Platform independent protein-based cell-of-origin subtyping of diffuse large B-cell lymphoma in formalin-fixed paraffin-embedded tissue

10 µm formalin-fixed paraffin-embedded tissue sections were deparaffinized with xylene; homogenization was performed in lysis-buffer (20 mM Tris-HCl, pH 8.8, 200 mM glycine, 200 mM DTT, 4% SDS)in an ultrasonic bath; samples were incubated under continuous agitation first at 99 °C for 30 min, then at 80 °C for 60 min, afterwards centrifuged at 12,000xg for 10 min. 50 µg of protein lysate were further processed according the GASP (Gel-assisted sample preparation) protocol; peptide identification (IDA, information dependent acquisition) and SWATH measurements by LC-MS/MS on TripleTOF5600 (AB Sciex)

将10 µm甲醛固定石蜡包埋组织切片经二甲苯脱蜡；采用裂解缓冲液（20 mM Tris-HCl，pH 8.8、200 mM 甘氨酸、200 mM 二硫苏糖醇（DTT）、4% 十二烷基硫酸钠（SDS））在超声水浴中完成匀浆处理；将样品置于持续振荡条件下，先于99 ℃孵育30分钟，再于80 ℃孵育60分钟，随后以12000×g离心10分钟。取50 µg蛋白质裂解液，按照凝胶辅助样品制备（Gel-assisted sample preparation，GASP）方案进行后续处理；肽段鉴定采用信息依赖性采集（information dependent acquisition，IDA）方式，并通过液相色谱-串联质谱（LC-MS/MS）在TripleTOF 5600质谱仪（AB Sciex公司）上完成SWATH检测。