Actively helping customers, employees and the global community during the coronavirus SARS-CoV-2 outbreak.  Learn more >>

Bright Tide Fluor™-Based Fluorescent Peptides and Their Applications In Drug Discovery and Disease Diagnosis

by Haitao Guo, Jinfang Liao, Q. Meng, Xing Han, Zhenjun Diwu, Chunmei Wei

Introduction


Fluorescent peptides are essential tools used extensively in drug discovery and disease diagnosis. Although EDANS, FAM, and TAMRA are widely used to label peptides, their short absorption wavelengths, low extinction coefficients, and pH-sensitive fluorescence have limited their use in developing sensitive fluorescent peptide probes. Alexa Fluor® (AF) dyes (Invitrogen) demonstrated improved fluorescence properties. However, the high cost of Alexa Fluors makes the AF dyes unaffordable for labeling peptides.

The strongly fluorescent and affordable Tide Fluor™ (TF) dyes eliminate the limitations mentioned above of the classic dyes. They are individually optimized for labeling peptides and nucleotides. As the Alexa Fluors, the TF dyes are bright, pH-insensitive, and photostable. Compared to Alexa Fluors, TF dyes are more stable to the peptide deprotection conditions.

We have used TF 2 and TF5 dyes to develop new fluorescent peptides for high throughput analysis of protease and protein kinase activities and screening of protease and protein kinase inhibitors. Excellent spectral properties and labeling efficiencies have also been observed for TF3, 4, 6, 7, and 8. Among them, TF 6, 7, and 8 have been used for developing fluorescent peptides that have emissions in the near-infrared or infrared range. These NIR and IR TF-based peptides have been successfully used in in-vivo imaging.

Materials and Methods


All the peptides were synthesized using the standard FMOC chemistry by the American Peptide Company. Some of the TF peptides were prepared using FMOC-Lys(TF)-OH, FMOCAsp(TF)-OH, or FMOC-Glu(TF)-OH amino acids. For the post-labeling of the premade peptides, TF NHS esters were used for labeling the N-terminal amino or ε–amino group of a lysine residue. TF maleimides or iodoacetamides were used for labeling the SH group of cysteine residues.

TF1, TF2, TF3, TF4, TF5, TF6, TF7, and TF8 acids, maleimides and NHS esters, FMOC-Lys(TF2-Boc)-OH, FMOCLys(TF3)-OH, FMOC-Asp(TF2-Boc)-OH, FMOC-Asp(TF3)-OH, FMOC-Glu(TF3)-OH, FMOCGlu(TF2-Boc)-OH and FMOC-Glu(TF3)-OH are now commercially available from ABD Bioquest.

All the enzyme assays were done with purified enzymes commercially available from Sigma, R&D Systems, EMD Chemicals. Absorption spectra were taken with Hitachi U-3010. Endpoint fluorescence assays were run on either BMG NovoStar or Gemini SpectraMax (Molecular Devices). Enzyme kinetic assays were run on FlexStation Molecular Devices).

Spectral Properties of Tide Fluor™ (TF) Dyes


Our TF series of fluorescent labeling dyes cover the entire visible spectrum with unusually high labeling efficiency. For example, TF2 has spectral properties essentially identical to AF 488, FAM, and FITC. It is three times brighter and four times more photostable than Fluorescein under physiological conditions or in cells. TF dyes have fluorescence insensitive to pH fluctuation from pH 4 to pH 10, while the fluorescence intensity of FAM and FITC is strongly dependent on pH in the same range.

