Labeling Of Proteins Research Articles

Nucleic Acid Covalent Tags.

The selective and site-specific chemical labeling of proteins has emerged as a pivotal research area in chemical biology and cell biology. An effective protein labeling typically meets several criteria, including high specificity, rapid and robust conjugation under physiological conditions, operation at low concentrations with biocompatibility, and minimal perturbation of the protein function and activity. The conjugation of nucleic acids with proteins has garnered significant attention recently due to the rapid advancements in nucleic acid probe technologies, leveraging the programmable nature of nucleic acids alongside the multifaceted functionalities of proteins. It helps to convert protein-specific information into nucleic acid signals, facilitating upstream versatile recognition and downstream signal amplification for the target protein. This review critically evaluates the recent progress in nucleic acid-based protein labeling methodologies, with a specific focus on covalent labeling using aptamer tags, protein fusion tags or the technique of metabolic oligosaccharide engineering. The tags establish covalent linkages with target proteins through various modalities such as small molecules or metabolic glycan engineering. The insights presented in the review highlight promising avenues for the development of highly specific and versatile protein labeling techniques, which is essential for the improvement of protein-targeted detection and imaging across diverse biological contexts.

An efficient and easily obtainable butelase variant for chemoenzymatic ligation and modification of peptides and proteins

The expanding field of site-specific ligation of proteins and peptides has catalyzed the development of novel methods that enhance molecular modification. Among these methods, enzymatic strategies have emerged as dominant due to their specificity and efficiency in modifying proteins under mild conditions. Asparaginyl endopeptidase is a group of cyclotide-producing cysteine proteases from plants. These plant cysteine proteases, known for their specificity, effectively recognize the tripeptide motif (Asx-Xaa-Yaa) and cleave at the C-terminal side of Asx residues, forming acyl-enzyme intermediates that facilitate transpeptidation. Butelase 1 stands out as the most efficient AEP for protein engineering, yet challenges in its expression and purification limit its accessibility for widespread research and industrial use. To address these challenges, we engineered a new, catalytically efficient variant of Butelase 1, Butelase AY, by mutating the gatekeeping residues Val237Ala and Thr238Tyr within the LAD-1 region. These modifications significantly enhanced the stability and yield of Butelase AY, allowing for successful application in various peptide and protein engineering tasks. Butelase AY was tested on the peptide GLGKY, the globular protein GFP, and the intrinsically disordered protein α-synuclein, effectively labeling them with a fluorescent probe. Notably, Butelase AY maintained its efficiency with substrates containing unnatural amino acids, making it a promising candidate for biorthogonal applications. Importantly, the mutations did not compromise the enzyme’s specificity, as it continued to process model peptides and native protein substrates with N-term NHV recognition motifs effectively. In conclusion, Butelase AY presents a novel recombinant tool for diverse protein labeling and modifications, particularly in biorthogonal strategies. This innovation has the potential to expand applications in biotechnology and therapeutic development, ultimately revolutionizing protein engineering and its utility in synthetic biology.

Small-Molecule Benzo-Phenoselenazine Derivatives for Multi-Subcellular Biomolecule Profiling.

Elucidating the subcellular localization of RNAs and proteins is fundamental to understanding their biological functions. Genetically encoded proteins/enzymes provide an attractive approach to target many proteins of interest, but are limited to specific cell lines. Although small-molecule-based methods have been explored, a comprehensive system for profiling multiple locations in living cells, comparable to fusion-protein techniques, is yet to be established. In this study, we introduce a novel proximity labeling strategy employing a suite of small molecules derived from benzophenoselenazine (e.g., selenium-containing Nile Blue [SeNB]), which achieves proximity labeling through singlet oxygen generation upon near-infrared light activation in the presence of propargylamine. These SeNB compounds allow forselective labeling of RNAs and proteins within living cells, exhibiting a distinct preference for organelle membranes, which are systematically investigated via in vitro, computational, and in cellulo examinations. Our findings highlight the capabilities of SeNB derivatives as wash-free and genetics-free approaches to illuminate the subcellular localization of biological molecules with deep penetration and high spatial resolution. Moreover, SeNB derivatives are capable of elucidating inter-organelle interactions at the molecular level, as evidenced by proteomic and transcriptomic analyses, thus holding significant potential for advancing our understanding of cellular processes related to disease progression and therapeutic development.

