Labeling Of Peptides Research Articles

Enhancing De Novo Protein Sequencing through the C-Terminal Labeling Strategy: Resolving Isobaric Ambiguities by Electron-Transfer/Higher Energy Collision Dissociation (EThcD).

De novo protein sequencing via a bottom-up approach requires various proteases to produce overlapping peptides. However, peptides generated by proteases other than trypsin, LysC, and ArgC often yield C-terminal fragments with suboptimal ionization in positive mode mass spectrometry (MS). This study introduces a novel peptide labeling strategy that involves modifying peptides at the C-terminal and at the carboxyl groups of Aspartic and Glutamic acid with arginine methyl ester (R-met) to improve peptide fragmentation and resolve isobaric ambiguities encountered during sequencing. An amidation reaction is used with coupling reagents to conjugate R-met to the peptide's C-terminal end, introducing a functional group that enhances the detectability of C-terminal peptide fragment ions by mass spectrometry. Subsequently, selecting a charge state of +2 or higher can facilitate optimal fragmentation of the derivatized peptides using electron-transfer/higher energy collision dissociation (EThcD), thereby generating essential w-ions to resolve common isobaric ambiguities. Demonstrating this strategy across diverse protein types, including albumin and antibodies and using different proteases for digestion, highlights the unique characteristics of combining the proposed amidation reaction with the specific proteases tested.

Circulating proteomic profiles in women with morbid obesity compared to normal-weight women

In this study, we aimed to evaluate circulating proteomic levels in women with morbid obesity (MO) compared to normal-weight (NW) women. Moreover, we have compared the proteomic profile between women with metabolically healthy (MH) MO and those with type 2 diabetes mellitus (T2DM). The study included 66 normal-weight (NW) women and 129 women with MO (54 MH and 75 with T2DM). Blood samples were processed for proteomics, involving protein extraction, quantification, digestion with peptide labelling and Nano (liquid chromatography (LC)-(Orbitrap) coupled to mass/mass spectrometry (MS/MS) analysis. Statistical analyses were performed. We identified 257 proteins. Women with MO showed significantly increased levels of 35 proteins and decreased levels of 45 proteins compared to NW women. Enrichment analysis of metabolic pathways revealed significant findings. Women with MO have an altered proteomic profile compared to normal-weight women, involving proteins significantly related to chylomicron assembly, complement cascade, clotting pathways and the insulin growth factor system. Regarding women with MO and T2DM compared to MHMO women, the proteomic profile shows alterations in mostly the same pathways associated with obesity. These findings confirmed in previous reports can help us better understand the pathophysiology of obesity and associated diseases. SignificanceWomen with morbid obesity (MO) exhibit substantial proteomic alterations compared to normal-weight (NW) women, involving 80 proteins. These alterations are linked to significant metabolic pathways, including chylomicron assembly, complement cascade, clotting pathways and the insulin growth factor system. Additionally, women with MO and type 2 diabetes mellitus (T2DM) compared to metabolically healthy MO women share similar proteomic changes than the first comparison. These findings enhance our understanding of the pathophysiology of obesity and associated diseases, offering potential targets for therapeutic intervention.

An optimised method to isotopically label pure synthetic peptides 'in-house' for absolute quantification in bottom-up proteomics.

Heavy-labelled internal standards increasingly represent the gold standard for absolute quantitation in mass spectrometry (MS)-based bottom-up proteomics. The biggest drawbacks of using these standards are that they have high costs and lengthy lead times. We describe an efficient, low-cost optimised method to enable 'in-house' heavy labelling of synthetic tryptic peptides for absolute quantification using tandem LC-MS/MS mass spectrometry. Our methodology uses 18O water in a trypsin-catalysed oxygen exchange reaction at the carboxyl terminus with the overall aim of reducing the costs and lead time associated with sourcing heavy standards from commercial vendors. Step-by-step instructions are provided on how to execute this protocol with high-throughput adaptations utilising a 96-well plate and a liquid-handling robot. Detailed notes on experimental setup, tips for troubleshooting and suggested improvements to maximise labelling efficiencies are highlighted to achieve the best results. Under optimum conditions, labelling efficiencies of peptides can reach from 95% to 100%. The application of the 'in-house' labelled standards in generating calibration curves to quantify endogenous peptide concentrations is just as effective as using the synthetically sourced standards while also having great cost reduction implications as well as saving time spent waiting for peptides to arrive. The protocol is highly adaptable and can be customized to fit the specific setup of any laboratory, maximizing achievable labelling efficiencies.

