Sinapinic acid matrix

Figure 4 MALDI-TOF mass spectra of bovine a-lactalbumin after 1 h incubation with 2% amidosulfobetaine-14 and (A) acrylamide, (B) 2-vinyipyridine, and (C) 4-vinyipyridine. in aii cases the reaction has been carried out at pH 7.0. Note, in both panels B and C, a single reaction channel of LCA with 2VP and 4VP, respectively. The higher-order peaks (m/z15 248 and 15 468 in B and C) represent adducts LCA with the MALDI matrix, sinapinic acid (unpublished data).

Fig. 17.3. Whole-body imaging MS of proteins from a f -day-old mouse pup. (a) FI E stained section from which numerous organs are clearly visible, (b) Serial section used for imaging MS on which matrix (sinapinic acid) has been automatically deposited in an array manner with a final center-to-center spacing of 200 xm. (c) Overlay of 12 individual organ or tissue-specific protein images, each presented with a different color. See text for details.

MALDI shares many features with FAB in that it employs a matrix which transfers energy to an analyte molecule to facilitate the ionization of polar and thermally labile, high-molecular-weight molecules. In this case, however, the energy is provided by a pulsed laser at a wavelength which may be absorbed by a matrix material such as nicotinic or sinapinic acids. The ability to obtain mass... 

For PMMA/additive dissolutions, it was not possible to identify any additive characteristic mass peaks, either by direct laser desorption or with matrix-assistance (dithranol, DHBA or sinapinic acid, 4-hydroxy-3,5-dimethoxy-cinnamic acid). This has again been ascribed to very strong interaction between PMMA and additives, which suppresses desorption of additive molecules. Also, partial depolymerisation of pho-tolytically labile PMMA by laser irradiation may play a role, which leads to saturation of the detector by PMMA fragment-ions and disappearance of additive mass peaks below noise level. Meyer-Dulheuer [55] has also reported MALDI-TOFMS analysis of a coating/2-ethylhexyldiphenylphosphate sample. Quantitative determination of the additives by means of MALDI-ToFMS proved impossible. Possibly the development of reproducible (automated) sample handling procedures or thin films might overcome this problem. 

Despite the large number whole-cell MALDI protocols that have been tested a single approach has not yet been widely adopted. There remain different and sometimes conflicting reports in the literature regarding optimum methodologies.3 In addition to the ferulic acid matrix mentioned above sinapinic acid, a-yano-4-hydroxycinnamic acid, 2,4-hydroxyphenyl benzoic... 

A lot of features of MALDI are conveyed by its name it is a desorption ionization, produced by a laser beam, and assisted by a matrix (Figure 2.5). The analyte (1 pmol or less) is mixed with a suitable matrix in a 1 1000 or higher molar ratio. The matrix is composed of a compound with a strong absorption at the wavelength of the laser used. These two factors, matrix excess and its strong absorption, ensure that the energy from the laser pulse is absorbed by the matrix and not by the analyte, thus avoiding its decomposition. Nicotinic acid, sinapinic acid (SA), 2,5-dihydroxy benzoic acid (2,5-DHB) and 2-(4-hydroxyphenylazo)benzoic acid (HABA) are some of the most commonly used matrices for MALDI. 

Figure 2.9. Schematic of a matrix-assisted laser desorption/ionization (MALDI) event. The SEM micrograph depicts sinapinic acid-equine myoglobin crystal from a sample prepared according to the dried drop sample preparation method. In the desorption event neutral matrix molecules (M), positive matrix ions (M+), negative matrix ions (M-), neutral analyte molecules (N), positive analyte ions (+), and negative analyte ions (-) are created and/or transferred to the gas phase. Reprinted from A. Westman-Brinkmalm and G. Brinkmalm (2002). In Mass Spectrometry and Hyphenated Techniques in Neuropeptide Research, J. Silberring and R. Ekman (eds.) New York John Wiley Sons, 47-105. With permission of John Wiley Sons, Inc.

Fig. 10.3. Threshold fluence for positive ions of (O) cytochrome c and ( ) sinapinic acid as function of the molar matrix-to-protein ratio. Reproduced from Ref [42] by permission. John Wiley Sons, 1994.

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]. 

Purification of peptide-amphiphile 15 was achieved using a Vydac 214TP152022 C4 column (15-20 pm particle size, 300-A pore size, 250 x22 mm) at a flow rate of 5.0 ml-min1. The elution gradient was 30-100% B in 70 min where A was 0.05% TFA in H20 and B was 0.05% TFA in iPrOH with detection at 280 nm. MALDI-TOF-MS was performed using a sinapinic acid matrix. 

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... 

The power of the method is greatly enhanced by using matrix assistance (matrix assisted laser desorption ionization, or MALDI). Two matrix materials, nicotinic acid and sinapinic acid, which have absorption bands coinciding with the laser employed, have found widespread use and sample molecular weights of up to two to three hundred thousand Da have been successfully analyzed. A few picomoles of sample are mixed with the matrix compound fol-... 

MALDI mass spectra were acquired on a Broker Reflex mass spectrometer equipped with a 337 nm nitrogen laser and a multiple sample stage source. The spectra were acquired in linear mode, represent the sum of 20 laser shots, and are un-smoothed. All ions were desorbed at a laser power just above threshold, at an ion extraction voltage of 30 kV. The matrix used was a saturated solution of 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid, Aldrich Chemicals) in a 1 1 water/acetonitrile solution. Low-mass matrix ions were deflected by the application of a voltage pulse. 

Mass spectrometry (matrix assisted laser desorption) of the cleavage products was done by dissolving the samples in 0.1% TFA then mixing 1 pi of sample solution with 1 pi internal standard solution (bovine insulin at 0.5 pmole/pl) and 1 pi saturated matrix solution (100 mM sinapinic acid). The mixture was loaded on the probe tip, air dried, and placed in the mass spectrometer. 

In order to determine the optimum sample preparation conditions, we initially tried several sample preparation methods in the absence of contaminants. The following peptide and protein mixture was prepared (peptides and protein were purchased from Sigma Chemicals, St. Louis, MO) 1 pmol/jul Parathyroid Hormone Fragment 39-68 (PHF), MW = 3285.7, 10 pmol/ul Pigeon Heart Cytochrome C (PHC), MW = 12,173, and 10 pmol/ul Bovine Serum Albumin (BSA), MW = 66,256. A saturated solution of Sinapinic acid in 1 1 ethanol water was used as matrix. 

Fig 1. 1 pmol Parathyroid Hormone Fragment 39-68 (PHF), lOpmoI Pigeon Heart Cytochrome C (PHC), 10 pmol Bovin Serum Albumin (BSA) in saturated sinapinic acid matrix, desorbed from a. Polyethylene Membrane 113-2 b. Polypropylene Membrane 1222 c. Type 61 Disposable IR Card d. C8 Extraction Disk e. C18 Extraction Disk. 

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. 

Short pulses of laser radiation are used, typically at 337 nm, (nitrogen laser) but UV or infrared (IR) lasers can also be employed, depending on the matrix compound selected. Common matrix compounds are 2,5-dihydroxybenzoic acid, nicotinic acid, sinapinic acid, and a-cyanocarboxylic acid.6... 

FIGURE 4.8 MALDI-TOF mass spectra of three proteins in a 10 mg/ml sinapinic acid and 0.1 to 0.3% acetic acid matrix (a) cytochrome-c (eqnine), =... 

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