A roadmap to cysteine specific labeling of membrane proteins for single-molecule photobleaching studies

Single-molecule photobleaching analysis is a useful approach for quantifying reactive membrane protein oligomerization in membranes. It provides a binary readout of a fluorophore attached to a protein subunit at dilute conditions. However, quantification of protein stoichiometry from this data requires information about the subunit labeling yields and whether there is non-specific background labeling. Any increases in subunit-specific labeling improves the ability to determine oligomeric states with confidence. A common strategy for site-specific labeling is by conjugation of a fluorophore bearing a thiol-reactive maleimide group to a substituted cysteine. Yet, cysteine reactivity can be difficult to predict as it depends on many factors such as solvent accessibility and electrostatics from the surrounding protein structure. Here we report a general methodology for screening potential cysteine labeling sites on purified membrane proteins. We present the results of two example systems for which the dimerization reactions in membranes has been characterized: (1) the CLC-ec1 Cl-/H+ antiporter, an Escherichia coli homologue of voltage-gated chloride ion channels in humans and (2) a mutant form of a member of the family of fluoride channels Fluc from Bordetella pertussis (Fluc-Bpe-N43S). To demonstrate how we identify such sites, we first discuss considerations of residue positions hypothesized to be suitable and describe the specific steps to rigorously assess site-specific labeling while maintaining the functional activity and robust single-molecule fluorescence signals. We find that our initial, well rationalized choices are not strong predictors of success, as rigorous testing of the labeling sites shows that only ≈30 % of sites end up being useful for single-molecule photobleaching studies

Modular Assembly of Heterotrifunctional Molecules Enabled by Iodosulfonylation of Allenes and Subsequent Amination.

Beyond the rapid achievements of therapeutic heterobifunctional molecules, some recent efforts have focused on constructing heterotrifunctional molecules, aiming at developing more potent and selective therapeutic agents or emerging additional functions to heterobifunctional molecules. However, the synthesis of these complex molecules requires a specific design and lengthy steps. We have developed a two-step strategy for the modular construction of heterotrifunctional molecules, enabled by the sustainable and convenient iodosulfonylation of allenes followed by SN2'-selective amination. This strategy successfully incorporates a broad range of biologically active molecules, labeling them with a fluorescent group. The applications of the obtained compounds in selective protein labeling, subcellular imaging, and targeted inhibition of tumor cells make this strategy highly appealing.

Diverse effects of fluorescent labels on alpha-synuclein condensate formation during liquid-liquid phase separation.

Liquid-liquid phase separation is an emerging field of study, dedicated to understanding the mechanism and role of biomolecule assembly into membraneless organelles. One of the main methods employed in studying protein and nucleic acid droplet formation is fluorescence microscopy. Despite functioning as an excellent tool for monitoring biomolecule condensation, a few recent reports have presented possible drawbacks of using fluorescently labeled particles. It was observed that fluorescent tags could alter the process of protein liquid-liquid phase separation and even promote their aggregation. In this study, we examined the influence of three different protein labels on alpha-synuclein phase separation in vitro and determined that the changes in droplet formation were related to both the type, as well as concentration of the fluorescently tagged alpha-synuclein. Both protein-based labels (mCherry and eGFP) induced the formation of significantly larger droplets, while fluorescein-tagged alpha-synuclein generated an abundance of small condensates or noticeably inhibited the process of LLPS. The study also revealed that alpha-synuclein with protein-based labels could self-associate at much lower concentrations than its untagged counterpart, forming either large droplets or protein aggregates.

Quantifying protein kinetics in vivo: Influence of precursor dynamics on product labeling.

Protein kinetics can be quantified by coupling stable isotope tracer methods with mass spectrometry readouts; however, inter-connected decision points in the experimental design affect the complexity of the workflow and impact data interpretations. For example, choosing between a single bolus (pulse-chase) or a continuous exposure protocol influences subsequent decisions regarding when to measure and how to model the temporal labeling of a target protein. Herein, we examine the merits of in vivo tracer protocols, we direct attention towards stable isotope tracer experiments that rely on administering a single bolus since these are generally more practical to use as compared to continuous administration protocols. We demonstrate how the interplay between precursor and product kinetics impacts downstream analytics and calculations by contrasting fast vs slow turnover precursors (e.g. 13C-leucine vs 2H-water, respectively). Although the data collected here underscore certain advantages of using longer lived precursors (e.g. 2H- or 18O-water) the results also highlight the influence of tracer recycling on measures of protein turnover. We discuss the impact of tracer recycling and consider how the sampling interval is critical for interpreting studies. Finally, we demonstrate that tracer recycling does not limit the ability to perform back-to-back studies of protein kinetics. It is possible to run experiments in which subjects are used as their own controls even though the precursor and product remain labeled following an initial tracer dosing.

