Digest Of Α-casein Research Articles

Enhanced detection of multiply phosphorylated peptides and identification of their sites of modification.

Phosphorylation is an important post-translational modification that rapidly mediates many cellular events. A key to understanding the dynamics of the phosphoproteome is localization of the modification site(s), primarily determined using LC-MS/MS. A major technical challenge to analysis is the formation of phosphopeptide-metal ion complexes during LC which hampers phosphopeptide detection. We have devised a strategy that enhances analysis of phosphopeptides, especially multiply phosphorylated peptides. It involves treatment of the LC system with EDTA and 2D-RP/RP-nanoUPLC-MS/MS (high pH/low pH) analysis. A standard triphosphorylated peptide that could not be detected with 1D-RP-nanoUPLC-MS/MS, even if the column was treated with EDTA-Na2 or if 25 mM EDTA-Na2 was added to the sample, was detectable at less than 100 fmol using EDTA-2D-RP/RP-nanoUPLC-MS/MS. Digests of α-casein and ß-casein were analyzed by EDTA-1D-RP-nanoUPLC, 2D-RP/RP-nanoUPLC, and EDTA-2D-RP/RP-nanoUPLC to compare their performance in phosphopeptide analysis. With the first two approaches, no tri- and tetraphosphopeptides were identified in either α- or ß-casein sample. With the EDTA-2D-RP/RP approach, 13 mono-, 6 di-, and 3 triphosphopeptides were identified in the α-casein sample, while 19 mono-, 8 di-, 4 tri-, and 3 tetraphosphopeptides were identified in the ß-casein sample. Using EDTA-2D-RP/RP-nanoUPLC-MS/MS to examine 500 μg of a human foreskin fibroblast cell lysate a total of 1,944 unique phosphopeptides from 1,087 unique phosphoproteins were identified, and 2,164 unique phosphorylation sites were confidently localized (Ascore ≥20). Of these sites 79% were mono-, 20% di-, and ∼1% were tri- and tetraphosphopeptides, and 78 novel phosphorylation sites in human proteins were identified.

Tuning of Ti-doped mesoporous silica for highly efficient enrichment of phosphopeptides in human placenta mitochondria

Extraction of phosphopeptides from rather complex biological samples has been a tough issue for deep and comprehensive investigation into phosphoproteomes. In this paper, we present a series of Ti-doped mesoporous silica (Ti-MPS) materials with tunable composition and controllable morphology for highly efficient enrichment of phosphopeptides. By altering the molar ratio of silicon to titanium (Si/Ti) in the precursor, the external morphology, Ti content, internal long-rang order, and surface area of Ti-MPS were all modulated accordingly with certain regularity. Tryptic digests of standard phosphoprotein α- and β-casein were employed to assess the phosphopeptide enrichment capability of Ti-MPS series. At the Si/Ti molar ratio of 8:1, the optimum enrichment performance with admirable sensitivity and capacity was achieved. The detection limit for β-casein could reach 10 fmol, and 15 phosphopeptides from the digest of α-casein were resolved in the spectrum after enrichment, both superior to the behavior of commercial TiO(2) materials. More significantly, for the digest of human placenta mitochondria, 396 phosphopeptides and 298 phosphoproteins were definitely detected and identified after enrichment with optimized Ti-MPS material, demonstrating its remarkable applicability for untouched phosphoproteomes. In addition, this research also opened up a universal pathway to construct a composition-tunable functional material in pursuit of the maximum performance in applications.