Phosphorylation is a common reversible post-translational modiﬁcation involved in the regulation of many essential biological processes. Phosphoproteins and phosphopeptides are usually present at very low concentrations and ionize poorly, making their detection by MS difﬁcult.
TiO2 Mag Sepharose magnetic beads simplify capture and enrichment of phosphopeptides by titanium dioxide (TiO2)-based chromatography (Figure 5.5). TiO2 has high afﬁnity for phosphopeptides and provides efﬁcient enrichment of phosphopeptides from complex samples.
Figure 5.5.TiO2 Mag Sepharose® is designed for efﬁcient small-scale enrichment of phosphopeptides.
Characteristics of TiO2 Mag Sepharose® beads are shown in Table 5.2.
Table 5.2. Characteristics of Mag Sepharose® beads
Two phosphorylated proteins (a-casein and b-casein) and one nonphosphorylated protein (bovine serum albumin) were reduced and alkylated with Tris(2-carboxyethyl) phosphine (TCEP) and iodoacetamide, respectively, followed by trypsin-digestion. A total of 50 pmol of each protein digest was mixed and applied to the magnetic beads. After enrichment, the eluates were lyophilized and dissolved in 20% acetonitrile with 0.1% triﬂuoroacetic acid (TFA, 20 µl) and analyzed by MALDI-ToF MS.
TiO2 Mag Sepharose® detected ﬁve peptides, with a ratio of 2.5 between phosphopeptides and nonphosphorylated peptides. Also, after a 100-fold dilution of the eluate, two phophopeptides could still be detected. The experimental conditions and mass spectrograms are shown in Figure 5.6.
Figure 5.6.MALDI-ToF MS analysis of trypsin-digested protein mix (50 pmol each of BSA, a-casein, and b-casein) enriched using three different chromatographic media. (A) Spotting from lyophilized eluates dissolved in 20 µl and (B) eluates diluted 100-fold before spotting. The spectra show start material (Panel I) and eluates from TiO2 Mag Sepharose® (Panel II). Identiﬁed phophorylated peptides are marked with asterisks*.
Performing a Separation
For complex samples, such as cell lysate digests, it is recommended to perform a desalting step by use of for example an RPC/C18 cartridge or similar for efﬁcient phosphopeptide enrichment.
Dilute the sample with a minimum of four volumes of binding buffer or dissolve lyophilized sample in binding buffer. Keep sample volumes small, preferably max 100 µl, however up to 250 µl may be used.
Use the magnetic rack with the magnet in place to attract the beads before each liquid removal step.
Eluates must be evaporated or neutralized with formic acid or triﬂuoroacetic acid before analysis with MALDI-ToF. Suitable solvent for evaporated samples is 20% acetonitrile acidiﬁed with 0.1% triﬂuoroacetic acid. For LC-MS analysis using reversed phase chromatography (RPC) the eluates must ﬁrstly be evaporated and resuspended in formic acid to a ﬁnal concentration of 0.1%.
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