. 2010 Jan 13;285(11):7929–7937. doi: 10.1074/jbc.M109.075010

TABLE 1.

Characterization of OPN fragments generated by thrombin, plasmin and cathepsin D

Treatments^a		Peptide^b	Observed mass^c	Calculated mass^d	Mass difference/comment^e
1st digest	2nd digest	Peptide^b	Observed mass^c	Calculated mass^d	Mass difference/comment^e
			Da	Da
T1		Ile¹–Arg¹⁵²	28,000†	16,891	11109 (P and O-glycans)
	Asp-N	Asp¹⁴⁵–Arg¹⁵²	908.48	908.48	Normal isotope pattern
T2		Ser¹⁵³–Arg²²⁸	9043.05†/8961.76†	8642.20	400.85 (5P)/319.56 (4P)
T3		Ser¹⁵³–Asn²⁹⁸	17,888.52†	16,843.08	1045.44 (13P)
T4		Leu²²⁹–Asn²⁹⁸	8864.33†	8219.90	644.43 (8P)
P1		Ile¹–Arg¹⁵²	28,200†	16,891	11,309 (P and O-glycans)
		Ile¹–Lys¹⁵⁴		17,106	11,094 (P and O-glycans)
	Asp-N	Asp¹⁴⁵–Arg¹⁵²	908.49	908.48	Normal isotope pattern
	Asp-N	Asp¹⁴⁵–Lys¹⁵⁴	1123.64	1123.60	Normal isotope pattern
P2		Ser¹⁵³–Lys¹⁸⁷	4325.60	4085.94	239.66 (3P)
P3		Ser¹⁵⁵–Lys¹⁸⁷	4110.60/4030.59	3870.82	239.78 (3P)/159.77 (2P)
P4		Phe¹⁵⁸–Lys¹⁸⁷	3767.47/3687.40	3527.59	239.88 (3P)/159.81 (2P)
P5		Arg¹⁶⁰–Lys¹⁸⁷	3464.16/3384.20	3224.43	239.73 (3P)/159.77 (2P)
P6		Ala¹⁸⁸–Lys²²⁵	4519.15†/4438.67	4198.91	317.78 (4P)/239.76 (3P)
P7		Ala¹⁸⁸–Lys²³¹	5294.22†/5214.29†	4977.28	316.94 (4P)/237.01 (3P)
P8		Arg²³²–Lys²⁸³	6620.45†	6142.62	477.83 (6P)
P9		Phe²⁸⁴–Asn²⁹⁸	1930.56	1690.80	239.76 (3P)
CD1		Ile¹–Thr²⁶	3078.35	2838.41	239.94 (3P)
CD2		Trp²⁷–Leu³⁸	1439.60	1439.72
CD3		Trp²⁷–Phe⁵³	3202.35	3042.44	159.91 (2P)
CD4		Leu³⁹–Leu¹⁵¹	22,200†	12,492	9708 (P and O-glycans)
		Lys⁵⁴–Leu¹⁵¹		10,888	11,312 (P and O-glycans)
	Asp-N	Asp¹⁴⁵–Leu¹⁵¹	752.29	752.38	Normal isotope pattern
CD5		Arg¹⁵²–Leu¹⁹⁵	5289.17	5049.49	239.68 (3P)
CD6		Asn¹⁹⁶–Leu²²⁹	4115.70/4037.73†	3875.74	239.96 (3P)/159.75 (2P)
CD7		Ser²⁵¹–Leu²⁶⁹	2594.90	2339.05	255.85 (3P, Mox)
CD8		Val²⁷⁰–Asn²⁹⁸	3643.30	3323.68	319.62 (4P)

^a Fractions were from the digestion of OPN with thrombin (T), plasmin (P), or cathepsin D (CD). In some cases, the fragments were further digested with endoproteinase Asp-N in buffer containing 50% H₂¹⁸O.

^b Peptides were identified by N-terminal sequence and MALDI-TOF-MS analyses.

^c Observed molecular masses (MH⁺) were determined by MALDI-TOF-MS. Unless mentioned otherwise, masses are monoisotopic. † means the molecular masses were determined in linear mode.

^d Theoretical calculated masses are shown.

^e Differences between observed and calculated masses are shown. The type of modification corresponding to the mass difference is given in parentheses as follows: phosphorylation (P) and oxidation of methionine (Mox). The C terminus of fragments secondarily digested with Asp-N in 50% H₂¹⁸O was identified by observation of peptides with a normal isotopic pattern.