Enhanced MALDI-TOF MS Analysis of Phosphopeptides Using an Optimized DHAP/DAHC Matrix

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 α-casein and β-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 α-casein and β-casein and to characterize two phosphorylation sites from the human histone H1 treated with Cyclin-Dependent Kinase-1 (CDK1) by MALDI-TOF/TOF MS.


Introduction
Reversible phosphorylation of serine, threonine, and tyrosine residues is one of the most common and important regulatory posttranslational modifications of proteins and plays a crucial role in many biological processes, including cellular signal transduction, regulation of enzyme activities, metabolic regulation, molecular recognition and biomolecular interaction, protein localization, cell differentiation and proliferation [1][2][3][4][5]. Therefore, identifying phosphorylated proteins is an important step to elucidate the structural and functional role of the protein's phosphorylation regulation.
2, 6-Dihydroxyacetophenone (DHAP) was first used by Gorman et al. as a matrix for MALDI analysis of fragile peptides, disulfide bonding, and small proteins, including 2 Journal of Biomedicine and Biotechnology phosphopeptides [24]. In recent years, Xu et al. reported that the DHAP/DAHC matrix combination was used as an effective MALDI matrix in detecting methyl esterified phosphopeptides by MALDI-TOF-MS [26,27]. After systematic optimization in this study, we found that contrary to previous reports, the matrix forms at a lower ratio of DHAP/DAHC and that this ratio shows greater efficiency in improving ionization ability of phosphopeptides. We investigated the characteristics of an optimal DHAP/DAHC matrix, and analyzed morphology and detection sensitivity of phosphopeptides. Using tryptic digests of αand β-casein, we demonstrated a CID MS/MS analysis of phosphopeptides by MALDI-TOF/TOF-MS with an optimized DHAP/DAHC matrix. Finally, we used this approach to analyze phosphorylation sites on human histone H1 treated with Cyclin-Dependent Kinase-1 (CDK1). were digested overnight at 37 • C with trypsin at an enzyme/substrate ratio of 1 : 25 (w/w). After digestion, TFA was added into the sample solution at 1.0% to quench the reaction. The digest was diluted with 60% acetonitrile containing 0.1% TFA to produce a series of sample solutions at concentrations of 1 pmol/μL, 200 fmol/μL, 50 fmol/μL, 20 fmol/μL, and 10 fmol/μL. A stock solution containing standard peptides pT (1 mM) and pY(1 mM) was prepared using ultra pure water. The solution was diluted with 60% acetonitrile containing 0.1% TFA to 1 pmol/μL, 200 fmol/μL, 50 fmol/μL, 20 fmol/μL, and 10 fmol/μL. All of the peptide solutions prepared for DHB/PA measurements were diluted with 1% phosphoric acid.

Human Histone H1 Phosphorylation and Digestion.
The phosphorylation reaction between histone H1 and CDK1 was carried out in vitro. Briefly, the reaction solution was prepared with 3 μL 10 × CDK1 reaction buffer, 0.15 μL CDK1 (150 U), 4 μL histone H1 (1 μg/μL), 100 μM ATP, and 13 μL water, then incubated at 30 • C for 45 minutes. The phosphorylated histone H1 was then separated using SDS-PAGE (12%) and stained with Coomassie Brilliant Blue using microwave-assisted methods [28]. The in-gel digestion of phosphorylated histone H1 was performed as follows: the band of histone H1 was excised from the polyacrylamide gel, washed twice with water, and destained with 40% acetonitrile/50 mM NH 4 HCO 3 . The gel pieces were dehydrated with 100% acetonitrile and dried for 5 min using a speedvac. Disulfide bonds were reduced with DTT (10 mM, 56 • C, 45 minutes), and the free sulfhydryl groups were alkylated with iodoacetamide (55 mM, 25 • C, 60 minutes in the dark). Gel pieces were washed with 50 mM NH 4 HCO 3 , 50% acetonitrile/50 mM NH 4 HCO 3 , and dehydrated with 100% acetonitrile. After drying with a speedvac, the gel was rehydrated using 100 ng/μL trypsin (50 mM NH 4 HCO 3 , pH 8.3) on ice for 30 minutes, and the digestion was carried out at 37 • C for 60 minutes and then quenched with 1.0% TFA. The tryptic peptides were extracted twice with 60% acetonitrile containing 0.1% TFA, and then the combined digest solution was concentrated to 10 μL under vacuum.
To prepare for MALDI-TOF-MS analysis, 1 μL of peptide solution was mixed with 3 μL of matrix solution in an Eppendorf tube, and 1 μL of the peptide/matrix solution was spotted onto the MALDI sample plate and then crystallized either under vacuum (when DHAP/DAHC was used as matrix) or in the air (when DHB/PA was used as matrix).

