Table 1.

Advantages and disadvantages of different techniques for membrane protease substrate identification.

	Advantages	Disadvantages
Glycocapture	• Large reduction of sample complexity when analyzing glycopeptides after PNGaseF release. • Possible with in vivo material	• Serum-free or serum-depleted medium required
SPECS	• Compatible with protein and serum supplements in the medium • Facilitates secretome and surfaceome analysis. • Many peptides for quantification	• No direct protease cleavage site identification (only semitryptic peptides can be used). • Only applicable for sheddases • Not suitable for in vivo analyses
AHA labeling	• Compatible with protein and serum supplements in the medium • Facilitates secretome and surfaceome analysis • Many peptides for quantification	• No direct protease cleavage site identification (only semitryptic peptides can be used) • Only applicable for sheddases • Titration of AHA concentration necessary to prevent toxicity • Not suitable yet for in vivo analyses
Surface Biotinylation	• Efficient pull-down of cell surface proteins • In vivo analyses are possible	• Secretome analysis difficult • No trypsin cleavage at biotinylated lysines
Murine CSF	• In vivo • Many peptides for quantification	• Low sample amount (5–15 μl) • Sampling is difficult (blood or cell contamination) • KO mice or inhibitor treatment of mice is necessary
TAILS	• Direct identification of protease cleavage sites • Also applicable for soluble proteases (e.g., in vitro incubation of protein lysate with protease of interest)	• Serum-free or serum-depleted medium required • TAILS is hard to establish (especially C-TAILS) • Few peptides for quantification → Additional whole secretome analysis of labeled peptides potentially required
COFRADIC	• Direct identification of protease cleavage sites • Also applicable for soluble proteases (e.g., in vitro incubation of protein lysate with protease of interest)	• Serum-free or serum-depleted medium required • HPLC is required for extensive sample fractionation • Histidine containing peptides are lost in the SCX depletion step • Few peptides for quantification → Additional whole secretome analysis of labeled peptides potentially required
Subtiligase	• Direct identification of protease cleavage sites • Also applicable for soluble proteases (e.g., in vitro incubation of protein lysate with protease of interest)	• Serum-free or serum-depleted medium required • Large sample amount required • Few peptides for quantification → Additional whole secretome analysis of labeled peptides potentially required