Enhanced MALDI-TOF MS analysis of phosphopeptides using an optimized DHAP/DAHC matrix

Abstract

Selecting an appropriate matrix solution is one of the most effective means of increasing the ionization efficiency of phosphopeptides in matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In this study, we systematically assessed matrix combinations of 2, 6-dihydroxyacetophenone (DHAP) and diammonium hydrogen citrate (DAHC), and demonstrated that the low ratio DHAP/DAHC matrix was more effective in enhancing the ionization of phosphopeptides. Low femtomole level of phosphopeptides from the tryptic digests of alpha-casein and beta-casein was readily detected by MALDI-TOF-MS in both positive and negative ion mode without desalination or phosphopeptide enrichment. Compared with the DHB/PA matrix, the optimized DHAP/DAHC matrix yielded superior sample homogeneity and higher phosphopeptide measurement sensitivity, particularly when multiple phosphorylated peptides were assessed. Finally, the DHAP/DAHC matrix was applied to identify phosphorylation sites from alpha-casein and beta-casein and to characterize two phosphorylation sites from the human histone H1 treated with Cyclin-Dependent Kinase-1 (CDK1) by MALDI-TOF/TOF MS.

Figure 1

The MALDI mass spectra of the tryptic digest of α-casein (250 fmol on target) measured in positive ion mode with DHAP/DAHC matrix at different ratios (10 : 1 to 1 : 80). The signal intensity is magnified 10-fold in the m/z range from 2575 to 3025. The number of sites on each phosphopeptide is equal to the number of asterisks (*) shown.

Figure 2

Top two views show the MALDI samples containing the phosphopeptide pT using (a) DHAP/DAHC matrix and (b) DHB/PA matrix. The two center pictures show the signal intensities of the phosphopeptides pT (250 fmol on target) measured with (c) DHAP/DAHC matrix and (d) DHB/PA matrix. The images only show a cut at signal intensity of 400 mv for clarity. The positions with signal intensities above 400 mv are indicated in purple. The bottom row (e) shows a schematic of the data acquisition method for investigating sample homogeneity of the matrix. In total, 196 positions were selected as a 14 × 14 spots array to cover the crystalline matrix/analyte layer, and 50 profiles (5 shots per profiles) were accumulated at each position.

Figure 3

The MALDI mass spectra of the tryptic digest of (a) and (c) α-casein and (b) and (d) β-casein measured in positive (upper) and negative (lower) ion modes with the DHAP/DAHC matrix. The amount of the tryptic peptides mixture ranged from 5 fmol to 250 fmol on target. The signal intensity is magnified 5-fold in the m/z range from 2400 to 3150 in section a.

Figure 4

The MALDI mass spectra of two synthesized phosphopeptides pT and pY mixed at equimolarity from 5 to 250 fmol on target, which were measured in positive (a) and negative (b) ion mode with DHAP/DAHC, respectively.

Figure 5

Tryptic digests of α- (upper) and β-casein (lower) (12.5–250 fmol on target) were detected with DHAP/DAHC (a) and (c) and DHB/PA (b) and (d) by MALDI-TOF MS in positive mode. DHAP/DAHC positions were selected randomly in the outer ring of the sample; for DHB/PA, only signals in hot spots were collected. In total, 200 profiles were accumulated for each sample. The number of sites on each phosphopeptide was equal to the number of asterisks (*).

Figure 6

The MALDI MS/MS spectra of phosphopeptides (a) Tβ1, (b) Tα3, and (c) Tβ2 (250 fmol for each on target) from α-casein and β-casein were obtained by MALDI-TOF/TOF MS. The sequential losses of H₃PO₄ from the parent ions are noted. The fragment patterns of the peptides are indicated in the magnified MS/MS spectra. “X” in the amino sequence shows a dehydroalanine residue converted from a phosphoserine residue by beta-elimination of H₃PO₄.

Figure 7

The MALDI MS analysis of phosphopeptides from CDK1-treated human histone H1. MS spectra of the tryptic peptides from CDK1-treated histone H1 with the untreated (Figure 7(a)) and alkaline phosphatase-treated histone H1 (Figure 7(b)). The two panels on the right show the magnified spectra to indicate the two phosphopeptides labeled with asterisks (*). The MALDI-TOF/TOF-MS analysis of phosphorylation sites on the two monophosphopeptides VApTPKKASKPK, m/z 1234.7 (c), APTKKPKApTPVKK, m/z 1473.9 (d) from the CDK1-treated human histone H1. The neutral-loss peak of phosphopeptide was noted as [MH-H₃PO₄]⁺. The fragment patterns of peptides were shown in the magnified MS/MS spectra. “B” in the amino sequence indicates a dehydroamino-2-butyric acid residue converted from a phosphothreonine residue by beta-elimination of H₃PO₄. All the spectra were detected using DHAP/DAHC matrix in the positive mode.