Tα1
Tα3 Tα2  Tα9 Tα5 Tα4 Tα8  Tα7  Tα6   T11 T10 T4 T3 T18 Measurements using DHAP/DAHC were performed by detecting 15-20 different positions on the broad outer zone (see discussion below) for each sample to accumulate a total of 200 profiles (5 shots per profile). For measurements with DHB/PA, a total of 200 profiles (5 shots per profile) were accumulated by searching for 10-15 hot spots for each sample. All experiments were repeated three times.
The standard peptide pT (250 fmol on target) was used to determine sample homogeneity of the matrix. On each sample spot, 196 (14 × 14) positions were selected manually to ensure a full overview over the sample preparation. For each position, 50 profiles (5 shots per profile) were accumulated in positive ion mode. The signal intensities of pT in each position were recorded for further analysis.
Mass spectrometry data were processed with Launchpad 2.7.1 software (Shimadzu/Kratos, Manchester, UK). The data of the CID MS/MS spectra of phosphopeptides from histone H1 were compared to the SwissProt 56.6 protein sequence database (Homo sapiens) using the on-line MAS-COT database search engine (Matrix Science, London, UK) with the following parameters: trypsin with three missed cleavages; average mass values; peptide mass tolerance: ±0.8 Da; fragment mass tolerance: ±1.0 Da; fixed modifications: Carbamidomethyl (C), variable modifications: Oxidation (M), phosphor (ST). Only the peptides with ions score above 38 were accepted as significant matches.

Optimization of DHAP/DAHC Matrix Combination.
The solution composition of the matrix is a factor that significantly influences the ionization efficiency and the quality of MALDI mass spectra [29]. Therefore, we prepared a series of different ratios of DHAP (10 mg/mL) and DAHC (20 mg/mL) matrix solutions (10 : 1, 5 : 1, 1 : 1, 1 : 4, 1 : 8, 1 : 16, 1 : 20, 1 : 30, 1 : 40, 1 : 50, 1 : 60, and 1 : 80 v/v) and investigated these matrix combinations using the tryptic digest of α-casein, which contains nine phosphopeptides (Tα1-Tα9) and some nonphosphopeptides (such as T4, T10, T11, T18) (see Table 1). Figure 1 shows a comparison of the MALDI-TOF-MS analysis of the tryptic digest of α-casein (250 fmol on target) using matrix DHAP/DAHC at different ratios in positive ion mode. The signal intensity of phosphopeptides (especially for multiple phosphorylated peptides) changed dramatically with the ratio of DHAP/DAHC. It was clear that all phosphopeptides could be easily detected with a low DHAP/DAHC matrix ratio in the range of 1 : 30 to 1 : 60. In an optimal matrixsolution, the concentration  of DHAP was 1.6-3.3 mg/mL, which is lower than the concentration of 10-15 mg/mL used by other groups [24,26,27]. Once the DHAP/DAHC ratio was down to 1 : 80, most multiple phosphorylated peptides could no longer be detected, likely because there were not sufficient numbers of DHAP molecules to transfer the laser energy to the peptides to be protonated.
Another interesting phenomenon apparent in Figure 1: the signal intensity of nonphosphopeptides was stable (T3), enhanced (T10) or weakened (T11, T4, T18) with the  Tα1   Tα3   Tα4  Tα6  Tα9   Tα5  Tα8   Tα7  Tα2   Tα1   Tα3   Tα6   Tα2   Tα1   Tα3  Tα6  Tα2   Tα1 Tα3  Tα6  Tα2   Tα1  Tα6   Tα4  Tα9   Tα5  Tα8   Tα7   Tα4  Tα9   Tα5 Tα8  Tα2   Tα6   Tα4   Tα9   Tα5  Tα8   Tα7   Tα4  Tα9   Tα5  Ta8   Ta7   Tα4  Tα9   Tα5   Tα8   Tα7   Tα4  Tα9   Tα7   Tα8   Tα1   Tα3   Tα2   Tα6   Tα1   Tα3  Tα2   Tα6   Tα1  Tα3   Tα6   Tα1   Tα3  Tα6 Signal intensity (mv)           (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-H 3 PO 4 ] + . 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 H 3 PO 4 . All the spectra were detected using DHAP/DAHC matrix in the positive mode. change of the DHAP/DAHC ratio. We calculated the Bull and Breese Index (BB Index) [19] of these peptides, an index generally used to reflect the hydrophobic/hydrophilic nature of peptides. Higher BB Index values are associated with more hydrophilic peptides. For the phosphopeptide, we calculated the BB Index by replacing the phosphoserine or phosphothreonine residues with glutamic acid [19]. By comparing the BB Index of peptides listed in Table 1, we found that the phosphopeptides were nearly hydrophilic peptides with high BB Indices and that the nonphosphopeptides for which signal intensity was weakened at low DHAP/DAHC ratio were highly hydrophobic with lower BB Index (T4 : −5530, T11 : −6480, T18 : −6900). The nonphosphopeptide T10 for which the ion signal was enhanced at lower ratios of DHAP/DAHC was found to be slightly hydrophobic with a BB Index of −750. These findings indicated that the optimal DAHC/DHAP matrix tended to detect more hydrophilic peptides. Most phosphopeptides are hydrophilic, so we propose that the low ratio of DHAP/DAHC matrix could increase cocrystallization between matrix and phosphopeptides and further improve ionization efficiency of phosphopeptide in MALDI MS.

