Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

392 Török et al.
Table 1
Peptide Fragments Isolated by HPLC on a Vydac Semipreparative Column;
Peptides were Analyzed by Electrospray and Nanospray Mass Spectrometry
Observed Modified Expected
Peak Average Mass Sequence Residues Average Mass
1 1092.5 T8 1093.1
(77–86)
2 521.5 T9 521.6
(87–90)
3 803.9 T4 804.9
(31–37)
4 1753.7 T10–11 1753.9
(91–106)
5 1562.8 T1 Acetyl Group 1562.8
(1–13) on Alanine 1
6 1843.9 T2–T3 1843.9
(14–30)
7 1901.9 75–86 *5-DTAF 1902
cyclized
8 2400.5 T12 (CH 3) 3 on 2402
(107–126) Lysine 115
9 864.1 75–77 5-DTAF on 863.6
Lysine 75
10 2489.1 T13 2490
(127–148)
11 2302.5 T2–T3 5-DTAF 2301.9
(14–30) on Lysine 21
12 a 2951 T13 5-DTAF 2948.7
(127–148) on Lysine 148
13 4070.7 T5 4071.5
(37–74)
a Doubly labeled only
intramolecularly cyclized between two amino groups by substitution of both
chlorines (see Fig. 6). Analysis of peak 9 by nanospray mass spectrometry shown
in Fig. 7A, shows a monoisotopic mass of 864.1, which identifies peptide fragment
75–77 with a 5-DTAF label on one of the two lysines. In Fig. 7B, we can
see the entire amino acid sequence and which lysine residue is labeled with
5-DTAF. In this case, the label is on Lys 75 and not on Lys 77 because the b ions
(fragmentation occurring at the amide backbone to produce acylium ions) and the
y” ions (C-terminal ions involving hydrogen rearrangement) would produce different
masses as outlined in Fig. 8 (see Note 2). The reaction mechanism that
gives these two different peptides is shown in Fig. 6. As aforementioned, in the