Fig. 1. An MS/MS spectrum of an acidic peptide selected by Ga(III) IMAC from the tryptic digest of milk. This peptide was concluded not to be phosphorylated based on the absence of the typical loss of a phosphate group from the parent ion in the MS/MS spectrum. This peptide was identified to be from β-lactoglobulin.

Fig. 2. An MS/MS spectrum of a phosphorylated peptide selected by Ga(III) IMAC from the glu-C digest of milk. The loss of three phosphates can be seen by the dominant peaks at multiples of 49 less than the molecular ion (charge state +2). The peptide was identified to be from αS1 casein.

Fig. 3. The MS/MS spectrum of a phosphorylated peptide from the glu-C digest of milk. It was concluded this peptide contained at least two phosphate groups based on the peaks at m/z 838 [M + H-98]⁺ and 740 [M + H-2(98)]⁺. Due to poor fragmentation, it was not possible to determine the sequence of this peptide.

Fig. 4. An MS/MS spectrum of an acidic peptide from the glu-C digest of milk. No loss of phosphate was observed in the fragmentation. This peptide contained three missed glu-C cleavages and as a consequence had five carboxyl groups. This is the apparent reason it bound to the Ga(III) IMAC column. The peptide was determined to be from αS1 casein.

Fig. 5. An MS/MS spectrum of a peptide from the glu-C digest of milk that non-specifically bound to the Ga(III) IMAC resin. Again no loss of phosphate was observed in the fragmentation. It was also determined to be from αS1 casein.

Fig. 6. The mass spectrum of differentially coded phosphopeptides from glu-C digests of β-casein. Experimental and control samples were differentially coded with d₃- and d₀-labeled acetate and mixed in a 3:1 ratio prior to selection and separation. Isotopic isoforms of the peptides were observed in roughly a 3:1 ratio in the mass spectrum based on peak height. The peptide isoforms were labeled both on their N-termini and -amino group of lysine, accounting for the 6 amu difference in their mass.

Fig. 7. A mass spectrum of a d₀/d₃-acetate coded peptide from glu-C digests of two milk samples. This peptide was present at a ratio of roughly 1.12:1 based on peak height. The 6 amu difference in the mass of the differentially coded peptides indicates that the peptide contains a lysine residue. This was later confirmed by MS/MS. The peptide was determined to be from αS1 casein.

Table 1. Phosphopeptides identified from a glu-C digest of three caseinsa

Table 2. Proteins identified from glu-C and tryptic digests of skim milk

The number in parentheses indicates the number of peptides that were phosphorylated out of the total number of peptides selected from that protein.