Sample Homogeneity.
In this study, the phosphopeptide pT (250 fmol on target) was used to investigate the sample homogeneity of optimized DHAP/DAHC matrix, and for a comparison, we chose matrix combination of 2,5-DHB with 1% phosphoric acid, a currently popular matrix used for detection of phosphopeptide [30][31][32]. It was apparent from the photographs of the MALDI sample spots that there was a dramatic difference between the optimized DHAP/DAHC (Figure 2(a)) and DHB/PA (Figure 2(b)). Small crystals formed in a DHAP/DAHC sample spot while larger, needle-like crystals formed in a DHB/PA sample spot. Furthermore, 196 (14 × 14) positions on the target were measured covering the crystalline layer for a full overview (Figure 2(e)), and the MS signal intensities of pT were monitored. The majority of the outer zones of the crystalline samples exhibited high signal intensities, except for a small center zone when DHAP/DAHC used as matrix (Figure 2(c)). In comparison, the crystalline DHB/PA matrix exhibited the hot-spot phenomenon, signals were primarily observed in the narrow, inhomogeneous ring at the edge of the sample layer, and the inner position almost did not exhibit any signal (Figure 2(d)). Therefore, the samples prepared with DHAP/DAHC were more homogeneous than those prepared with a DHB/PA matrix, reducing the need for time-consuming hot-spot searches. Based on the sample homogeneity of DHAP/DAHC and DHB/PA, in the next experiments, the data acquisition with DHAP/DAHC was carried out by randomly measuring the outer zone of the sample layer, while data were acquired from the DHB/PA matrix by searching for hot spots of sample.

Sensitivity Measurement of Phosphopeptides with the DHAP/DAHC Matrix.
In order to further evaluate the measurement sensitivity of phosphopeptides with the optimal DHAP/DAHC matrix, the tryptic digest of α-casein and βcasein with 2.5-250 fmol on the target were detected by MALDI-TOF MS in both positive and negative ion mode. Figure 3 shows that all eleven phosphopeptides from αcasein and β-casein could be detected with signal-to-noise (S/N) ratios greater than 3 at the low amount of 12.5 fmol in the positive mode (a) and (b) and detected at a lower amount of 5 fmol in the negative mode (c) and (d). The monophosphopeptides (Tα1 and Tβ1) could still be observed at 2.5 fmol on target in negative ion mode (Figure 3(c)). Phosphopeptides generally exhibited better S/N ratios in the MS spectra in negative ion mode than that in positive ion mode, although, consistent with previous reports [19], the absolute signal intensity of phosphopeptide was usually lower in negative ion mode. Because all tryptic phosphopeptides from α-casein and β-casein are serine-phosphorylated peptides, a threonine-phosphorylated peptide pT and a tyrosinephosphorylated peptide pY (2.5-250 fmol on target) were used to investigate whether the DHAP/DAHC matrix had a generic nature in detection of phosphopeptides. As expected, pT and pY could be readily detected down to 5 fmol on the target in both positive and negative modes ( Figure 4).
We further directly compared optimized DHAP/DAHC to DHB/PA. Tryptic digests of α-casein and β-casein (12.5-250 fmol on target) were detected using these two matrices ( Figure 5). When using the DHAP/DAHC matrix, all the phosphopeptides could readily be detected at three different sample amounts. By comparison, when DHB/PA was used as the matrix, although all the phosphopeptides in 250 fmol digest (on target) could be detected with DHB/PA, the amount of digest went down to 50 fmol (on the target), and only Tα1, Tα2, Tα3, Tα6, Tβ1, and Tβ2 could be observed. Furthermore, when the amount of digest went down to 12.5 fmol, only Tα1 and Tβ1 were detected, with weak signals. Comparing these results, it was clear that compared with DHB/PA, the optimized DHAP/DAHC had comparative sensitivity in the detection of singly phosphorylated peptides (Tα1 and Tβ1), but greater sensitivity in the detection of multiply phosphorylated peptides, such as Tβ2 (4p) and Tα4 (5p). Herein, we suggest that sample should be analyzed immediately after sample preparation when optimized DHAP/DAHC matrix used, as the crystals might not last too long under atmospheric conditions and in vacuo.

