A two‐matrix system for MALDI MS analysis of serine phosphorylated peptides concentrated by Fe 3 O 4 /Al 2 O 3 magnetic nanoparticles

̨-Cyano-4-hydroxycinnamic acid (CHCA) is often selected as the matrix-assisted laser desorption/ionization (MALDI) matrix for peptide analysis. However, CHCA is recognized as a hot matrix,1–5 which is seldom used in the analysis of phosphopeptides. This is because extensive fragmentation from intact analytes is frequently observed in MALDI mass spectra when CHCA is used as the matrix.6,7 However, fragmentation derived from phosphopeptides can provide data for confirmation of phosphorylation of unknown ions7,8 after enrichment by the affinity probes. These results suggested an approach for confirmation of the presence of phosphorylated peptides simply by using CHCA as the MALDI matrix. 2,5-dihydroxybenzoic acid (DHB) is the most common matrix for MALDI MS analysis of phosphopeptides.9,10 The detection limit of phosphopeptides using 2,5-DHB as the matrix is generally better than that of using CHCA. Unlike analytes homogeneously distributing among CHCA crystalline, inhomogeneous crystalline of 2,5-DHB/analyte, i.e. ‘sweet spots’, is often observed during MALDI sample preparation. Although it is time-consuming to search ‘sweet spots’ to obtain analyte signals, analytes are generally concentrated on specific spots, which, therefore, lower the detection limit of analytes when 2,5-DHB is used as the MALDI matrix. Two matrix-systems have been used for improvement of MALDI MS results.11,12 Laugesen and Roepstorff11 have demonstrated that the mass spectral results with increased sequence coverage and spot-to-spot reproducibility for peptide mass mapping were obtained when the mixture of CHCA and 2,5-DHB was used as the MALDI matrix. Cotter and coworkers12 have reported that homogeneous sample deposition, improved peak intensity and good mass resolution were obtained in MALDI MS analysis by modifying the MALDI target to be hydrophobic and using CHCA/2,5-DHB mixture as the MALDI matrix. We herein examine the results of using the mixture of 2,5-DHB and CHCA as the MALDI matrix for MALDI MS analysis of serine phosphopeptides after enrichment from tryptic digests of proteins using Fe3O4/Al2O3 as the affinity probes, which have been demonstrated very effectively in enriching phosphopeptides selectively from complex samples.13 We expect that a low concentration of phosphopeptides could be characterized by using this two-matrix system for MALDI MS analysis. The details of the preparation steps of Fe3O4/Al2O3 have been described elsewhere.13 Proteins (1 mg) were prepared in an ammonium bicarbonate (Riedel-de Haën, Germany) aqueous solution (50 mM, 0.25 ml) containing urea (Riedel-de Haën, Germany) (8 M), followed by the incubation at 38 °C for 30 min. Dithiothreitol (0.4 mg) (Sigma, St. Louis, MO) was added to the solution and the mixture reacted at 45 °C for 1 h. After cooling to room temperature, the mixture was mixed with iodoacetic acid (1 mg) (Sigma, St. Louis, MO) prepared in aqueous ammonium bicarbonate (50 mM) in an ice bath. The sample vial was wrapped with aluminum foil and vortex-mixed for 2.5 h at room temperature. After completion of the reaction, an aqueous ammonium bicarbonate (50 mM, 0.5 ml, pH 3⁄48.2) was added into the above mixture. The denatured protein solution (1 mg/ml D 3⁄44 ð 10 5 M) obtained above was digested enzymatically by mixing with trypsin (0.1 mg/ml) (Sigma, St. Louis, MO) prepared in ammonium bicarbonate (50 mM, pH 3⁄48.2), at a weight ratio of 50 : 1, and incubated at 37 °C for 18 h. The protein digest with a concentration of 10 6 M was prepared directly by dilution with 0.15% trifluoroacetic acid (TFA)/water solution. However, the sample with a concentration of 5 ð 10 11 M was prepared by diluting the original tryptic digest (4 ð 10 5 M) to 5 ð 10 10 M by ammonium bicarbonate (50 mM) followed by 10-fold dilution with 0.15% TFA/water solution. Fe3O4/alumina magnetic nanoparticles (40 mg/ml) were prepared in 0.15% TFA(Riedel-de Haën, Germany)/water under sonication for 30 min. The magnetic