Multimaterial 3D Laser Printing of Cell-Adhesive and Cell-Repellent Hydrogels.

Here, a straightforward method is reported for manufacturing 3D microstructured cell-adhesive and cell-repellent multimaterials using two-photon laser printing. Compared to existing strategies, this approach offers bottom-up molecular control, high customizability, and rapid and precise 3D fabrication. The printable cell-adhesive polyethylene glycol (PEG) based material includes an Arg-Gly-Asp (RGD) containing peptide synthesized through solid-phase peptide synthesis, allowing for precise control of the peptide design. Remarkably, minimal amounts of RGD peptide (<0.1wt%) suffice for imparting cell-adhesiveness, while maintaining identical mechanical properties in the 3D printed microstructures to those of the cell-repellent, PEG-based material. Fluorescent labeling of the RGD peptide facilitates visualization of its presence in cell-adhesive areas. To demonstrate the broad applicability of the system, the fabrication of cell-adhesive 2.5D and 3D structures is shown, fostering the adhesion of fibroblast cells within these architectures. Thus, this approach allows for the printing of high-resolution, true 3D structures suitable for diverse applications, including cellular studies in complex environments.

Functional Insights into the Sphingolipids C1P, S1P, and SPC in Human Fibroblast-like Synoviocytes by Proteomic Analysis.

The (patho)physiological function of the sphingolipids ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P), and sphingosylphosphorylcholine (SPC) in articular joints during osteoarthritis (OA) is largely unknown. Therefore, we investigated the influence of these lipids on protein expression by fibroblast-like synoviocytes (FLSs) from OA knees. Cultured human FLSs (n = 7) were treated with 1 of 3 lipid species-C1P, S1P, or SPC-IL-1β, or with vehicle. The expression of individual proteins was determined by tandem mass tag peptide labeling followed by high-resolution electrospray ionization (ESI) mass spectrometry after liquid chromatographic separation (LC-MS/MS/MS). The mRNA levels of selected proteins were analyzed using RT-PCR. The 3sphingolipids were quantified in the SF of 18 OA patients using LC-MS/MS. A total of 4930 proteins were determined using multiplex MS, of which 136, 9, 1, and 0 were regulated both reproducibly and significantly by IL-1β, C1P, S1P, and SPC, respectively. In the presence of IL-1ß, all 3 sphingolipids exerted ancillary effects. Only low SF levels of C1P and SPC were found. In conclusion, the 3 lipid species regulated proteins that have not been described in OA. Our results indicate that charged multivesicular body protein 1b, metal cation symporter ZIP14, glutamine-fructose-6-P transaminase, metallothionein-1F and -2A, ferritin, and prosaposin are particularly interesting proteins due to their potential to affect inflammatory, anabolic, catabolic, and apoptotic mechanisms.

In-depth phosphoproteomic profiling of the insulin signaling response in heart tissue and cardiomyocytes unveils canonical and specialized regulation

