A binary matrix for improved detection of phosphopeptides in matrix‐assisted laser desorption/ionization mass spectrometry

Abstract

Application of matrix-assisted laser-desorption/ionization mass spectrometry (MALDI MS) to analysis and characterization of phosphopeptides in peptide mixtures may have a limitation, because of the lower ionizing efficiency of phosphopeptides than nonphosphorylated peptides in MALDI MS. In this work, a binary matrix that consists of two conventional matrices of 3-hydroxypicolinic acid (3-HPA) and alpha-cyano-4-hydroxycinnamic acid (CCA) was tested for phosphopeptide analysis. 3-HPA and CCA were found to be hot matrices, and 3-HPA not as good as CCA and 2,5-dihydroxybenzoic acid (DHB) for peptide analysis. However, the presence of 3-HPA in the CCA solution with a volume ratio of 1:1 could significantly enhance ion signals for phosphopeptides in both positive-ion and negative-ion detection modes compared with the use of pure CCA or DHB, the most common phosphopeptide matrices. Higher signal intensities of phosphopeptides could be obtained with lower laser power using the binary matrix. Neutral loss of the phosphate group (-80 Da) and phosphoric acid (-98 Da) from the phosphorylated-residue-containing peptide ions with the binary matrix was decreased compared with CCA alone. In addition, since the crystal shape prepared with the binary matrix was more homogeneous than that prepared with DHB, searching for 'sweet' spots can be avoided. The sensitivity to detect singly or doubly phosphorylated peptides in peptide mixtures was higher than that obtained with pure CCA and as good as that obtained using DHB. We also used the binary matrix to detect the in-solution tryptic digest of the crude casein extracted from commercially available low fat milk sample, and found six phosphopeptides to match the digestion products of casein, based on mass-to-charge values and LIFT TOF-TOF spectra.

Carbon Dots and 9AA as a Binary Matrix for the Detection of Small Molecules by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

“…Recently, some binary matrices have been proposed to overcome the above drawbacks of singular matrix and improve the quality of D/I process via suppressing the matrix clusters and fragments [25], producing homogeneous sample-matrix co-crystallization [26], expanding the detection range [27], and increasing the detection sensitivity [28]. However, the binary matrices generated with carbon-based materials have never been proposed with increased sensitivity and reproducibility.…”

Intact spore MALDI-TOF mass spectrometry and proteomic analysis of Puccinia pathogenic fungi

“…The best results as regards to the homogeneity of cocrystals were achieved for the binary and ternary mixtures (CA:SA, FA:SA a CA:FA:SA). This is in accordance with previous findings that the use of mixed matrices has an advantage in obtaining fine cocrystals with the sample, which show a homogeneous on-target distribution [15,[30][31][32]. The number of signals and their intensity was evaluated in three m/z regions: 1200-3500; 3500-5000; 5000-10,000.…”

Sample Preparation for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

“…To mitigate the effects of sample spot inhomogeneity, binary matrices that include two matrix materials, such as CHCA/DHB, 22,23 DHB/aminopyrazine, 24 CHCA/3-HPA for phosphopeptides, 25 and DHB/CHCA or DHB/SA for glycans and glycoproteins, 26 have been developed. Recently, a combination of three materials (DHB, CHCA, and SA) in a single matrix was used to improve performance in analyses of polyethylene glycol (PEG) polymers.…”