Amine-Functionalized Sol–Gel-Based Lab-in-a-Pipet-Tip Approach for the Fast Enrichment and Specific Purification of Phosphopeptides in MALDI-MS Applications

Abstract

Amine-functionalized sol-gels were investigated for the enrichment and purification of phosphopeptides from digested protein mixture solutions. Tetramethylorthosilicate (TMOS) and N'[3-(trimethoxysilyl)-propyl]-diethylenetriamine (TPDA) were used in a 1:1 mole ratio in the production of amine-functionalized sol-gels. The sol-gel network was then used for phosphopeptide enrichment. Phosphopeptide enrichment onto the synthesized amine-functionalized sol-gels was performed using an enolase digested peptide mixture, a β-casein digested peptide mixture, as well as these digested peptide mixtures contaminated 50-fold with bovine serum albumin (BSA). Moreover, phosphopeptide enrichment was successfully performed using nonfat milk as a highly contaminated and complex material. In each phosphopeptide enrichment and purification process, only phosphopeptides were enriched and separated from the other digested peptides. Phosphopeptides were adsorbed onto the amine-functionalized sol-gels at pH 4.0 and eluted at pH 1.0 using trifluoroacetic acid (TFA). For phosphopeptide analysis by MALDI-MS, a 2,5-dihydroxybenzoic acid matrix containing 1.0% phosphoric acid was used to overcome the degradation of phosphopeptides and provide high intensity phosphopeptide protonated molecular ion signal intensities. It was also found that phosphopeptide detection limits were improved to approximately 10 femtomoles. For rapid and specific phosphopeptide enrichment and purification, sol-gel materials were placed in a 10 μL pipet tip with glass wool on either side. Phosphopeptide enrichment from digested peptide mixtures was performed in a very short time (less than 1 min) at subpicomole levels using this novel lab-in-a-pipet-tip approach.

Amine-functionalized TiO 2 nanoparticles for highly selective enrichment of phosphopeptides

“…Some other absorption bands at 3400-3150, 1650-1400 and 1300-1200 cm À 1 are assigned to N-H and C-N stretching vibration and N-H bending vibration of amine groups located on the alkyl chain present in TPDA, respectively. Two strong absorption bands at 2930 cm À 1 and 2848 cm À 1 attributed to aliphatic carbon-carbon stretching vibrations on the alkyl chain in the structure of TPDA have been also observed [22,23]. The results from infrared spectroscopy suggest that TPDA is attached to the surface of TiO 2 through Ti-O-Si bonds, and amine groups located on the alkyl chain could be retained in the TiO 2 @TPDA structure.…”

Ti (IV) attached‐phosphonic acid functionalized capillary monolith as a stationary phase for in‐syringe‐type fast and robust enrichment of phosphopeptides

“…The protein solution was interacted with trypsin using a protein–enzyme ratio of 40:1 w/w, for 16 h at 37°C. The digestion was finally stopped using TFA solution (1%, v/v) (Atakay, Çelikbıçak, & Salih, ; Çelikbıçak, Atakay, Güler, & Salih, ).…”

Hydrophilic modification of titania nanomaterials as a biofunctional adsorbent for selective enrichment of phosphopeptides

“…22,23 To further evaluate the enrichment specificity of the NH 2 @TiO 2 nanohybrids toward phosphopeptides, a more complex peptide mixture of β-casein and BSA at a molar ratio of 1 : 1 and 1 : 10 was used as a mimic biological sample for the test. The NH 2 @TiO 2 nanohybrids with good water solubility could reduce the loss of phosphopeptides through the strong hydrophilic interaction between the amino groups on the surface of TiO 2 and phosphopeptides.…”