Analysis of effect of casein phosphopeptides on zinc binding using mass spectrometry

Abstract

Phosphorylation is one of the key events in signal transduction and zinc plays an important catalytic and/or structural role in many biological systems. The binding of Zn to a phosphopeptide will alter the physiological functions of a peptide. The binding of casein phosphopeptides (CPPs) to Zn has been analyzed using nanospray mass spectrometry. Electrospray ionization (ESI) spectra of peptides produced by tryptic digestion of alpha-casein incubated with Zn show both free and Zn-bound phosphopeptides. The interaction of CPPs and the corresponding dephosphorylated peptides with zinc is compared. This study demonstrates that the phosphorylation state of a peptide dramatically affects Zn binding, with the decrease in Zn-bound forms of peptide paralleling the decrease in phosphorylation as casein is chemically dephosphorylated, although, in some cases, a small amount of residual Zn-binding capacity remains in the completely dephosphorylated peptide. The observed fragmentation patterns of the Zn-bound CPPs support the thesis that nonphosphorylated residues are involved in the metal binding.

Marginal Maternal Zinc Deficiency in Lactating Mice Reduces Secretory Capacity and Alters Milk Composition ,

“…To our knowledge, only one study has explored effects of maternal Zn deficiency on milk nutrient concentration; that study noted that moderate maternal Zn deficiency increased the b-casein concentration in rat milk (13). This observation may be physiologically relevant to the nutrition of the offspring, because b-casein can chelate Zn (14) and calcium (15) and thus alter Zn bioavailability in the developing infant.…”

“…Furthermore, casein phosphopeptides have been associated with antithrombotic, antihypertensive, and opioid activities, which may contribute to sleeping behavior and fluid transport in the small intestine of breast-fed infants [reviewed in (58)]. Caseins are also important Zn-binding proteins (14); thus, a reduction in milk caseins may alter the pool of calcium and Zn that is available for absorption during infancy. WAP is a Zn-dependent protease inhibitor that is found in milk.…”

HPLC separation of casein components on a diol-bonded silica column with MALDI TOF/TOF MS identification

“…10,11 The matrix-assisted laser desorption/ionization mass spectrometry technique (MALDI MS) is one of the most useful tools in proteomics, 12 enabling the identication of proteins and peptides and the analysis of their post-translational modications. 13 The purpose of the present study was to study the separation of the components of bovine casein using laboratory-made diolbonded silica stationary phases. Casein from bovine milk was separated in a reversed-phase (RP) mode and fractionated.…”

Assaying pharmacodynamic endpoints with targeted therapy: Flavopiridol and 17AAG induced dephosphorylation of histone H1.5 in acute myeloid leukemia

“…The direct analysis of the casein tryptic digests using phosphopeptide enrichment, nano‐ESI‐LC‐MS 3 combined with hierarchical MS 2 /MS 3 database search resulted in the identification of 70 unique casein peptides including 26 unique casein phosphopeptides (Table 1) with no matches returned from the decoy database. Two phosphorylation sites were identified by our workflow which were not reported 26, 27, 40, 60, 71, 72. One is phosphorylation at T56 from the peptide 48 FQpSEEQQQpTEDELQDK 63 of β casein 26, 27, 40 (Table 1 and Supporting Information Fig.…”

“…Overall, hierarchical data analysis significantly reduced the number of false positives matching resulting in improved sensitivity and specificity. For sake of comparison, peptides identified in other reports are also listed in Table 1 26, 27, 40, 60, 71, 72.…”