FAPM: Functional Annotation of Proteins using Multi-Modal Models Beyond Structural Modeling.

Assigning accurate property labels to proteins, like functional terms and catalytic activity, is challenging, especially for proteins without homologs and "tail labels" with few known examples. Previous methods mainly focused on protein sequence features, overlooking the semantic meaning of protein labels. We introduce FAPM, a contrastive multi-modal model that links natural language with protein sequence language. This model combines a pretrained protein sequence model with a pretrained large language model to generate labels, such as Gene Ontology (GO) functional terms and catalytic activity predictions, in natural language. Our results show that FAPM excels in understanding protein properties, outperforming models based solely on protein sequences or structures. It achieves state-of-the-art performance on public benchmarks and in-house experimentally annotated phage proteins, which often have few known homologs. Additionally, FAPM's flexibility allows it to incorporate extra text prompts, like taxonomy information, enhancing both its predictive performance and explainability. This novel approach offers a promising alternative to current methods that rely on multiple sequence alignment for protein annotation. The online demo is at: https://huggingface.co/spaces/wenkai/FAPM_demo. Supplementary data are available at Bioinformatics online.

Endogenous Cu(II) Labeling for Distance Measurements on Proteins by EPR.

In-cell measurements of the relationship between structure and dynamics to protein function is at the forefront of biophysics. Recently, developments in EPR methodology have demonstrated the sensitivity and power of this method to measure structural constraints in-cell. However, the need to spin label proteins ex-situ or use noncanonical amino acids to achieve endogenous labeling remains a bottleneck. In this work we expand the methodology to endogenously spin label proteins with Cu(II) spin labels and describe how to assess in-cell spin labeling. We quantify the amount of Cu(II)-NTA in cells, assess spin labeling, and account for orientational effects during distance measurements. We compare the efficacy of using heat-shock and hypotonic swelling to deliver spin label, showing that hypotonic swelling is a facile and reproducible method to efficiently deliver Cu(II)-NTA into E. coli. Notably, over six repeats we accomplish a bulk average of 57 μM spin labeled sites, surpassing existing endogenous labeling methods. The results of this work open the door for endogenous spin labeling that is easily accessible to the broader biophysical community.

A rapid and versatile reverse genetic approach and visualization animal models for emerging zoonotic pseudorabies virus

Pseudorabies virus (PRV), a member of the Alphaherpesvirinae subfamily and a causative pathogen of Aujeszky's disease, has a broad host range including domestic and wild animals. PRV has been reported as a causative agent in patients with acute encephalitis in 2021, which suggests PRV might be a novel animal-origin virus in terms of zoonotic spillover and spread potential. To manage current PRV epidemics in pigs and prepare for future pandemics in other species including humans. Fundamental techniques essential for procuring such knowledge on prevention and therapy of PRV. Here, PRV CD22 strain was isolated and phylogenetic analysis showed that PRV CD22 belongs to the current epidemic strains in China. PRV CD22 was highly lethal to mice and piglets in vivo. Moreover, a rapid and efficient system to generate recombinant PRV was constructed based on PRV CD22 genomic DNA fosmid library. Using this system, a recombinant PRV strain expressing engineered labeling protein was rescued for visualization of viral infection in mouse model. Our study allows the generation of PRV that can be used for downstream treatment analyses. Once experimental or surveillance samples are obtained, PRV can be generated and treated efficiently based on our study.

Tunable Activated Esters Enable Lysine-Selective Protein Labeling and Profiling.

Lysine residues on protein surfaces are abundant and often found in enzyme active sites, making them critical targets for studying undruggable proteins. However, the varied microenvironment surrounding lysine residues results in a wide range of pKa values, complicating site-specific covalent binding. In this study, we address the challenges posed by the diverse reactivity of amino side chains by modulating the amide reaction activity of heteroaromatic activated esters. By fine-tuning the type, position, and number of heteroatoms, we successfully rationalized the regulation of their amide reaction activity, leading to the design of probes for selective lysine labeling within the proteome for profiling purposes. Systematic optimization of these esters' reactivity and selectivity has yielded a series of effective probes suitable for both in vitro and cellular applications. These findings significantly enhance our understanding of protein functions and mechanisms, facilitated by the precise identification and analysis of protein labeling and profiling.

