Effective enrichment and mass spectrometry analysis of phosphopeptides using mesoporous metal oxide nanomaterials

Abstract

Mass spectrometry (MS)-based phosphoproteomics remains challenging due to the low abundance of phosphoproteins and substoichiometric phosphorylation. This demands better methods to effectively enrich phosphoproteins/peptides prior to MS analysis. We have previously communicated the first use of mesoporous zirconium dioxide (ZrO(2)) nanomaterials for effective phosphopeptide enrichment. Here, we present the full report including the synthesis, characterization, and application of mesoporous titanium dioxide (TiO(2)), ZrO(2), and hafnium dioxide (HfO(2)) in phosphopeptide enrichment and MS analysis. Mesoporous ZrO(2) and HfO(2) are demonstrated to be superior to TiO(2) for phosphopeptide enrichment from a complex mixture with high specificity (>99%), which could almost be considered as a "purification", mainly because of the extremely large active surface area of mesoporous nanomaterials. A single enrichment and Fourier transform MS analysis of phosphopeptides digested from a complex mixture containing 7% of alpha-casein identified 21 out of 22 phosphorylation sites for alpha-casein. Moreover, the mesoporous ZrO(2) and HfO(2) can be reused after a simple solution regeneration procedure with comparable enrichment performance to that of fresh materials. Mesoporous ZrO(2) and HfO(2) nanomaterials hold great promise for applications in MS-based phosphoproteomics.

Figure 1. Structural characterization of the mesoporous metal oxide nanomaterials

TEM micrographs for mesoporous TiO₂ (a), ZrO₂ (c), and HfO₂ (e); and the SAXS diffraction patterns for mesoporous TiO₂ (b), ZrO₂ (d), and HfO₂ (f).

Figure 2. Mesoporous metal oxide for phosphopeptide enrichment from a tryptic α-casein digest

Positive ion mode ESI/FTMS spectra of peptide mixtures before enrichment (a), and after enrichment with mesoporous TiO₂ (b), ZrO₂ (c), and HfO₂ (d). Circle, double circle, triangle, square, and star indicate singly, doubly, triply, quadruply and quintuply phosphorylated peptides, respectively. Phosphopeptides labeled with numbers are identified and shown in Table 1.

Figure 3. Comparison of mesoporous metal oxide for phosphopeptide enrichment from a tryptic α-casein digest

Negative ion mode ESI/FTMS spectra of peptide mixtures before enrichment (a), and after enrichment with mesoporous TiO₂ (b), ZrO₂ (c), and HfO₂ (d). Circle, double circle, triangle, square, and star indicate singly, doubly, triply, quadruply and quintuply phosphorylated peptides, respectively. Phosphopeptides labeled with numbers are identified and shown in Table 1. Insets are expanded MS spectra at m/z 1365–1369; a singly charged non-phosphopeptide at m/z 1366 in (a), and doubly charged quintuply phosphorylated peptide (QMEAES*IS*S*S*EEIVPNS*VEQK), p14, at m/z 1367 in (b, c, d).

Figure 4. Comparison of mesoporous metal oxide for phosphopeptide enrichment from a tryptic digest of the 6-protein mixture

Negative ion mode ESI/FTMS spectra of peptide mixtures before enrichment (a), and after enrichment with mesoporous TiO₂ (b), ZrO₂ (c) and HfO₂ (d). Circle, double circle, triangle, square, and star indicate singly, doubly, triply, quadruply and quintuply phosphorylated peptides, respectively. Phosphopeptides labeled with numbers are identified and shown in Table 1. Insets are expanded MS spectra at m/z 962–964; a singly charged non-phosphopeptide at m/z 962.47 in (a) and a doubly charged bisphosphopeptide (DIGS_pES_pTEDQAMEDIK ) at m/z 962.33 in (b, c, d).

Figure 5. Reuse of mesoporous metal oxides

Negative ion mode ESI/FTMS spectra of blank, and peptide mixtures digested from α-casein with trypsin enriched with mesoporous ZrO₂ (a, b) and HfO₂ (c,d) materials that were regenerated with concentrated NH₄OH and ACN. Asterisks indicate identified phosphopeptides shown in Table 1. NL: normalized intensity level of the most abundant peak.

Scheme 1

Mesoporous metal oxide materials synthesis scheme.