Gastric cancer (GC) has significant morbidity and mortality worldwide and especially in China. Its molecular pathogenesis has not been thoroughly elaborated. The acknowledged biomarkers for diagnosis, prognosis, recurrence monitoring and treatment are lacking. Proteins from matched pairs of human GC and adjacent tissues were analyzed by a coupled label-free Mass Spectrometry (MS) approach, followed by functional annotation with software analysis. Nano-LC-MS/MS, quantitative real-time polymerase chain reaction (qRT-PCR), western blot and immunohistochemistry were used to validate dysregulated proteins. One hundred forty-six dysregulated proteins with more than twofold expressions were quantified, 22 of which were first reported to be relevant with GC. Most of them were involved in cancers and gastrointestinal disease. The expression of a panel of four upregulated nucleic acid binding proteins, heterogeneous nuclear ribonucleoprotein hnRNPA2B1, hnRNPD, hnRNPL and Y-box binding protein 1 (YBX-1) were validated by Nano-LC-MS/MS, qRT-PCR, western blot and immunohistochemistry assays in ten GC patients’ tissues. They were located in the keynotes of a predicted interaction network and might play important roles in abnormal cell growth. The label-free quantitative proteomic approach provides a deeper understanding and novel insight into GC-related molecular changes and possible mechanisms. It also provides some potential biomarkers for clinical diagnosis.

Manganese superoxide dismutase (MNSOD) is one of the major scavengers of reactive oxygen species (ROS) in mitochondria with pivotal regulatory role in ischemic disorders, inflammation and cancer. Here we report oxidative modification of MNSOD in human renal cell carcinoma (RCC) by the shotgun method using data-dependent liquid chromatography tandem mass spectrometry (LC-MS/MS). While 5816 and 5571 proteins were identified in cancer and adjacent tissues, respectively, 208 proteins were found to be up- or down-regulated (p < 0.05). Ontological category, interaction network and Western blotting suggested a close correlation between RCC-mediated proteins and oxidoreductases such as MNSOD. Markedly, oxidative modifications of MNSOD were identified at histidine (H⁵⁴ and H⁵⁵), tyrosine (Y⁵⁸), tryptophan (W¹⁴⁷, W¹⁴⁹, W²⁰⁵ and W²¹⁰) and asparagine (N²⁰⁶ and N²⁰⁹) residues additional to methionine. These oxidative insults were located at three hotspots near the hydrophobic pocket of the manganese binding site, of which the oxidation of Y⁵⁸, W¹⁴⁷ and W¹⁴⁹ was up-regulated around three folds and the oxidation of H⁵⁴ and H⁵⁵ was detected in the cancer tissues only (p < 0.05). When normalized to MNSOD expression levels, relative MNSOD enzymatic activity was decreased in cancer tissues, suggesting impairment of MNSOD enzymatic activity in kidney cancer due to modifications. Thus, LC-MS/MS analysis revealed multiple oxidative modifications of MNSOD at different amino acid residues that might mediate the regulation of the superoxide radicals, mitochondrial ROS scavenging and MNSOD activity in kidney cancer.

Histone acetylation adds an acetyl group on the lysine residue commonly found within the N-terminal tail protruding from the histone core of the nucleosome, and is important for chromosome structure and function in gene transcription and chromatin remodeling. Acetylation may also occur on other residues additional to lysine, but have not been thoroughly investigated at the proteomics level. Here we report a wide tolerance acetylation study mimicking the addition of 42 ± 0.5 Da delta mass modification on undefined amino acid residues of histones by shotgun proteomics using liquid chromatography–tandem mass spectrometry. A multi-blind spectral alignment algorithm with a wide peptide tolerance revealed frequent occurrence of 42 ± 0.5 Da modifications at lysine (K), serine (S) and threonine (T) residues in human histones from kidney tissues. Precision delta mass analysis identified acetylation (42.011 ± 0.004 Da) and trimethylation (42.047 ± 0.002 Da) modifications within the delta mass range. A specific antibody was produced to validate the acetylated T22 of human histone H3 (H3T22ac) by immune assays. Thus, we demonstrated that the wide tolerance acetylation approach identified histone acetylation as well as modification variants commonly associated with acetylation at undefined residues additional to lysine.

Chimeric antigen receptor (CAR)-T cell therapy for colorectal cancer (CRC) faces three major barriers: antigen heterogeneity, off−tumor toxicity, and the efficacy−safety trade−off. To overcome these obstacles, next−generation engineering strategies have emerged, including: (1) novel CRC−associated targets with improved tissue restriction; (2) architectural innovations such as armored CARs, optimized signaling (1XX, 28−ΔIL2RB−z(YXXQ)), and cytokine−arming; (3) combinatorial antigen−sensing circuits (AND/OR/NOT gates, SUPRA, synNotch); and (4) CRISPR−based editing for exhaustion−related knockouts (e.g., PD−1, Fas, TGFBR2) and site−specific CAR knock−in. Clinical evidence has demonstrated objective responses and disease stabilization in subsets of patients. However, the immunosuppressive tumor microenvironment—including inhibitory cells, dense stroma, and metabolic dysfunction—remains a critical hurdle limiting CAR−T persistence. Future directions should prioritize molecular typing−guided intervention, universal CAR−T, multicellular platforms (CAR−NK, CAR−M), and interdisciplinary collaboration. This review provides a framework for designing next−generation CAR−T therapies capable of achieving durable remissions in advanced CRC.