Comprehensive Evaluation of Different TiO2-Based Phosphopeptide Enrichment and Fractionation Methods for Phosphoproteomics

Abstract

Protein phosphorylation is an essential post-translational modification that regulates multiple cellular processes. Due to their low stoichiometry and ionization efficiency, it is critical to efficiently enrich phosphopeptides for phosphoproteomics. Several phosphopeptide enrichment methods have been reported; however, few studies have comprehensively compared different TiO2-based phosphopeptide enrichment methods using complex proteomic samples. Here, we compared four TiO2-based phosphopeptide enrichment methods that used four non-phosphopeptide excluders (glutamic acid, lactic acid, glycolic acid, and DHB). We found that these four TiO2-based phosphopeptide enrichment methods had different enrichment specificities and that phosphopeptides enriched by the four methods had different physicochemical characteristics. More importantly, we discovered that phosphopeptides had a higher deamidation ratio than peptides from cell lysate and that phosphopeptides enriched using the glutamic acid method had a higher deamidation ratio than the other three methods. We then compared two phosphopeptide fractionation methods: ammonia- or TEA-based high pH reversed-phase (HpH-RP). We found that fewer phosphopeptides, especially multi-phosphorylated peptides, were identified using the ammonia-based method than using the TEA-based method. Therefore, the TEA-based HpH-RP fractionation method performed better than the ammonia method. In conclusion, we comprehensively evaluated different TiO2-based phosphopeptide enrichment and fractionation methods, providing a basis for selecting the proper protocols for comprehensive phosphoproteomics.

Characterization ofN-glycosylation and its Functional Role in SIDT1-Mediated RNA Uptake

“…The purified samples were digested into peptides as previously described (53). Briefly, samples were reduced with DTT at a final concentration of 10 mM at 30 °C for 1 hour.…”

Rapid Characterization of Phospholipids from Biological Matrix Enabled by Indium Tin Oxide (ITO) Coated Slide Assisted Enrichment MALDI Mass Spectrometry

“…[33] ITO-coated material like other metal oxides utilized in metal oxide affinity chromatography (MOAC) has also been reported to possess a similar characteristic to electrostatically interact with polar species. [31,34,35] Compared to other purification methods, ITO material can be subjected to a wider pH range, which creates more possibilities for versatile sample handling processes and different pH-buffer treatments. [35] In previous studies, phosphopeptides were comprehensively investigated and proven to be retained by ITO-coated glass slide via the acid-base rinsing procedure and the phosphorylation sites were able to be deciphered with this approach.…”

“…[31,34,35] Compared to other purification methods, ITO material can be subjected to a wider pH range, which creates more possibilities for versatile sample handling processes and different pH-buffer treatments. [35] In previous studies, phosphopeptides were comprehensively investigated and proven to be retained by ITO-coated glass slide via the acid-base rinsing procedure and the phosphorylation sites were able to be deciphered with this approach. [35] It has been reported that the ITO-SPE method offers a faster manner to assist processing the samples, which we can save time preparing columns, loading samples, washing, and eluting the extracts with high pH solvents.…”

“…[35] In previous studies, phosphopeptides were comprehensively investigated and proven to be retained by ITO-coated glass slide via the acid-base rinsing procedure and the phosphorylation sites were able to be deciphered with this approach. [35] It has been reported that the ITO-SPE method offers a faster manner to assist processing the samples, which we can save time preparing columns, loading samples, washing, and eluting the extracts with high pH solvents. By adopting this method, the extracts can be simply obtained by pipetting serum or biological specimens onto the ITO-coated planar surface and analyzing extracts after washing and matrix application.…”

“…By adopting this method, the extracts can be simply obtained by pipetting serum or biological specimens onto the ITO-coated planar surface and analyzing extracts after washing and matrix application. [35] Herein, we proposed a simple and rapid enrichment workflow for lipidomic analysis of complex human serum samples. The optimizations of sample loading, washing, and selections of organic matrices are discussed.…”