Protein phosphorylation introduces negative charges on the hydroxyl groups of serine, threonine, and tyrosine residues, reducing the ionization efficiency of phosphorylated peptides. The low abundance of phosphorylated peptides often diminishes their detection using mass spectrometry. To enhance the identification of the low-abundance peptides, an enrichment step was often used, which complicated the high-throughput analysis of phosphorylated proteomes. In this study, we developed a titanium dioxide surface-modified macroporous silicon encapsulated micropipette tips, loaded with trypsin, to integrate rapid enzymatic protein hydrolysis with selective enrichment and extraction of phosphorylated peptides within a microfluidic enzyme reactor. This streamlined approach simplified the protein sample preparation process, combining enzymatic hydrolysis, selective enrichment and separation while maintaining high efficiency. The method enabled comprehensive analysis of complex cancer cell line samples in 1–2 h. Successful detection of phosphorylated peptides from protein mixtures was achieved using matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry. This application may provide the potential for high-throughput phosphoproteomics and advance the study of protein modifications.
The low-temperature C–H bond activation of alkanes remains a big challenge in alkane dehydrogenation. In this work, ethylbenzene (EB) oxidative dehydrogenation has been investigated on rutile(R)-TiO2(110) under both ultrahigh vacuum (UHV) and ambient conditions. Under UHV conditions, styrene is produced with nearly 100% selectivity in a stepwise manner, in which the first C–H bond dissociation of EB occurs at <285 K with the help of surface O22– species, followed by the second C–H bond dissociation at about 400 K. However, styrene, acetophenone, and 2,3-diphenylbutane products are produced from EB oxidative dehydrogenation under ambient conditions, suggesting that α-H dissociation is the initial step of EB oxidative dehydrogenation. This may be also possible for EB oxidative dehydrogenation on R-TiO2(110) under UHV conditions. The different pathways of EB oxidative dehydrogenation under UHV and ambient conditions may originate from different intermediates and O2 concentrations. This work provides new insight into the fundamental understandings of the low-temperature C–H bond activation of alkyl chains of aromatic hydrocarbons, which may promote the development of new catalysts for efficient styrene production from EB oxidative dehydrogenation under mild conditions.
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