BackgroundInsulin signaling regulates cardiac substrate utilization and is implicated in physiological adaptations of the heart. Alterations in the signaling response within the heart are believed to contribute to pathological conditions such as type-2 diabetes and heart failure. While extensively investigated in several metabolic organs using phosphoproteomic strategies, the signaling response elicited in cardiac tissue in general, and specifically in the specialized cardiomyocytes, has not yet been investigated to the same extent.MethodsInsulin or vehicle was administered to male C57BL6/JRj mice via intravenous injection into the vena cava. Ventricular tissue was extracted and subjected to quantitative phosphoproteomics analysis to evaluate the insulin signaling response. To delineate the cardiomyocyte-specific response and investigate the role of Tbc1d4 in insulin signal transduction, cardiomyocytes from the hearts of cardiac and skeletal muscle-specific Tbc1d4 knockout mice, as well as from wildtype littermates, were studied. The phosphoproteomic studies involved isobaric peptide labeling with Tandem Mass Tags (TMT), enrichment for phosphorylated peptides, fractionation via micro-flow reversed-phase liquid chromatography, and high-resolution mass spectrometry measurements.ResultsWe quantified 10,399 phosphorylated peptides from ventricular tissue and 12,739 from isolated cardiomyocytes, localizing to 3,232 and 3,128 unique proteins, respectively. In cardiac tissue, we identified 84 insulin-regulated phosphorylation events, including sites on the Insulin Receptor (InsrY1351, Y1175, Y1179, Y1180) itself as well as the Insulin receptor substrate protein 1 (Irs1S522, S526). Predicted kinases with increased activity in response to insulin stimulation included Rps6kb1, Akt1 and Mtor. Tbc1d4 emerged as a major phosphorylation target in cardiomyocytes. Despite limited impact on the global phosphorylation landscape, Tbc1d4 deficiency in cardiomyocytes attenuated insulin-induced Glut4 translocation and induced protein remodeling. We observed 15 proteins significantly regulated upon knockout of Tbc1d4. While Glut4 exhibited decreased protein abundance consequent to Tbc1d4-deficiency, Txnip levels were notably increased. Stimulation of wildtype cardiomyocytes with insulin led to the regulation of 262 significant phosphorylation events, predicted to be regulated by kinases such as Akt1, Mtor, Akt2, and Insr. In cardiomyocytes, the canonical insulin signaling response is elicited in addition to regulation on specialized cardiomyocyte proteins, such as Kcnj11Y12 and DspS2597. Details of all phosphorylation sites are provided.ConclusionWe present a first global outline of the insulin-induced phosphorylation signaling response in heart tissue and in isolated adult cardiomyocytes, detailing the specific residues with changed phosphorylation abundances. Our study marks an important step towards understanding the role of insulin signaling in cardiac diseases linked to insulin resistance.Graphical

Quenching Trypsin Is Unnecessary in Filter-Based Bottom-Up Proteomics.

Quenching digestions in proteomics prior to analysis is routine in order to eliminate residual protease activity. Residual activity leads to overdigestion, nonspecific star-activity, and back-exchange in isotopic 18O quantitation. Chemical and isobaric labeling (e.g., TMT/iTRAQ) of proteins or peptides for mass spectrometry-based proteomics is generally incompatible with ubiquitous postdigestion acidification. This necessitates buffer exchange and pH adjustments. We demonstrate that quenching is unnecessary with peptides generated from protein filter-traps, as trypsin activity and intact trypsin are negligible in the eluate from these preparations. Labeling can be directly performed on enzymatic digests from these methods, improving recovery, throughput, and ease of automation.

Hetero-Diels-Alder and CuAAC Click Reactions for Fluorine-18 Labeling of Peptides: Automation and Comparative Study of the Two Methods.

Radiolabeled peptides are valuable tools for diagnosis or therapies; they are often radiofluorinated using an indirect approach based on an F-18 prosthetic group. Herein, we are reporting our results on the F-18 radiolabeling of three peptides using two different methods based on click reactions. The first one used the well-known CuAAC reaction, and the second one is based on our recently reported hetero-Diels-Alder (HDA) using a dithioesters (thia-Diels-Alder) reaction. Both methods have been automated, and the 18F-peptides were obtained in similar yields and synthesis time (37-39% decay corrected yields by both methods in 120-140 min). However, to obtain similar yields, the CuAAC needs a large amount of copper along with many additives, while the HDA is a catalyst and metal-free reaction necessitating only an appropriate ratio of water/ethanol. The HDA can therefore be considered as a minimalist method offering easy access to fluorine-18 labeled peptides and making it a valuable additional tool for the indirect and site-specific labeling of peptides or biomolecules.

Peptide derived from plant defensins: A promising 68Ga radiolabelled agent for diagnostic of infection foci in PET.

The development of new radiopharmaceuticals for the detection of hidden infection foci has great relevance for early detection and the selection of the correct treatment, particularly in immunosuppressed patients. In that sense, the labelling of antimicrobial peptides (AMPs) that are capable of binding specifically to the pathogenic microorganism which causes the infection, should provide a sufficiently specific agent, able to distinguish an infection from a sterile inflammation. Defensins are particularly interesting molecules with antimicrobial activity, the EcgDf1 defensin was identified from the genome of a Uruguayan native plant, Erythrina crista-galli, the 'Ceibo' tree. Our group has previously reported a synthetic biologically active short analogue EcgDf21 (ERFTGGHCRGFRRRCFCTKHC) successfully labelled with 99mTc. Herein we present a shorter analogue which also preserves the γ-core domain, as a pharmacophore for a potential infection detection agent. This peptide was derivatized with the bifunctional chelating agent 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) through a lysine linker in the amino-terminal group (NOTA-KGHCRGFRRRC) and radiolabelled with 68Ga ([68Ga]Ga-NOTA-K-EcgDf1(10)). The [68Ga]Ga-NOTA-K-EcgDf1(10) labelling procedure rendered a product with high radiochemical purity and stability in the labelling milieu. The Log P value indicated that the complex has a hydrophilic behaviour, confirmed by the biodistribution profile. The [68Ga]Ga-NOTA-K-EcgDf1(10) complex demonstrated specific binding to cultures of Candida albicans and Aspergillus niger. Its biodistribution showed renal elimination and low accumulation in the rest of the body. It was possible to successfully differentiate sterile inflammation from infection by PET images in nude mice with a target/non-target ratio of 3.3 for C. albicans and 3.7 for A. niger, respectively.

