Table 1.

Endoproteinase Asp-N proteolysis of microprotein S: Identification of fragments

Digestion time, min	Retention time, min	Peak area, %	Mass, Da		Identity
			Observed	Calculated
0	17.13	100	13,608	13,607	Ala1 –Asp116
90	15.08	30	4,792	4,793	Ala1 –Thr37
	15.58	70	4,302*	4,303	Asp78 –Asp116
			9,341*	9,340†	Ala1 –Pro77‡
			4,565*	4,565†	Asp38 –Pro77

Microprotein S (1 mg/ml) was cleaved with endoproteinase Asp-N (10 μg/ml) at pH 8.0. Digests were applied to HPLC and peaks were analyzed by using ESI-MS. 

^*These fragments all co-eluted in a heterogeneous peak at 15.58 min. 

^† Calculated mass is based on hydrolysis of the peptide bond at Asp⁶⁸ (+18) in the TSR module (Fig. 1). 

^‡ The Ala¹–Pro⁷⁷ fragment was detectable during the early phase of proteolysis but disappeared after prolonged incubation (150 min).