Charlesworth, Mary Cristine

ABSTRACT T‐cell receptor (TCR) signaling plays a crucial role in various biological processes and is usually studied using global mass spectrometry‐based phosphoproteomic studies. Despite advancements in targeted mass spectrometry‐based assays for protein quantification, their application in studying signaling processes, for example, reproducible measurements of post‐translational modifications (PTMs) such as phosphorylation, remains limited. Tyrosine phosphorylation is critical for many signaling pathways but presents challenges due to the low abundance of phosphotyrosine‐containing peptides. Conventional untargeted methods often encounter data gaps when analyzing large sample sets, particularly for low‐abundance peptides. To address this issue, a targeted proteomics method called “SureQuant” was employed, which relies on triggered data acquisition with heavy isotope‐labeled peptides. This method has been shown to provide sensitive and reproducible quantification of low‐abundance peptides. Here we describe the development of a SureQuant‐based method to quantify phosphotyrosine peptides that are involved in the TCR signaling pathway. To monitor the change in phosphotyrosine signals upon activation, the T‐cells were stimulated with anti‐CD3/CD28 antibodies. We successfully quantified changes in important phosphotyrosine peptides in primary T‐cells upon stimulation with anti‐CD3/CD28 antibodies. This study showcases the ability of the SureQuant approach to accurately quantify low‐abundance phosphotyrosine peptides, highlighting its broader potential to study a diverse set of PTMs in physiological or clinical settings. Summary T‐cell receptor (TCR) signaling plays a fundamental role in immune responses, regulating T‐cell activation, differentiation, and function. While tyrosine phosphorylation is a key regulatory mechanism in this pathway, the low abundance of phosphotyrosine peptides presents a major challenge for their detection and quantification in complex biological samples. By employing the SureQuant targeted mass spectrometry approach, we achieved highly sensitive and reproducible quantification of key phosphotyrosine sites involved in T‐cell activation. This study provides a systematic view of TCR signaling dynamics, revealing distinct phosphorylation patterns across different activation timepoints. Our findings demonstrate the effectiveness of SureQuant in quantifying low‐abundance, post‐translationally modified peptides, offering a valuable tool for studying signaling pathways with greater precision. Additionally, this methodological framework can be extended to investigate other signaling networks, immune cell functions, and disease‐associated phosphotyrosine modifications.

Craniofacial reconstruction of critical bone defects typically requires a bone graft. As graft availability may be restricted by disease or comorbidities, tissue engineering approaches are actively sought. The pericranium could provide new bone graft material. During development and repair, bone transitions through a chondrogenic phase. However, with tissue engineering, pluripotent cells can differentiate directly into bone cells. Does ability to recapitulate bone formation in vitro affect osteogenesis and vascularization of pericranium grafts? To answer this, we obtained tissue from nine patients with preplanned craniotomy surgery and studied three-dimensional osteogenesis and angiogenesis of pericranium-derived spheroids. First, we established growth and differentiation conditions on Matrigel. For each spheroid sample, we investigated (i) continuous osteogenic differentiation (COD) and (ii) osteogenic differentiation preceded by chondrogenesis (CD → OD). The effect of vascular endothelial growth factor (VEGF) was compared to VEGF supplemented with fibroblast growth factor, interleukin (IL)-1, IL-6, platelet-derived growth factor, and tumor necrosis factor-α, a growth factor mix (GFM) with possible synergistic effects. In this limited sample, we observed no age- or sex-related differences in cell expansion. Similarly, no statistically significant differences in osteogenic or angiogenic scores between COD or CD → OD spheroids were noted with regular media. In COD, however, VEGF statistically significantly increased angiogenesis compared to control media ( p = 0.007). Also, in COD, both VEGF and VEGF + GFM increased osteogenesis ( p = 0.047 and p = 0.038, respectively). By contrast, in CD → OD, neither VEGF nor VEGF + GFM yielded statistically significant angiogenesis or osteogenesis scores compared to control media. To understand these results, we characterized spheroid protein expression by nanoliquid chromatography coupled to tandem mass spectrometry. Nine angiogenic proteins were either uniquely expressed or upregulated in COD compared to CD → OD: (i) endothelial markers JUP, PTGIS, PTGS2, and TYMP, (ii) tissue remodeling factors CHI3L1 and MMP14, and (iii) metabolic pathways modulators ANGPTL4, ITGA5, and WNT5A. ANGPTL4, ITGA5, PTGIS, PTGS2, and WNT5A define a conserved angiogenic network and were >2-fold increased in VEGF compared to VEGF + GFM. Finally, we examined bone formation on printable poly-(propylene-fumarate) (PPF) scaffolds for individualized grafting. Under COD + VEGF conditions, PPF scaffolds loaded with pericranium-derived cells displayed hallmarks of spongiform-like bone formation. Thus, the human pericranium may be a potential repository for bone-generating cells with applications in craniofacial bone repair using tissue printing.

Objectives/HypothesisVocal fold (VF) paralysis by sectioning the recurrent laryngeal nerve dramatically impacts the life of thyroidectomy patients. Volume‐expanding materials can temporarily restore VF medialization. To prolong this benefit, adipose mesenchymal stem cells (ADSCs) and micronized acellular dermis (MACD) were co‐injected in a rabbit model of injection medialization laryngoplasty. Biomarkers of in situ proliferation were identified by mass spectrometry proteomics and pathway analysis to guide future efforts to increase the length of benefit.MethodsADSCs were expanded and/or differentiated into chondrocytes (CHON) as collagen microspheres. After VF paralysis rabbits received MACD, MACD + undifferentiated ADSC, or MACD+CHON, ADSCs differentiated into chondrocytes. After 12 weeks, animals were sacrificed and 5‐µm paraffin‐embedded cryosections were prepared from larynges for hematoxylin and eosin visualization and nanoflow liquid chromatography electrospray‐ionization tandem‐mass spectrometry analysis of tissue collections. Validated proteins were processed by Venn subtraction and gene ontology (GO) overrepresentation analysis to identify unique pathways and biomarkers.ResultsConfirmed proteins numbered 147 (MACD), 1,243 (MACD+ADSC), and 1,033 (MACD+CHON). Totally, 333 proteins were uniquely found in the MACD+ADSC group, including mesenchymal surface markers CD9, CD44, fibronectin, and vimentin. Over 70% of proteins belonged to catalytic activity and binding GO categories, with the histone (H) family being overrepresented (P < 0.05). Histone variants H3.3, H2A.V, and H2A.Z (associated with open chromatin states) were overrepresented in the MACD+ADSC group, whereas structural histones H2A, H2B, and H4 were not.ConclusionBiomarkers, including atypical histones, are associated with in vivo proliferation of ADSCs and an expanded VF medialization volume.Level of EvidenceNA Laryngoscope, 128:E402–E408, 2018