Table 1.

Commonly applied techniques to study proteolytic reactions.

Method	Description Application	Protease	Substrate	Inhibitor	Process
General techniques for protease research
Yeast two-hybrid systems	Yeast libraries carrying cloned open reading frames (ORF)¹⁴ Sequencing of positive PCR amplification products Identification of protein interactions on a large scale Two target ORFs are analyzed per experiment	•	•	•
Phage-display	Affinity selection of clones and screening of peptide libraries⁹ DNA sequencing of remaining amplification products High throughput screening of protein interactions and inhibitors One target protein or peptide is analyzed per experiment	•	•	•
2D differential gel electrophoresis (2D DIGE)	Gel-based protein separation of fluorescent labeled samples¹⁵ Proteins are imaged by fluorescence and identified by MS Quantitation of proteins and posttranslational modifications Comparison of two samples with internal standards	•	•
Multidimensional protein identification technique (MudPIT)	Peptide products are separated by SCX and RP-HPLC¹³ Identification of peptide products by MS Comparison of an unlimited number of samples	•	•
Isotope-coded affinity tag (ICAT)	Labeling of cysteine-residues in proteins followed by digestion¹⁶ Relative quantitation of peptide products by MS Comparison of two samples		•
Isobaric tags for relative and absolute quantitation (iTRAQ)	Isobaric amine-specific tagging of peptide products¹⁷ Relative and absolute quantitation of protease activity by MS Comparison of up to eight samples		•
Stable isotope labeling with amino acids in cell culture (SILAC)	Expressed proteases/peptide products labeled by amino acids¹⁸ Identification and quantitation of peptide products by MS Comparison of an unlimited number of samples	•
Multiple reaction monitoring (MRM)	Multiple reaction monitoring of known peptide fragments by targeted MS¹⁹ Absolute quantitation of known peptides Comparison of an unlimited number of samples		•	•
Techniques specifically designed for protease research
Cellular libraries of peptide substrates (CLiPS)	Combinatorial approach to measure substrate hydrolysis¹¹ Identification of substrates by quantitative screening of whole-cell fluorescence		•		•
Positional scanning synthetic libraries	Screening of tetra-peptide libraries by proteolysis-dependent signal intensities¹⁰ Screen for P1–P4 substrate specificities		•
Colloidal barcoding bead- based protease profiling	Screening of combinatorial libraries using polyelectrolyte-coated fluorescent silica reporter particles²⁰ Identification of consensus proteolytic cleavage sites	•	•	•	•
Near infrared (NIR) fluorogenic reporters	NIR fluorescence signal upon cleavage of protease-sensitive peptide linkers²¹ In vivo imaging and quantitation of protease activities	•	•	•	•
C- and N-term enrichment of cleavage products	Negative or positive enrichment of C- or N-terminal peptide cleavage products²² Identification of proteolytic peptides and cleavage sites Comparison of two samples		•
Activity-based probes (ABPs)	Chemical probes with affinity and fluorescent tags²³ Report on the structure and reactivity of enzyme active sites in cells and tissues	•		•	•
Proteinase activity labeling employing ¹⁸O- enriched water (PALeO)	¹⁸O-labeling of proteolytic peptides during hydrolysis²⁴ Quantitation and identification of protease activity, peptide substrates and cleavage products Comparison of an unlimited number of samples	•	•	•	•