Sequencing of Phosphopeptides with High Energy CID MS/MS.
We employed the AXIMA-TOF 2 MALDI mass spectrometer (Shimadzu/Kratos, Manchester, UK) to perform high energy CID MS/MS of phosphopeptides to characterize phosphorylation sites. Figure 6 shows the MS/MS spectra of three phosphopeptides Tβ1, Tα3, and Tβ2 from αcasein and β-casein (250 fmol on target) with DHAP/DAHC as matrix. The number of [MH-98] + or [MH-80] + peaks in the MS/MS spectra indicated the number of phosphate groups in each phosphorylated peptide, and the fragment ions including a, b, and y ions clearly show the phosphorylation sites of the phosphopeptides Tβ1, Tα3, and Tβ2. These results demonstrated the feasibility of MALDI-TOF/TOF MS with the DHAP/DAHC matrix for the analysis of the phosphorylation sites of phosphopeptides, including singly and multiply phosphorylated peptides.

Application of the Optimized DHAP/DAHC Matrix in the Characterization of Phosphorylated Human Histone H1.
Further investigating the practicality of the DHAP/DAHC matrix, we used this approach to characterize human histone H1 phosphorylated with CDK1. Experimentally, phosphorylated histone H1 was separated by SDS-PAGE and ingel digested by trypsin. In order to generate appropriate peptide sizes for MALDI-MS analysis, since the sequence of histone proteins is known to contain many lysine and arginine residues, the trypsin digestion time was optimized and reduced to 1 hour. Figure 7(a) shows the results of the MALDI-TOF-MS analysis of the tryptic digest of the phosphorylated histone H1 without the desalting step, directly detected with the optimized DHAP/DAHC matrix in positive ion mode. After performing data processing using the on-line MASCOT search engine, 31 peptides, including 2 possible phosphopeptides (in Figure 7(a) labeled with " * "), were assigned to human histone H1, and 81% of the sequence was covered. These two peptides corresponded to the two monophosphopeptides of histone H1, comprising amino acids 117-127 (VATPKKASKPK, 1234.7 m/z; possible phosphorylation sites indicated with underlined letters) and 133-145 (APTKKPKATPVKK, 1473.9 m/z). The tryptic sample of H1 was also analyzed with DHB/PA, but the two peaks of the phosphopeptide were lower than those in Figure 7(a) (see Figure S1). To validate the MASCOT searching result for the two phosphorylated peptides, we compared the mass spectra of tryptic peptides of H1 before and after treatment with alkaline phosphatase, which can cleave the phosphate group from phosphopeptides. It was clear that the two peptide peaks (marked with asterisks in Figure 7(a)) had disappeared and two new peaks with a mass shift of 80 Da (HPO 3 = 80 Da) were visible (Figure 7(b)). This demonstrated that the two peptides (marked with asterisks in Figure 7(a) were singly phosphorylated peptides. To identify the phosphorylation sites, we used MALDI-TOF/TOF MS to perform MS/MS analysis of the two peptides (Figure 7(c) and 7(d)). Only one neutral-loss peak corresponding to [MH-H 3 PO 4 ] + indicated that both phosphopeptides were monophosphopeptides. After a MASCOT search, the phosphorylation sites of the two phosphopeptides were determined to be APTKKPKApTPVKK (pT indicating the phosphothreonine) and VApTPKKASKPK with ion scores of 53 and 50, respectively (scores exceeding 38 were accepted as significant matches).
In general, the protein phosphorylation sites by a particular protein kinase shared a set of "consensus sequence", which is necessary and sufficient for recognition by the kinase [33]. To further validate the phosphorylation sites of histone H1 identified with MALDI-TOF/TOF MS, we compared the two phosphorylated peptide sequences with the consensus sequence pS/pT-P-X-R/K, most frequently recognized by CDK1 [34,35], and found that both phosphopeptide sequences were consistent with the consensus sequence.
These results thus demonstrate that the DHAP/DAHC matrix was robust and effective in analyzing the protein phosphorylation of the biological sample by MALDI-MS.

Conclusion
By optimizing the matrix solution composition, we found that a low ratio of DHAP/DAHC formulation was more efficient in detecting phosphopeptides than earlier protocols. Compared with DHB/PA, the optimized DHAP/DAHC exhibited higher sample homogeneity, and the phosphopeptides in tryptic digests of αand β-casein without desalting and phosphopeptide enrichment could be measured with a higher sensitivity. Further, the optimized DHAP/DAHC showed high energy CID MS/MS analysis of the phosphorylation sites of phosphopeptides, including multiple phosphorylated peptides, and we successfully applied this method to the characterization of the phosphorylation sites of CDK1-treated human histone H1.