Highly efficient and selective enrichment of phosphopeptides using porous anodic alumina membrane for MALDI-TOF MS analysis

Abstract

Because of its good biocompatibility, high surface-to-volume ratio, and distinct surface electrical properties, porous anodic alumina (PAA) membrane has been used to selectively enrich phosphopeptides from a mixture of synthetic peptides and tryptic digest product of ␤-casein by a direct MALDI-TOF MS analysis. As we reported previously, PAA membrane has strong incorporation ability to the phosphate anion. Herein, we describe the application of PAA membrane as a selective sampling absorbent for phosphopeptides. The PAA membrane could enrich phosphopeptides with high efficiency and selectivity; for example, the tryptic digest product of ␤-casein at a concentration as low as 4 ϫ 10 Ϫ9 M can be satisfactorily detected. Compared to that from the nonenriching peptide mixture, the MS signal of the phosphorylated peptides enriched by the PAA membrane is remarkably improved. In addition, acidic peptides have insignificant influence on the enriching process. Results show that the adsorption of phosphate anions on the PAA membrane plays a determining role in achieving highly selective enriching capacity toward phosphopeptides. The feasibility of PAA membranes as specific absorbents for phosphopeptides is also demonstrated. (J Am Soc Mass Spectrom 2007, 18, 1387-1395

The adsorption of globular proteins onto a fluorinated PDMS surface

“…In order to determine the effect of both oxidation and fluorination of the PDMS polymers on protein desorption, experiments were carried out in which the relative amounts of a specific protein remaining after washing with water/methanol solution was determined using MALDI-MS. Fig. 1a shows the S/N ratios [24] arising from the cytochrome c protein remaining on unmodified, oxidized and fluorinated PDMS surfaces following washing with methanol/water mixtures of varying concentrations. At low methanol concentrations, cytochrome c is readily desorbed from all three surfaces.…”

Nanodiamonds Used as a Platform for Studying Noncovalent Interaction by MALDI‐MS

“…Since phosphorylation plays a critical role in regulating biological functions as a post-translational modification of proteins, kinds of metal affinity-based methods have been approached to improve the ion signals of phosphopeptides. [21][22][23][24][25][26][27][28][29] As Figure 3a shows, direct analysis of trypic digest product of β-casein (6 nmol/L) gives four peaks at m/z 1396, 2061, 2186 and 3121, among which the peaks of 2061 and 3121 are phosphopeptide residues with one and four phosphorylated serines respectively (Table 1). And Figure 3b shows the results got with 4 µg of PL-bound diamonds in 100 µL of 6 nmol/L solutions.…”

“…Thus the application makes the PL-coated nanodiamond as a new effective approach for enrichment of phosphopeptides selectively from enzymatic digest product of proteins, with its detectable concentration level of phosphopeptides lower than or comparative to other affinity-based methods. [21][22][23][24][25][26][27][28][29] It has been demonstrated here that the nanodiamonds can be used as a platform to study noncovalent interaction by MALDI-MS. The method is quite fast, easy- Table 1.…”

The use of liquid phase deposition prepared phosphonate grafted silica nanoparticle‐deposited capillaries in the enrichment of phosphopeptides

“…This method is limited by high cost and the availability of specific antibodies 12, 13. (3) Immobilized metal‐ion affinity (IMAC) 14–18 and metal oxide affinity chromatography (MOAC) 19–25 are based on specific interactions between the metal ion and phosphopeptides.…”