Clickable HaloTag ligands for live cell labeling and imaging

Self-labeling protein (SLP) tags, such as HaloTag, have gained considerable interest as advanced tools for live cell labeling. However, the chloroalkane-based substrates that can be directly used for protein labeling are limited. Here, we report two bioorthogonal small molecule linkers, chloroalkane-tetrazine (CA-Tz) and chloroalkane-azide (CA-N3), which can penetrate cell membranes and facilitate click chemistry-based labeling in live cells. We compare their labeling capability using two clickable silicon rhodamine dyes (SiR-PEG3-TCO and SiR-PEG4-DBCO). Confocal imaging results demonstrate that using CA-Tz and SiR-PEG3-TCO dye exhibits superior intracellular labeling with low nonspecific signals. We subsequently compared the photostability of SiR dyes with that of green fluorescent proteins (mEmerald). Total internal reflection fluorescence (TIRF) imaging indicates that SiR dyes exhibit superior photostability under identical excitation conditions, making them suitable for long-term cell imaging. Furthermore, SiR dyes labeling also shows high structure retention for the fourth-order super-resolution optical fluctuation imaging (SOFI) compared to fluorescent proteins. This study presents clickable HaloTag linkers as effective tools for live cell labeling and imaging, highlighting the high-quality labeling of chloroalkane linkers and clickable dyes for live cell imaging.

Ac34deoGlcNAz: A Selective Probe for Identifying O-GlcNAc-Modified Proteins in Prostate Cancer.

O-GlcNAcylation is a prevalent protein modification in eukaryotic cellsandincreasing evidences indicated that over-expressed O-GlcNAcylation are intimately linked to the development and prognosis of prostate cancer.Thus, exploring this modification in the context of prostate cancer is vital for understanding the underlying mechanisms and hopefully used forfuture targeted therapies. In this paper, we use our previously established metabolic probes to comprehensively compare the labeling efficiency ofO-GlcNAc modified proteins in PC3 cells. Our results demonstrated that all the tested probes were non-toxic to PC3 cells and only Ac4GlcNAz, Ac4GalNAzand Ac34deoGlcNAzexhibited robust labeling signals amongst the probes. Further investigations by western blot and flow cytometry analysis revealed that Ac34deoGlcNAzwas a specific and efficient probe for intracelluar protein labeling with negligible S-glyco-modification signal. In addition, proteomic analysis further confirmed that 94% of the proteins identified byAc34deoGlcNAzwere in the form of O-linked GlcNAc, these enriched O-GlcNAcylated proteins were mainly involved in the regulated processes of prostate cancer. Our results here together prove Ac34deoGlcNAzis a safe and reliable probefor metabolic labeling O-GlcNAc modified proteins in prostate cancer, providing a mean to fully exploitthe regulatory mechanism of O-GlcNAcylation in the process of prostate cancer.

Immunoproteomics: Approach to Diagnostic and Vaccine Development.

The study of large protein sets (proteomics) involved in the immunological reaction is known as immunoproteomics. The methodology of immunoproteomics plays a major role in identifying possible vaccine candidates that could protect against pathogenic infection. The study of immunogenic proteins that are expressed during the outset of infection is the focus of the crosstalk between proteomics and immune protection antigens utilizing serum. Peptide presentation by MHC provides the new 'window' into changes that occur in the cell. Thus, there is strong, intense pressure on the pathogen that has been mutated in such an unusual manner that it can bypass the MHC peptide presentation by the MHC molecule. The pathogen's ability to evade the immune system is strongly restricted by the two unique distinct properties of MHC molecules, i.e., polygenic and polymorphic properties. MHC-I restriction epitope identification has traditionally been accomplished using genetic motif prediction. The study of immune system proteins and their interactions is the main emphasis of the specialist field of immunoproteomics within proteomics. Methodologies include mass spectrometry (MS), SRM assay, MALDI-TOF, Chromatography, ELISA, 2DG PAGE, and bioinformatics tools. Challenges are the complexity of the immune system, protein abundance and dynamics, sample variability, post-translational modifications (PTMs), and data integration. Current advancements are enhanced mass spectrometry techniques, single-cell proteomics, artificial intelligence and machine learning, advanced protein labeling techniques, integration with other omics technologies, and functional proteomics. However, the recently emerging field of immunoproteomics has more promising possibilities in the field of peptide-based vaccines and virus-like particle vaccines. The importance of immunoproteomics technologies and methodologies, as well as their use in the field of vaccinomics, are the main topics of this review. Here, we have discussed immunoproteomics in relation to a step towards the future of vaccination.