Uncovering mussel foot protein (Mfp-3)'s binding secrets: Enhanced biomimetic adhesives via mass spectrometry and residue labeling

This study delves into the development of biomimetic adhesives, taking inspiration from the impressive underwater adhesion of mussels. In a departure from the traditional focus on DOPA, we explore a more comprehensive range of amino acids crucial for adhesion in wet environments. By utilizing amino acid labeling and mass spectrometry-based peptide mapping, we identify key peptide sequences within Mfp-3 responsible for effective aquatic adhesion. Of particular interest is the sequence YNRYARGY, which exhibits robust adhesion on both polar/acidic and hydrophobic surfaces, with an adsorption-free energy of approximately -4.5 kcal/mol (as determined by QCM). Incorporating this sequence (1% w/v), with tyrosine substitution for DOPA, into a standard PVA binder significantly enhances its adhesion capabilities. Lap-shear tests demonstrate a significant increase in PVA adhesion from 2.5 to 5 kPa on SiO2 and from 2.1 to 3.3 kPa on HDPE. These findings highlight the practical implications of our mass spectrometry approach with side chain modification in elucidating the mechanisms of mussel adhesion, thereby offering a promising strategy for advancing polymer adhesion technology.

Early postovulatory aging reveals the first proteomic markers of egg quality in pikeperch

This study explored the molecular mechanisms underlying egg quality deterioration due to in vivo postovulatory aging in pikeperch (Sander lucioperca L.), a key species in aquaculture. We employed tandem mass tag (TMT) peptide labeling coupled with LC–MS/MS quantitative proteomics to analyze eggs collected at various postovulation intervals. Our research revealed four distinct proteomic markers (Gins4, Atrx, DnaJB14, and Mrpl10) that are differentially expressed in response to early aging, shedding light on their potential roles in DNA replication, chromatin organization, protein folding, and mitochondrial function. The study confirmed that eggs maintain morphological integrity up to 5 h postovulation but exhibit compromised fertilization capacity, underscoring the importance of timely egg utilization in aquaculture practices. These findings enhance the understanding of egg aging at the molecular level, offering insights for improving reproductive success and larval quality in pikeperch aquaculture. The data are available via ProteomeXchange with the identifier PXD048349.

Ideas Behind the Tryptophan-Mediated Petasis Reaction (TMPR) Concept for Peptide Stapling.

This Concept short review offers an insightful analysis of pivotal research papers and explores the key synthetic ideas behind the intersection of two realms in peptide chemistry: using tryptophan and Petasis multicomponent reactions for macrocyclisation and labelling of peptides. The recently published tryptophan-mediated Petasis reaction (TMPR) concept represents a critical junction between these two worlds, highlighting how combining such methodologies leads to more effective and versatile synthetic strategies, setting a potentially new direction for future research in the field of peptide-drug conjugates.

Fluorescence Labeling of Peptides: Finding the Optimal Protocol for Coupling Various Dyes to ATCUN-like Structures.

Labeling of peptides and proteins with fluorescent dyes is a key step in functionalizing these structures for a wide array of biological assays. However, coupling strategies of such dyes have not been optimized for the most common compounds, while this step is typically the most precious and costly of the whole synthesis. We searched for the best conditions for attachment of the most widely used fluorescent dyes such as 6-carboxyfluorescein, Rhodamine B, and BODIPY-FL to peptides, where amino terminal Cu(II) and Ni(II) binding site (ATCUN) peptides were used as a model system. Surprisingly, conventional methods of dye attachment proved to not be satisfactory and yielded poor efficiency results. We have discovered that when labeling primary amines on peptides, the uncommon synthesis of activated pentafluorophenol (PFP) esters is the most efficient strategy, expedited by microwave irradiation. Coupling to secondary amines is achieved most efficiently through conventional coupling reagents such as HATU and PyBOP. Furthermore, we have employed our fluorescently labeled ATCUN peptides in studies for Cu(II) and Ni(II) sensing, showing that changing the fluorophore does not significantly affect the fluorescence quenching process and discovering the optimal linker length between the ATCUN core and the dye, expanding the repertoire of fluorophores that can be used in this application.

