HCCA matrix

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... 

Our laboratory was the first to report the reactivity of sinapinic acid matrix with proteins from bacterial cell lysates and the apparent lack of reactivity of HCCA matrix [26,27]. The differential reactivity of sinapinic... 

For two matrices, DHB and sinapinic acid, the enhancement was smaller on a gold substrate. HCCA matrix gave a similar enhancement on both metals. This pattern is consistent with a two-photon ionization model based on interaction of matrix orbitals with the conduction band of the metal. 

Figure 4a MALDI-TOF mass spectral analysis of H-Thr-Arg-Asn-Leu-Ala-Asp-Gln-Glu-Asp-NH2. The spectrum was acquired on a TofSpec SE (Micromass) mass spectrometer equipped with a nitrogen laser and a reflectron, using a peptide solution of approximately 10 pmol//u,L and HCCA (Hewlett-Packard) as the matrix.

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

HCCA (0.67 mg/mL in ethanol acetone, 2 1). Dissolve approximately 1 mg HCCA in 500 pL acetone to make a 2-mg/mL stock solution. The matrix usually takes 5-10 min to completely dissolve at room temperature. We usually start the magnetic beads fractionation while waiting for the matrix to dissolve. Then add one part of above stock solution to two parts ethanol (e.g. 100 pL stock solution to 200 pL ethanol). Prepare the matrix solution daily. 

Matrix mixtures have also been used for improved MALDI-MS performance for both proteins and peptides. For example, a mixture of DHB and HCCA was found to improve spot-to-spot reproducibility for peptide mass mapping and to increase the protein sequence coverage [20]. For examples of other matrices useful for peptide analysis, see Table 3.2. Membrane proteins can be efScientiy analyzed using optimized sample preparation methods and detergents, as described by Cadene and Chait [22]. Also, in situ liquid-liquid extraction using ethyl acetate and HCCA may enable the analysis of integral membrane proteins by MALDI-MS [23]. For additional information on the choice of matrix and sample preparation, see also Chapter 1, Section 1.8. 

Nanosubstrate, rather than particle, approaches have been utilized in a handful of experiments [33, 34]. HCCA has been crosslinked to SU-8 photoresist polymer via cationic photoiortization, forming a hydrophobic surface. When aqueous sample droplets are applied to this support, surface tension during evaporation essentially concentrates the samples and thus improves the analysis sensitivity [34]. Another engineering approach has been to pre-deposit CHCA matrix crystals by vacuum sublimation onto an ultra-phobic surface. The resultant disposable chips contain an array of matrix spots which concentrate analytes from aqueous matrixes during the drying process. The approach has been applied to the quantitation of drug compounds in biofluids such as serum or urine [31]. 

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. 

Our laboratory used a number of different MALDI matrices (e.g., HCCA, sinapinic acid, ferulic add) for this work. HCCA is considered a hot matrix and favors the lower mass range generating singly, doubly. 

For routine detection of P-lactamase activity, a fresh bacterial culture is mixed with a p-lactam solution (meropenem or ertapenem) (Hrabak et al. 2011 Burckhardt and Zimmermarm 2011 Hrabak et al. 2013, 2014 Studentova et al. 2015) (Fig. 12.2). After incubation at 35-37 °C for 2 h, the reaction mixture is centrifuged and the supernatant is apphed onto a MALDI target, covered by a matrix solution (HCCA or DHB), and measured in a positive ion mode. In the first descrip-... 

On each defined spot, 20 nl of HCCA/ANI solution is applied. Five droplets of 100 pi are deposited at each spot per cycle, then 40 iterations are necessary to obtain the total volume. For slides after digestion, the matrix is deposited with the same array than the one used for trypsin deposition. In this case matrix is deposited exactly at the same position than the trypsin. 

Whole tissue matrix deposition is performed by applying a solution of HCCA/ANI using a micropipette ( 10-20 rl according to the surface to cover). Careful attention must be drawn on this step to avoid touching the tissue with the micropipette tip and not induce tissue damages. 

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

For classical analysis, 1 xl of sample solution and 1 (xl of matrix solution (HCCA/ANI) were mixed on the MALDI plate using the dried-droplet technique as a standard control for the different Tag-Mass molecules before imaging. 

Sinapinic acid is the matrix of choice for protein measurements. For peptides and lipids it is better to use 2,5-dihydroxybenzoic acid (DHB) or apha-cyano-4-hydroxycinnamic acid (HCCA). The latter has the advantage of giving very small crystals, therefore allowing highest lateral resolution. 

MALDl-MS owes much of its popularity to its usefulness as an analytical tool for the characterization of peptides and proteins. While an extremely large number of matrix candidates have been investigated for their applicability in peptide and protein analysis, a relatively small group has proven reliable over the past 15 + years. The members of this small group include 2,5-dihydroxybenzoic acid (2,5-DHB), a-cyanohydroxycinnamic acid (CHCA or HCCA), and sinapinic acid (SA). 

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