SPOT: spatial proteomics through on-site tissue-protein-labeling

BackgroundSpatial proteomics seeks to understand the spatial organization of proteins in tissues or at different subcellular localization in their native environment. However, capturing the spatial organization of proteins is challenging. Here, we present an innovative approach termed Spatial Proteomics through On-site Tissue-protein-labeling (SPOT), which combines the direct labeling of tissue proteins in situ on a slide and quantitative mass spectrometry for the profiling of spatially-resolved proteomics.Materials and MethodsEfficacy of direct TMT labeling was investigated using seven types of sagittal mouse brain slides, including frozen tissues without staining, formalin-fixed paraffin-embedded (FFPE) tissues without staining, deparaffinized FFPE tissues, deparaffinized and decrosslinked FFPE tissues, and tissues with hematoxylin & eosin (H&E) staining, hematoxylin (H) staining, eosin (E) staining. The ability of SPOT to profile proteomes at a spatial resolution was further evaluated on a horizontal mouse brain slide with direct TMT labeling at eight different mouse brain regions. Finally, SPOT was applied to human prostate cancer tissues as well as a tissue microarray (TMA), where TMT tags were meticulously applied to confined regions based on the pathological annotations. After on-site direct tissue-protein-labeling, tissues were scraped off the slides and subject to standard TMT-based quantitative proteomics analysis.ResultsTissue proteins on different types of mouse brain slides could be directly labeled with TMT tags. Moreover, the versatility of our direct-labeling approach extended to discerning specific mouse brain regions based on quantitative outcomes. The SPOT was further applied on both frozen tissues on slides and FFPE tissues on TMAs from prostate cancer tissues, where a distinct proteomic profile was observed among the regions with different Gleason scores.ConclusionsSPOT is a robust and versatile technique that allows comprehensive profiling of spatially-resolved proteomics across diverse types of tissue slides to advance our understanding of intricate molecular landscapes.

In-Depth Proteomic Analysis of Paraffin-Embedded Tissue Samples from Colorectal Cancer Patients Revealed TXNDC17 and SLC8A1 as Key Proteins Associated with the Disease.

A deeper understanding of colorectal cancer (CRC) biology would help to identify specific early diagnostic markers. Here, we conducted quantitative proteomics on FFPE healthy, adenoma, and adenocarcinoma tissue samples from six stage I sporadic CRC patients to identify dysregulated proteins during early CRC development. Two independent quantitative 10-plex TMT experiments were separately performed. After protein extraction, trypsin digestion, and labeling, proteins were identified and quantified by using a Q Exactive mass spectrometer. A total of 2681 proteins were identified and quantified after data analysis and bioinformatics with MaxQuant and the R program. Among them, 284 and 280 proteins showed significant upregulation and downregulation (expression ratio ≥1.5 or ≤0.67, p-value ≤0.05), respectively, in adenoma and/or adenocarcinoma compared to healthy tissue. Ten dysregulated proteins were selected to study their role in CRC by WB, IHC, TMA, and ELISA using tissue and plasma samples from CRC patients, individuals with premalignant colorectal lesions (adenomas), and healthy individuals. In vitro loss-of-function cell-based assays and in vivo experiments using three CRC cell lines with different metastatic properties assessed the important roles of SLC8A1 and TXNDC17 in CRC and liver metastasis. Additionally, SLC8A1 and TXNDC17 protein levels in plasma possessed the diagnostic ability of early CRC stages.

Specificity determinants of pathogens in forest

Abstract Host‐specific pathogens have long been suggested to act as a major driver of species diversity in tropical forests. However, determining the degree of host specificity of potential pathogens coupled to key cellular characteristics of infection is difficult and time‐consuming. These challenges have delayed progress in relating the functional specificity of pathogenic fungi to ecological consequences. We tested the pathogenicity of 27 (of 215) fungi that were isolated from surface sterilized roots of seedlings from four common tree species in a diverse subtropical forest. Inoculation experiments showed that six fungi exhibited strong pathogenicity on the host seedlings. Five of these only infected their specific hosts (i.e. host‐specific pathogen species). Green fluorescent protein labelling revealed that in three host‐specific pathogens fungal hyphae were able to grow into the vascular tissues only in their specific host plant, in contrast to other fungal‐host combinations that exhibit non‐pathogenic interactions. This fluorescent labelling technique allows direct tracking of the progress of fungal infection in different host tissues. Synthesis. By coupling the green fluorescent protein technique with standard host inoculation experiments, we determined the developmental differences between infections by pathogenic and non‐pathogenic fungi in a relatively simple and straightforward way. Our work provides a useful tool for rapidly screening and categorizing host–fungal interactions, reflecting the functional basis of host specificity of pathogenic fungi. More broadly, this technique can contribute to understanding the roles of pathogens in species coexistence and biodiversity maintenance in forest communities.