A Method for Rigorously Selective Capture and Simultaneous Fluorescent Labeling of N-Terminal Glycine Peptides.

Although chemical methods for the selective derivatization of amino acid (AA) side chains in peptides and proteins are available, selective N-terminal labeling is challenging, especially for glycine, which has no side chain at the α-carbon position. We report here a double activation at glycine's α-methylene group that allows this AA to be differentiated from the other 19 AAs. A condensation reaction of dibenzoylmethane with glycine results in the formation of an imine, and subsequent tautomerization is followed by intramolecular cyclization, leading to the formation of a fluorescent pyrrole ring. Additionally, the approach exhibits compatibility with AAs possessing reactive side chains. Further, the method allows for selective pull-down assays of N-terminal glycine peptides from mixtures without prior knowledge of the N-terminal peptide distribution.

An all-in-one tetrazine reagent for cysteine-selective labeling and bioorthogonal activable prodrug construction

The prodrug design strategy offers a potent solution for improving therapeutic index and expanding drug targets. However, current prodrug activation designs are mainly responsive to endogenous stimuli, resulting in unintended drug release and systemic toxicity. In this study, we introduce 3-vinyl-6-oxymethyl-tetrazine (voTz) as an all-in-one reagent for modular preparation of tetrazine-caged prodrugs and chemoselective labeling peptides to produce bioorthogonal activable peptide-prodrug conjugates. These stable prodrugs can selectively bind to target cells, facilitating cellular uptake. Subsequent bioorthogonal cleavage reactions trigger prodrug activation, significantly boosting potency against tumor cells while maintaining exceptional off-target safety for normal cells. In vivo studies demonstrate the therapeutic efficacy and safety of this prodrug design approach. Given the broad applicability of functional groups and labeling versatility with voTz, we foresee that this strategy will offer a versatile solution to enhance the therapeutic range of cytotoxic agents and facilitate the development of bioorthogonal activatable biopharmaceuticals and biomaterials.

An efficient peptide ligase engineered from a bamboo asparaginyl endopeptidase.

In recent years, a few asparaginyl endopeptidases (AEPs) from certain higher plants have been identified as efficient peptide ligases with wide applications in protein labeling and cyclic peptide synthesis. Recently, we developed a NanoLuc Binary Technology (NanoBiT)-based peptide ligase activity assay to identify more AEP-type peptide ligases. Herein, we screened 61 bamboo species from 16 genera using this assay and detected AEP-type peptide ligase activity in the crude extract of all tested bamboo leaves. From a popular bamboo species, Bambusa multiplex, we identified a full-length AEP-type peptide ligase candidate (BmAEP1) via transcriptomic sequencing. After its zymogen was overexpressed in Escherichia coli and self-activated in vitro, BmAEP1 displayed high peptide ligase activity, but with considerable hydrolytic activity. After site-directed mutagenesis of its ligase activity determinants, the mutant zymogen of [G238V]BmAEP1 was normally overexpressed in E. coli, but failed to activate itself. To resolve this problem, we developed a novel protease-assisted activation approach in which trypsin was used to cleave the mutant zymogen and was then conveniently removed via ion-exchange chromatography. After the noncovalently bound cap domain was dissociated from the catalytic core domain under acidic conditions, the recombinant [G238V]BmAEP1 displayed high peptide ligase activity with much lower hydrolytic activity and could efficiently catalyze inter-molecular protein ligation and intramolecular peptide cyclization. Thus, the engineered bamboo-derived peptide ligase represents a novel tool for protein labeling and cyclic peptide synthesis.

Steady State and Time-Dependent Fluorescent Peptide Assays for Protein Kinases.

