Secreted proteins serve as pivotal messengers of intricate intercellular interactions. These proteins govern a spectrum of important biological processes including cell–cell interaction, immune responses, and cellular functional regulation, etc. Particularly, cytokines are a class of vital molecular messengers that exert substantial influence on immune responses. As imperative signaling transduction proteins, cytokines regulate immune reactions [1], elicit apoptosis effects against tumor cells [2], bolster inflammatory cascades [3], and intricately influence cellular metabolism [4]. Therefore, cytokines hold paramount significance in elucidating immune regulatory mechanisms, advancing disease prevention, and refining diagnostic and therapeutic interventions [5], [6], [7].

The emergence of single-cell analysis has paved the way to elucidate inter-cellular disparities, encompassing transcriptomic [8], [9], [10], [11], [12], [13], proteomic [14], genomic [15], [16], and epigenetic profiles [17], which often remain concealed or overlooked by conventional bulk-based methods. Particularly, single-cell protein secretion analysis provides critical insights into the immune cell functionality and the evaluation of tumor immunotherapies [18], [19]. Usually, flow cytometry has been extensively utilized to detect secreted proteins labeled with fluorescent antibodies or isotopes [20], [21], [22], presenting notable advantages including high throughput capabilities and versatility. But the pre-processing steps, such as cell fixation, may potentially impact the accuracy. On the other hand, Enzyme-Linked Immunospot Assay (ELISpot) has also been employed to capture secreted proteins from cultured cells using antibodies immobilized on a plate [23], [24], offering simplicity in operation, general applicability, and independence from complex equipment. Besides, microarray-based strategies [25], [26], [27], [28], [29], [30] and microfluidic-based strategies [31], [32], [33], [34], [35] have been developed for high-throughput analysis of secreted proteins based on fluorescent images of secreted proteins.

While the importance of analyzing secreted proteins is well recognized, it remains challenging to decipher the intricate correlation between gene expressions and protein secretion at the single-cell level. The intricate interplay serves as a cornerstone of cellular communication, influencing diverse biological processes ranging from intercellular signaling to disease progression. Integrated characterization of single-cell transcriptomes and secreted proteins enables precise dissection of cellular types or states and facilitates a nuanced understanding of cellular dynamics under diverse physiological conditions. The advent of single-cell RNA sequencing (scRNA-seq) has transformed our ability to characterize the transcriptomes of individual cells. More importantly, the incorporation of barcoding DNA-antibody complexes inspired by CITE-seq and REAP-seq has facilitated simultaneous analysis of transcriptome and proteins [36], [37]. In these cases, barcode antibodies can be captured by barcode beads in a similar way as mRNA, shared the same cell index with transcriptome during reverse transcription, and finally deciphered by high-throughput sequencing. Although these strategies have found extensive applications in surface proteins and intracellular proteins [38], [39], but few has been utilized for secreted proteins. The challenge lies in how to simultaneously identify individual cell with corresponding secreted proteins. To anchor capture antibodies on cells, strategies such as using avidin/biotin interactions [21], [40] or bispecific antibodies [34], [41], [42], [43] have been proposed. Although accessible, they are susceptible to either the interference of non-specific binding or engineering complexity and limited universal surface marker to anchor antibodies.

Herein, we proposed a novel cell-antibody conjugate-based sequencing methodology (Cellab-seq) that enabled simultaneous analysis of protein secretion and transcriptome. Cellab-seq leveraged a chemoenzymatic strategy to construct cell-antibody conjugates using fucosyltransferase [44], [45], which enabled the transfer of capture antibodies to the glycan on cell surfaces. In this way, capture antibodies could be easily anchored on the cell surface in one-step, leading to the construction of cell-antibody complexes. Consequently, secreted proteins can be captured on cell surface, facilitating the formation of on-cell immunosandwich structure with the incorporation of barcoded detection antibodies. Finally, these distinctive antibody signals were translated into sequencing data. Taking natural killer (NK) cells, a subset of innate immune cells that play a pivotal role in immune surveillance and tumor killing, as the example, we correlated their transcriptome and protein secretion levels across diverse stimulated status. The results revealed the transcriptomic signatures underpinning with protein secretion of NK cells across varying stimulation status, and indicated asynchronous patterns between mRNA and protein expression levels. We anticipated that this strategy could forge a pathway to a more comprehensive understanding of cellular behavior and elevate our comprehension of immune cell orchestration.