Localization of albumin with correlative super resolution light- and electron microscopy in the kidney

The functioning of vertebrate life relies on renal filtration of surplus fluid and elimination of low-molecular-weight waste products, while keeping serum proteins in the blood. In disease, however, there is leak of serum proteins and tracing them to identify the leaking position within tissue with a nanometer resolution poses a significant challenge. Correlative microscopy integrates the specificity of fluorescent protein labeling into high-resolution electron micrographs. Using chemical tagging of albumin with synthetic fluorophores we achieve protein-specific labeling that preserve their post-embedding fluorescence after high-pressure freezing and freeze-substitution of murine kidney tissue. Using advanced registration techniques for super-resolution correlative light and electron microscopy, we can localize the labeled albumin with a high precision in the x-y plane of electron micrographs and cartograph its distribution. Thereby we can quantify the albumin concentration and measure a linear reduction gradient across the kidney filtration barrier. Our study shows the feasibility of combining different microscopy contrasts for tracing fluorescently labeled protein markers with super resolution in various tissue samples and opens new perspectives for correlative imaging in volume electron microscopy.

Selective pH-Responsive Conjugation between a Pair of De Novo Discovered Peptides.

There is a demand for site-selective peptide/protein conjugation chemistry that is fully reversible in a stimulus-responsive manner. The contemporary methods for site-selective protein modification enable the preparation of homogeneous protein-small molecule conjugates, which are indispensable for drug delivery and chemical biology purposes, but such chemistries are usually irreversible. In contrast, the existing reversible protein labeling techniques are generally not site-selective. Here, we report an mRNA display-enabled de novo discovery of a pair of peptides which selectively react with each other to form a conjugate that is stable under neutral conditions (pH 7.5) but rapidly dissociates into the constituents at pH 10. A Cys thiol of peptide CP1 rapidly reacts (k1 = 340 M-1·s-1) with the isothiocyanate moiety in partner ITC6 to furnish a stable dithiocarbamate (t1/2 = 6 h at pH 7.5). We show that the pH-responsive nature of the reaction (conjugate's t1/2 = 5 min at pH 10) can be leveraged to utilize ITC6 (1) as a pull-down handle to selectively isolate CP1 from cell lysates and (2) as a temporary protecting group to protect CP1 from nonspecific Cys labeling reagents such as iodoacetamide. Altogether, the chemistry developed here complements the existing approaches and is applicable in a variety of chemical biology settings where selective reversible reactions are needed.

Enhancing substrate specificity of microbial transglutaminase for precise nanobody labeling

Streptomyces mobaraenesis transglutaminase (smTG) can be used for site-specific labeling of proteins with chemical groups. Here, we explored the use of modified smTG for the biosynthesis of nanobody-fluorophore conjugates (NFC). smTG catalyzes the conjugation of acyl donors containing glutamine with lysine-containing acceptors, which can lead to non-specific cross-linking. To achieve precise site-specific labeling, we employed molecular docking and virtual mutagenesis to redesign the enzyme's substrate specificity towards the peptide GGGGQR, a non-preferred acyl donor for smTG. Starting with a thermostable and highly active smTG variant (TGm2), we identified that single mutations G250H and Y278E significantly enhanced activity against GGGGQR, increasing it by 41 % and 1.13-fold, respectively. Notably, the Y278E mutation dramatically shifted the enzyme's substrate preference, with the activity ratio against GGGGQR versus the standard substrate CBZ-Gln-Gly rising from 0.05 to 0.93. In case studies, we used nanobodies 1C12 and 7D12 as labeling targets, catalyzing their conjugation with a synthetic fluorophore via smTG variants. Nanobodies fused with GGGGQR were successfully site-specifically labeled by TGm2-Y278E, in contrast to non-specific labeling observed with other variants. These results suggest that engineering smTG for site-specific labeling is a promising approach for the biosynthesis of antibody-drug conjugates.