TF Dyes Excitation Emission Replacement for
TF1 353 nm 442 nm AF350, AMCA, EDANS
TF2 498 nm 520 nm AF488; DL488, FITC, FAM, Cy2
TF3 560 nm 575 nm AF546, AF555, Cy3, DL549, TAMRA
TF4 585 nm 605 nm AF594, DL594, Texas Red®, ROX
TF5 650 nm 670 nm AF633, AF647, DL633, DL649, Cy5
TF6 685 nm 700 nm AF680, DL680, Cy5.5
TF7 755 nm 780 nm AF750, DL750, Cy7
TF8 785 nm 800 nm DL800, IRDye CW800

Legends: AF = Alexa Fluor® (Invitrogen); DL = DyLight™ (Thermo Fisher); IRDye CW800 (Li-Cor); TF = Tide Fluor™ (ABD Bioquest)

Detection of Matrix Metalloproteinase (MMP) Activities Using TF-Based FRET Substrates


Matrix metalloproteinases (MMPs) are involved in the degradation of components of the extracellular matrix and play an important role in apoptosis, embryogenesis, reproduction, tissue remodeling, and repair. The following TF-based FRET substrates were synthesized and screened for detecting MMP activities.

#Sub Peptide Sequence Excitation Emission
1 TQ2-Gaba-Pro-Cha-Abu-Smc-His-AlaDab(5-FAM)-Ala-Lys-NH2 492 nm 514 nm
2 TQ2-Gaba-Pro-Cha-Abu-Smc-His-AlaDab(TF2)-Ala-Lys-NH2 495 nm 520 nm
3 TQ3-Gaba-Pro-Cha-Abu-Smc-His-AlaLys(TF3)-Ala-Lys-NH2 560 nm 575 nm
4 TQ4-Gaba-Pro-Cha-Abu-Smc-His-AlaLys(TF4)-Ala-Lys-NH2 585 nm 605 nm
5 TQ5-Gaba-Pro-Cha-Abu-Smc-His-AlaLys(FT5)-Ala-Lys-NH2 650 nm 670 nm
6 TQ6-Gaba-Pro-Cha-Abu-Smc-His-AlaLys(FT6)-Ala-Lys-NH2 685 nm 700 nm
7 TQ7-Gaba-Pro-Cha-Abu-Smc-His-AlaLys(TF7)-Ala-Lys-NH2 755 nm 780 nm
8 TQ8-Gaba-Pro-Cha-Abu-Smc-His-AlaLys(TF8)-Ala-Lys-NH2 785 nm 800 nm

Detection of MMP Activities (Continued)

Figure 1. TF-based MMP substrates were incubated with MMP-1 (50 units/ml) at room temperature for 15 min. All the substrates were used at 5 uM, and the reaction rate is set at 100% with Sub #2. The fluorescence increase was measured with the excitation and emission wavelengths, respectively, listed in the above table.

Detection of Generic Protease Activities Using TF-Labeled Casein Substrates


Monitoring of various protease activities has become a routine task for many biological laboratories. TF dyes-labeled casein conjugates are proven to be generic substrates for a broad spectrum of proteases. In the intact substrates, casein is heavily labeled with a TF dye, resulting in significant fluorescence quenching. Protease-catalyzed hydrolysis relieves its quenching effect, yielding brightly fluorescent TF dye-labeled peptide fragments. The increase in fluorescence intensity of TF dye-labeled peptide is directly proportional to the protease activity.

 

#Sub Peptide Substrate Ex/Em Rel. Kcat/Km
1 TF1-Casein (~6 Dyes/Casein) 492/514 nm 98%
2 TF2-Casein (~6 Dyes/Casein) 495/520 nm 92%
3 TF3-Casein (~6 Dyes/Casein) 560/575 nm 100%
4 TF4-Casein (~5 Dyes/Casein) 585/605 nm 73%
5 TF5-Casein (~5 Dyes/Casein) 650/670 nm 81%
6 TF6-Casein (~4 Dyes/Casein) 685/700 nm 76%
7 TF7-Casein (~4 Dyes/Casein) 755/780 nm 56%
8 TF8-Casein (~4 Dyes/Casein) 785/800 nm 43%


Figure 2. Proteolytic cleavage of Substrate #3 trypsin. Substrate #3 was incubated with trypsin at room temperature. The control wells had protease substrate only without trypsin. The fluorescence signal was measured starting from Time 0 (when trypsin was added) using FlexStation.



Original created on December 17, 2019, last updated on December 17, 2019
Tagged under: Fluorescence Resonance Energy Transfer (FRET), Oligonucleotide Labeling, Peptide Labeling