Fluorescent Dyes for Peptide Labeling

Fluorescent markers are widely used in protein function research and drug screening. Fluorescently labeled peptides are often used to detect the activity of target proteins. The high-throughput activity screening methods developed from this are applied to drug screening and drug development of disease-related target proteins (such as various kinases, phosphatases, and peptidases). Therefore, fluorescent modification of peptides is an important aspect in the field of peptide synthesis.

What is Fluorescent Peptide?

In customized peptide synthesis, there are various labeling and modification options, including N-terminal labeling or modification, N-terminal fluorescein labeling, photosensitizer modification, lysine modification, PEG modification, and conjugation. These modifications are widely applied in the research of peptide-based drugs, peptide biology, peptide antibodies, and peptide synthesis reagents. Peptide fluorescent labeling is a biological labeling technique in which fluorescent dyes or probes are covalently attached to peptide molecules, typically by incorporating the dye directly during peptide synthesis. Labeling can also be done indirectly using biotinylated amino acids. Both methods allow for site-specific labeling, as the dye can be incorporated at any desired position rather than being randomly distributed across the molecule. Site-specific labeling enables researchers to study specific peptide regions or domains and understand how they interact with other molecules. It also allows for fine-tuning the level of fluorescent signal, adjusting detection sensitivity to meet specific needs. This process is commonly used to study the function, structure, and biological behavior of peptides, helping scientists track and quantify peptide distribution and dynamic changes in biological systems by detecting fluorescent signals.

Fig. 1. Fluorescent labeling of peptides.

Peptide Fluorescent Labeling

The chemistry of peptide fluorescent labelling involves attaching fluorescent molecules to peptides by chemically reacting with those peptides to covalently bind the fluorescent molecule to the specific position in the peptide chain. The fluorescent dyes when exposed to a wavelength of light take up the energy and then instantly move back from excited to ground state and emit that energy in the form of light (florescence). That fluorescent signal is then spotted by fluorescence microscopes, flow cytometers or spectrophotometers. Also, fluorescent labelling is stable and generally non-toxic, so it is ideal for monitoring biology in vitro and in vivo. Amino acid residues in peptides are typical labelling points: amino acid of lysine, thiol of cysteine, hydroxyl groups of serine and threonine. Also, fluorescent groups can be introduced at the N- or C-terminus of peptides using chemical or biological synthesis.

Fluorescent Peptide Synthesis

There are various methods for peptide fluorescent labeling, and the choice of method depends primarily on the chemical properties of the reaction, the selection of labeling sites, and experimental requirements. Below are some common peptide fluorescent labeling techniques:

Peptide Fluorescent Probes

Active fluorescent dyes are used to mark proteins, peptides, nucleic acids and other biomolecules in life sciences experiments like fluorescence microscopy, flow cytometry, fluorescence in situ hybridization (FISH), fluorescence resonance energy transfer (FRET), receptor binding, and enzyme detection. Choose the appropriate fluorescent dye that is very important to the performance of peptide labelling and it can be classified into emission wavelength, photostability and sensitivity. Here are some of the most popular fluorescent dyes:

• Fluorescein Dyes

Fluorescein dyes (e.g., FITC) are among the most widely used in biological experiments due to their high absorbance and strong fluorescence emission, typically emitting green light in the visible spectrum. Carboxyfluorescein requires activation before use, while FITC is more reactive. FITC primarily reacts with thiols or primary amines, such as the side chain of reduced cysteine or amines in peptides or proteins. In many applications, the fluorescent group can be introduced during chemical synthesis, and after selective deprotection, FITC can react with lysine or ornithine side chains, or with the N-terminus of peptides. During N-terminal labeling, an alkyl spacer group, such as aminohexanoic acid (Ahx), is often introduced between the amine and the isothiocyanate to prevent cyclization reactions under acidic conditions, which could otherwise remove the final amino acid during cleavage from the resin. Sometimes, spacers are necessary to overcome steric hindrance, allowing FITC to conjugate to peptides without obstruction.

