Aminoacyl tRNA synthetases (aaRSs) have long been viewed as mere housekeeping proteins and have therefore often been overlooked in drug discovery. However, recent findings have revealed that many aaRSs have non-canonical functions and several of the aaRSs have been linked to autoimmune diseases, cancer and neurological disorders. Deciphering these roles has been challenging due to a lack of tools to enable their study. To help solve this problem, we have generated recombinant high-affinity antibodies for a collection of thirteen cytoplasmic and one mitochondrial aaRSs. Selected domains of these proteins were produced recombinantly in Escherichia coli and used as antigens in phage display selections using a synthetic human single-chain fragment variable (scFv) library. All targets yielded large sets of antibody candidates that were validated through a panel of binding assays against the purified antigen. Furthermore, the top performing binders were tested in immunoprecipitation followed by mass spectrometry (IP-MS) for their ability to capture the endogenous protein from mammalian cell lysates. For antibodies targeting individual members of the multi-tRNA synthetase complex (MSC), we were able to detect all members of the complex, co-immunoprecipitating with the target, in several cell types. The functionality of a sub-set of binders for each target was also confirmed using immunofluorescence. The sequences of these proteins have been deposited in publicly available databases and repositories. We anticipate this open source resource, in the form of high quality recombinant proteins and antibodies, will accelerate and empower future research of the role of aaRSs in health and disease.

Systemic Lupus Erythematosus (SLE) is a heterogeneous autoimmune disease, which currently lacks specific diagnostic biomarkers. The diversity within the patients obstructs clinical trials but may also reflect differences in underlying pathogenesis. Our objective was to obtain protein profiles to identify potential general biomarkers of SLE and to determine molecular subgroups within SLE for patient stratification. Plasma samples from a cross-sectional study of well-characterized SLE patients ( n = 379) and matched population controls ( n = 316) were analyzed by antibody suspension bead array targeting 281 proteins. To investigate the differences between SLE and controls, Mann–Whitney U -test with Bonferroni correction, generalized linear modeling and receiver operating characteristics (ROC) analysis were performed. K-means clustering was used to identify molecular SLE subgroups. We identified Interferon regulating factor 5 (IRF5), solute carrier family 22 member 2 (SLC22A2) and S100 calcium binding protein A12 (S100A12) as the three proteins with the largest fold change between SLE patients and controls (SLE/Control = 1.4, 1.4, and 1.2 respectively). The lowest p -values comparing SLE patients and controls were obtained for S100A12, Matrix metalloproteinase-1 (MMP1) and SLC22A2 (p adjusted = 3 × 10 −9 , 3 × 10 −6 , and 5 × 10 −6 respectively). In a set of 15 potential biomarkers differentiating SLE patients and controls, two of the proteins were transcription factors, i.e., IRF5 and SAM pointed domain containing ETS transcription factor (SPDEF). IRF5 was up-regulated while SPDEF was found to be down-regulated in SLE patients. Unsupervised clustering of all investigated proteins identified three molecular subgroups among SLE patients, characterized by (1) high levels of rheumatoid factor-IgM, (2) low IRF5, and (3) high IRF5. IRF5 expressing microparticles were analyzed by flow cytometry in a subset of patients to confirm the presence of IRF5 in plasma and detection of extracellular IRF5 was further confirmed by immunoprecipitation-mass spectrometry (IP-MS). Interestingly IRF5, a known genetic risk factor for SLE, was detected extracellularly and suggested by unsupervised clustering analysis to differentiate between SLE subgroups. Our results imply a set of circulating molecules as markers of possible pathogenic importance in SLE. We believe that these findings could be of relevance for understanding the pathogenesis and diversity of SLE, as well as for selection of patients in clinical trials.

We describe a mass spectrometry-based approach for validation of antibody specificity. This method allows validation of antibodies or antibody fragments, against their endogenous targets. It can assess if the antibody is able to bind to its native antigen in cell lysates among thousands of other proteins, DNA, RNA, and other cellular components. In addition, it identifies other proteins the antibody is able to immunoprecipitate allowing for the assessment of antibody specificity and selectivity. This method is easily scalable, adaptable to different cell lines and conditions and has been shown to be reproducible between multiple laboratories.