Protein kinases catalyze the phosphorylation of proteins most commonly on Ser, Thr, and Tyr residues and regulate many cellular events in eukaryotic cells, such as cell cycle progression, transcription, metabolism, and apoptosis. Protein kinases each have a conserved ATP-binding site and one or more substrate-binding site(s) that exhibit recognition features for different protein substrates. By bringing ATP and a substrate into proximity, each protein kinase can transfer the γ phosphate of the ATP molecule to a hydroxyl group of the target residue on the substrate. In such a way, signaling pathways downstream from the substrate can be regulated based on the phosphorylated versus dephosphorylated status of the substrate. Although there are a number of ways to assay the activity of protein kinases, most of them are technically cumbersome and/or are indirect or based on quenched reactions. This protocol describes an assay employing a fluorescent peptide substrate to detect phosphorylation by protein kinases in real time. The assay is based on the principle that the phosphorylation of the peptide substrate leads to an increase in the fluorescence emission intensity of an appended fluorophore. We extend the application of this assay to an example of how to assess time-dependent covalent inhibition of kinases as well. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Measuring protein kinase activity using fluorescent peptides Alternate Protocol: Measuring protein kinase activity using a fluorescence plate reader Support Protocol: Labeling peptides with sox fluorophore Basic Protocol 2: Measuring time-dependent ATP-competitive inhibition of protein kinases using fluorescent peptides.

Quantitative N‐ or C‐Terminal Labelling of Proteins with Unactivated Peptides by Use of Sortases and a d‐Aminopeptidase

AbstractQuantitative and selective labelling of proteins is widely used in both academic and industrial laboratories, and catalytic labelling of proteins using transpeptidases, such as sortases, has proved to be a popular strategy for such selective modification. A major challenge for this class of enzymes is that the majority of procedures require an excess of the labelling reagent or, alternatively, activated substrates rather than simple commercially sourced peptides. We report the use of a coupled enzyme strategy which enables quantitative N‐ and C‐terminal labelling of proteins using unactivated labelling peptides. The use of an aminopeptidase in conjunction with a transpeptidase allows sequence‐specific degradation of the peptide by‐product, shifting the equilibrium to favor product formation, which greatly enhances the reaction efficiency. Subsequent optimisation of the reaction allows N‐terminal labelling of proteins using essentially equimolar ratios of peptide label to protein and C‐terminal labelling with only a small excess. Minimizing the amount of substrate required for quantitative labelling has the potential to improve industrial processes and facilitate the use of transpeptidation as a method for protein labelling.

Quantitative N- or C-Terminal Labelling of Proteins with Unactivated Peptides by Use of Sortases and a d-Aminopeptidase.

Quantitative and selective labelling of proteins is widely used in both academic and industrial laboratories, and catalytic labelling of proteins using transpeptidases, such as sortases, has proved to be a popular strategy for such selective modification. A major challenge for this class of enzymes is that the majority of procedures require an excess of the labelling reagent or, alternatively, activated substrates rather than simple commercially sourced peptides. We report the use of a coupled enzyme strategy which enables quantitative N- and C-terminal labelling of proteins using unactivated labelling peptides. The use of an aminopeptidase in conjunction with a transpeptidase allows sequence-specific degradation of the peptide by-product, shifting the equilibrium to favor product formation, which greatly enhances the reaction efficiency. Subsequent optimisation of the reaction allows N-terminal labelling of proteins using essentially equimolar ratios of peptide label to protein and C-terminal labelling with only a small excess. Minimizing the amount of substrate required for quantitative labelling has the potential to improve industrial processes and facilitate the use of transpeptidation as a method for protein labelling.

Visualization of the Plasmid DNA Delivery System by Complementary Fluorescence Labeling of Arginine-Rich Peptides.

Cell-penetrating peptides, such as arginine-rich peptides, encapsulate nucleic acid drugs and deliver them to intracellular compartments. Comprehensive tracking of drug delivery systems (DDSs) provides information about the behavior of the drug as well as the fate of the drug carrier after drug release, which is crucial for minimizing side effects. In this study, we labeled peptides designed to carry plasmid DNA with two types of dyes, traditional dye fluorescein and aggregation-induced emission (AIE) dye tetraphenylethylene, and subsequently tracked the DDS through the complementary ON and OFF fluorescence behaviors of the dyes. Traditional fluorescent dyes are susceptible to aggregation-caused quenching during bioimaging, a problem that is mitigated by using AIE dyes. However, by using both of these contrasting fluorescent labels, we were able to clearly visualize the DDS at different stages of its deployment, from drug transport and delivery to carrier dissociation and migration, demonstrating the feasibility of accurate DDS visualization by complementary fluorescence labeling.