• Rhodamine Dyes

Rhodamine (TRITC, for example) dyes give orange or red fluorescence and are more stable and bright than fluorescein dyes, which is a good choice for experiments that will take longer. Rhodamine dyes are also water and cell-permeable, so they're perfect for labeling inside cells. A popular rhodamine dye is tetramethylrhodamine (TAMRA), which is commonly used in FRET probes, and probes that are based on TAMRA have even distribution in cells and diffuse well into the cytoplasm. TAMRA also has some advantages over FITC like more resistance to photobleaching. FITC is excited at 494 nm and fluoresces at 520 nm; TAMRA has the maximum absorption wavelength (546 nm) and emission wavelength (580 nm). What's more, TAMRA's additional positive charge could help enhance binding of probes to target molecules. Like Cy3, TAMRA is stable and fluorescent, a great fluorescent group. In addition, FITC and TAMRA could both be applied to the same experiment (because of their different excitation and emission wavelengths) without each other being killed off.

• Cyanine Dyes

Cyanine dyes (e.g., Cy3, Cy5) are known for their long emission wavelengths, high fluorescence brightness, and slow photobleaching, making them ideal for experiments requiring high sensitivity. Cy3 emits orange fluorescence, while Cy5 emits red fluorescence, making them suitable for multiplex labeling experiments. In dual-labeling experiments, Cy5 is often used with another short-wavelength dye, such as FITC, for multiple labeling. For example, in research, Cy5-labeled peptides can be used with FITC-labeled antibodies, and multiple fluorescence microscopy imaging techniques can detect different targets in cells. Cy5 is also widely used in in vivo imaging experiments to track the biodistribution of targeted peptides in mouse models.

• BODIPY Dyes

BODIPY dyes (boron-dipyrromethene) are a popular class of fluorescent probes due to their high photostability, narrow emission spectra, and low sensitivity to environmental changes. They are widely used in biological labeling and fluorescence imaging. BODIPY dyes have a high quantum yield, and their emission wavelength can be adjusted through chemical modifications, making them ideal for labeling peptides and proteins. BODIPY dyes are commonly used in studies of peptide behavior in cell membranes or liposomes. Due to their excellent optical properties, BODIPY-labeled peptides help researchers investigate peptide penetration mechanisms or interactions with membrane proteins. Additionally, BODIPY dyes perform well in fluorescence lifetime imaging microscopy (FLIM), enabling precise measurements of the microenvironment of labeled peptides in cells or tissues.

• Alexa Fluor Dyes

Alexa Fluor dyes are a series of fluorescent dyes derived from fluorescein and rhodamine, offering improved photostability and fluorescence intensity. They are suitable for live-cell imaging and long-term fluorescence detection. This series covers a wide range of excitation/emission wavelengths, from ultraviolet to infrared, with popular examples including Alexa Fluor 488, Alexa Fluor 555, and Alexa Fluor 647. Compared to traditional FITC and rhodamine, Alexa Fluor dyes show significantly improved water solubility, photostability, and fluorescence intensity. Alexa Fluor dyes are frequently used in live-cell and fixed-cell imaging studies. For example, Alexa Fluor 488-labeled peptides are often used to monitor protein transport in living cells or to study peptide interactions with cell membrane receptors. Alexa Fluor 647 is commonly used in dual or multiple labeling experiments, leveraging its far-red emission properties to minimize fluorescence signal overlap and produce clearer images when combined with other dyes.

• ATTO Dyes

Atto dyes can be a better choice than traditional fluorescent dyes and are more photostable, ozone-resistant, signal long-lives and less background for greater sensitivity. They're great for using visible and near-infrared wavelengths of emission. ATTO 488, for example, is a great substitute for fluorescein and Alexa Fluor 488, and ATTO 550 replaces rhodamine dyes, Cy3, and Alexa Fluor 550, to give conjugates that are better photostable and more fluorescent.

What are Fluorescent Peptides Used For?

Fluorescently labeled peptides are widely used in modern medicine, not only for protein interaction studies but also for cellular localization research, the development of novel disease models, high-throughput screening in peptide synthesis, biosensing, and more. Whether in basic biological research or drug development and clinical diagnostics, fluorescent peptides are valuable tools for investigating molecular processes and biomolecular interactions.

