Matrix cyano-4-hydroxy-cinnamic acid

Matrix assisted laser desorption ionization time-of-flight (MALDI-TOE) mass spectrometry was carried out with a PerSeptive Biosystems Voyager-DE-RP MALDl-TOF mass spectrometer. A 337-nm UV nitrogen laser producing 3-ns pulses was used in the reflectron mode. The samples were prepared by mixing 10 pi of a 0.1 M HAc solution of the sample with 20 pi of a solution of 3 mg/1 a-cyano-4-hydroxy cinnamic acid in wafer. One pi of that solution was loaded on the gold-sample plate. 

Matrix/Energy Absorbing Molecule (EAM) a-cyano-4-hydroxy cinnamic acid (CHCA), 5 mg/tube (Bio-Rad Laboratories). 

About 10% of the sample was concentrated in a vacuum centrifuge to give an estimated concentration of 1 pmol/pl if possible. Samples were not dried down completely to avoid sample loss. The MALDI matrix was a saturated solution of a-cyano-4-hydroxy cinnamic acid dissolved in water/acetonitrile (7 3) [8]. A 0.5-pl aliquot of the matrix solution was placed on the stainless steel probe and mixed with an equal volume of sample solution. The mixture was left to dry at room temperature prior to introduction into the mass spectrometer. A new sample preparation method which decouples matrix surface preparation and sample handling was also used [9]. 

MALDI-MS was performed using a Kratos Kompact MALDI III mass spectrometer fitted with a standard 337 nm nitrogen laser, and operated in the linear mode at an accelerating voltage of 20 kV. Two sample preparation methods were used (1) for collected peptides, 0.3 pL aliquots of sample and matrix (a-cyano-4-hydroxy cinnamic acid, Biomolecular Separations, Inc.) were mixed on the probe slide and allowed to air dry or (2) for unfiactionated digests, a thin polycrystalline film was prepared according to (11) (with modifications for use on a probe slide (12)), matrix and sample aliquots were mixed (usually 0.3 pL each) on this surface and prior to drying, rinsed twice with 2 pL of deionized water. 

Peptides were sequenced using either an Applied Biosystems Model 470 or 477 or a Hewlett Packard GIOOOA protein sequencer, each equipped with narrow bore RP-HPLC for on-line analysis of the PTH-amino acids. Mass analysis of peptides was performed using matrix-assisted laser desorption/ionization mass spectrometry on a KRATOS MALDl 111 with a-cyano-4 hydroxy-cinnamic acid as the matrix. 

Materials. Trypsin, Lys C, chymotrypsin and adrenocorticotropic hormone fragment (18-39) were purchased from Sigma (St. Louis, Mo). Tris(hydroxymethyl)aminomethane (Tris), sucrose, acetonitrile, HPLC grade water and acetic acid were purchased from Fisher Scientific (Pittsburgh, PA). Matrix (a-cyano 4-hydroxy-cinnamic acid) was purchased from Hewlett Packard (Palo Alto, CA). The low-molecular weight calibration standard was purchased from Bio-Rad (Richmond, CA). 

Figure 3. MALDI mass spectrum of the total supernatant from the tryptic digest of the H,K-ATPase-enriched tubulovesicles. The H,K-ATPase was digested with tiypsin and the vesicles centrifuged to separate supernatant from the pellet. An aliquot of the supernatant was analyzed by MALDl/MS in the reflectron ion mode using a-cyano 4-hydroxy cinnamic acid as a matrix. The signals are denoted by numbers and were assigned to a-subunit peptides (Table 1).

Dissolve the peptide ladder in 3 to 5 ml 50% aq. acetonitrile/0.1% (v/v) TFA and ultrasonicate for 5 minutes. Pipette several aliquots (0.3 to 0.5 al) one after the other onto the carrier and let them air dry for 5 minutes each. Finally, pipette 0.3 il matrix solution (1% (w/v) a-cyano-4-hydroxy cinnamic acid in 50% aq. acetonitrile/0.1% (v/v)/TFA) onto the dried sample and air dry again. Insert this preparation into the MALDI mass spectrometer and analyze it. Important ... 

The selection of the matrix is of major importance in MALDI because the amount of energy transferred to the analyte is matrix-specific. Examples of matrices include dihydroxybenzoic acid, alpha-cyano-4-hydroxy-cinnamic acid, and sinapinic acid (also called sinapic acid). Alpha-cyano is the choice for peptides up to 5 kDa, while sinapinic acid is used for larger peptides and proteins. The reason for the changeover from one matrix to the other is that alpha-cyano transfers larger amounts of energy to analytes than does sinapinic acid, resulting in the destruction of larger peptides and proteins. Samples and matrices are preferably prepared in the same solvents, e.g., an acetonitrileiwater (1 1 v/v) mixture that may contain 0.1-1% trifluoroacetic acid. The concentration of the matrix is 10 mg/ml. The simplest approach with alpha-cyano and sinapinic acid, and an acetonitrile water solvent mixture, is to prepare saturated... 

Mix equal volumes of bacterial cell supernatant and saturated solution MALDI matrix (67% water, 33% acetonitrile, 0.1-0.2% TFA) in an Eppendorf tube. Typical MALDI matrices for this application such as a-cyano-4-hydroxy-cinnamic acid (HCCA),sinapinic acid, and ferulic acid are mixed in an Eppendorf tube. 

I. 2 V vs. Ag/AgCl in the presence of mvRuOx. Before electrolysis, a peak related to glutathione appears at 308.1 m/z. The peak at 355.4 m/z is related to the cysteic acid analogue of glutathione. Other peaks are from the a-cyano-4-hydroxy cinnamic acid matrix. (From... 

This results in co-crystaUization of matrix and sample and therefore higher sample concentration focused at the distinct hydrophilic anchor points [16]. One of the most commonly used matrices is a saturated solution of a-cyano-4-hydroxy-cinnamic acid in 97% acetone/3% water containing a small amoimt of TFA to support the ionization of the sample molecules in the MALDI source. The matrix is directed to the AnchorChip target and this is then dried. The sample fractionation after HPLC can be performed automatically using special sample fractionation systems (e.g., Probot, LC Packings Dionex). The target is introduced to a special desk and can be adjusted in the three XYZ planes so that the small nano-LC volume is placed directly onto the matrix. Using this set-up, fractions of volume less than 40 nL can be collected automatically. After collection, the individual fractions are recrystallized from 0.01% TFA, 30% acetone, and 60% ethanol in water and analyzed. 

Peptide ladder samples were analyzed on a matrix-assisted laser desorption time-of-flight mass spectrometer constructed at The Rockefeller University and described elsewhere (8,9). The individual peptides (9 residues to 18 residues in one vial and 19 to 32 residues in another vial) were mixed in approximately equal amounts and dissolved in water. The ladder mixtures were added to the matrix material (4-hydroxy-a-cyano-cinnamic acid (4HCCA) in formic acid/water/isopropanol 1 3 2) to a final concentration of 1-5 pM for each peptide component. The complete peptide ladder, which ranged from the 9 mer to the 32 mer (except 16 mer and 28 mer) was measured from 4HCCA in water/acetonitrile 2 1. The final concentration of each peptide component was in the range of 0.2-1 pM. Bovine insulin emd substance P were used as internal calibrants. 

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