concentration for UTLC is stated to be 2.66 nmol mm of a-cyano-4-hydroxycinnamic acid, which is about 10 times less than compared to HPTLC (22.3 nmol mm ) [6]. 

MALDI analysis kits (e.g. Sequazyme peptide mass standards kit from AB Sciex) are a convenient way to obtain matrix, peptide standards, and solvents however, these items can easily be purchased independently. Any peptides which span a reasonable proportion of the useful mass range can be used for calibration, provided their accurate molecular masses are known, a-cyano-4-hydroxycinnamic acid is the most common matrix used for peptide analysis. Matrix solution is prepared by dissolving 5 mg of matrix in 1 mL of 4 1 acetonitrile/water containing 0.1% TFA A mixture of peptide calibration standards, e.g. des-Arg -bradykinin (monoisotopic mass 904.4681), Angiotensin 1 (monoisotopicmass 1,296.6853), ACTH (1-17 clip) (monoisotopic mass 2,093.0867), ACTH (18-39 clip) (monoisotopic mass 2,465.1989) is made to a concentration of 2 pmol/pL each in 0.1% TFA in water. This solution is then mixed 1 1 with matrix solution to give the final peptide calibration mixture. 

Neubert H, Halket JM, Ocana MF, Patel RKP. MALDI post-source decay and LIFT-TOF/TOF investigation of A-Cyano-4-Hydroxycinnamic acid cluster interferences. J Am Soc Mass Spectrom. 2004 15(3) 336 3. 

Table 11.1 Overview of studies for profiling human pathogens with MALDI-TOF MS at the strain level Examples of strain categorization (A), strain differentiation (B), and strain identification (C). MRSA methicillin-resistant Staphylococcus aureus, MSSA methicillin-sensitive S. aureus, L library-based approach, P proteomics-based approach, CHCA a-cyano-4-hydroxycinnamic acid, CMBT 5-chloro-2-mercaptobenzothiazole, DHB 2-hydroxy-5-methoxy benzoic acid, FA ferulic acid, HABA 2,4-hydroxyphenylazobenzoic acid, SA 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid), THAP 2,4,6-trihydroxyacetophenon. Adapted and modified from Sandrin et al. (2013)... 

HCCA/ANI solution 1.5 equivalent of aniline (ANI) was added to a solution containing 10 mg/ml of a-Cyano-4-hydroxycinnamic acid (HCCA) in acetonitrile/aqueous TFA 0.1% (6 4, v/v) see Note 3). Aniline and TFA are toxic, so work in the hood. 

MALDI matrix used for antibody detection a-Cyano-4-hydroxycinnamic acid (HCCA) and 3-hydroxypicolinic acid (3-HPA) (Sigma-Aldrich). 

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