• Protein Interaction Research

Fluorescently labeled peptides play a crucial role in studying biomolecular interactions. As recognition and binding molecules for proteins, enzymes, or nucleic acids, peptides are often used to analyze molecular interactions. Fluorescence labeling provides a highly sensitive tool for quantitatively and qualitatively detecting these interactions. For example, fluorescence resonance energy transfer (FRET) allows researchers to monitor changes in protein-protein, protein-nucleic acid, or protein-ligand interactions through energy transfer between two fluorescent groups based on their distance. By measuring changes in fluorescence emission, FRET can detect subtle molecular interactions and even track conformational changes in molecular complexes. This technique can be used not only in vitro but also to study interactions in live cells, making it highly relevant for physiological conditions.

• Cellular Imaging and Tracking

Fluorescent peptides are extensively used in cellular imaging, a key technique in cell biology. Through fluorescence labeling, researchers can observe the distribution, localization, and dynamic behavior of fluorescent peptides in cells in real time, gaining insights into their roles in biological processes. Fluorescent peptides can enter cells directly, and through imaging technologies like microscopy, their fluorescent signals reveal their activity inside living cells. This application is particularly critical for drug delivery systems. By labeling peptides that deliver drugs or carriers, researchers can dynamically monitor their uptake, distribution, intracellular release, and metabolism in cells. This is essential for developing targeted drugs and evaluating the efficacy of new treatments. For instance, in cancer drug development, fluorescent peptides can be used to assess whether drug molecules effectively enter tumor cells and their distribution in the tumor microenvironment. Additionally, research on cell-penetrating peptides (CPPs) relies on fluorescent labeling to track how these peptides help drugs or biomolecules cross cell membranes, aiding in the development of new drug delivery platforms.

• Drug Screening and Enzyme Kinetics Research

Fluorescent peptides play a critical role in drug screening and enzyme kinetics research. In drug screening, researchers often need to evaluate how small-molecule drugs affect the activity of specific enzymes or proteins. Fluorescently labeled substrate peptides provide direct feedback, as the fluorescence signal changes when enzymes act on the substrate, making drug and enzyme activity assessments rapid and efficient. For example, FRET probes are commonly used to monitor enzyme reactions in real-time. By incorporating two different fluorophores into a substrate peptide, researchers can track enzymatic processes through FRET. When the enzyme cleaves the substrate, the distance between the fluorophores changes, altering the fluorescence signal. This method is ideal for high-throughput screening of enzyme inhibitors or activators, especially for enzymes like proteases and kinases with well-defined substrates.

• Diagnostics and Biosensing

Fluorescent peptides also show great potential in medical diagnostics and biosensing. Fluorescent peptide probes can bind specific biomarkers, such as tumor cell surface receptors or pathogen antigens, allowing for the early diagnosis of diseases. In cancer detection, for instance, fluorescently labeled peptides can selectively bind to targets on cancer cells, making tumor tissues easier to identify in imaging, either in vivo or in vitro. This non-invasive detection technology offers new possibilities for early cancer diagnosis and treatment monitoring. In infectious disease detection, fluorescent peptide-labeled biosensors can rapidly detect pathogens. By designing fluorescent peptide sensors that bind specifically to viral or bacterial antigens, rapid and sensitive detection of pathogens can be achieved. Biosensor technologies combined with fluorescently labeled peptides simplify complex detection procedures while improving sensitivity and specificity.

• New Drug Discovery

Fluorescent peptides are widely used in drug discovery for studying targeted drug delivery and drug-target interactions. Fluorescence labeling allows researchers to track the distribution, metabolism, and target engagement of peptide drugs in the body. For example, in the development of antibody-drug conjugates (ADCs), fluorescence labeling is commonly used to monitor the targeting of the conjugates to tumors, evaluating delivery efficiency and release processes. This helps optimize drug design, improve efficacy, and reduce side effects. Additionally, fluorescently labeled peptides can be used to study drug interactions with cell membrane receptors, ion channels, and other biomolecular targets, providing critical experimental data for developing targeted drugs. In drug screening platforms, fluorescent peptides as substrates or probes accelerate the drug discovery process, enhancing the accuracy and efficiency of screening.

• Molecular Probes and Disease Biomarker Detection

Fluorescent peptides are also widely used as molecular probes for detecting disease biomarkers. By designing fluorescent peptide probes that bind specifically to disease-related proteins, enzymes, or metabolites, researchers can quickly and quantitatively detect these markers. For example, in Alzheimer's disease research, fluorescently labeled peptide probes are used to detect amyloid protein aggregation, providing new methods for studying disease mechanisms and screening potential therapeutic drugs.

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