Mass spectrometry for SUMO site detection from 3×Flag-SUMO-T86K expressing cells

Anti-Flag immunoprecipitated 3×Flag-SUMO-T86K proteins on beads were washed once and suspended in 50 mM ammonium bicarbonate, followed by the addition of 1 µg Lys-C and an overnight incubation at 37ºC. The digests were reduced, alkylated, diluted 10-fold with HBS (50 mM HEPES-NaOH pH7.5, 150 mM NaCl), and incubated with PTMScan HS K-ε-GG Remnant Magnetic Immunoaffinity Beads (Cell Signaling Technology) for 2 h at 4ºC. After washing with HBS three times, peptides were eluted with 200 µL of 0.1% TFA and 60% ACN. The eluates were evaporated, dissolved in 0.1% TFA, and desalted with GL-Tip SDB. After evaporation, the resultant peptides were dissolved in 0.1% TFA and 3% ACN and analyzed by an EASY-nLC 1200 UHPLC connected to an Orbitrap Fusion mass spectrometer. The peptides were separated on a 75-µm inner diameter × 150 mm C18 reversed-phase column with a linear 4-32% ACN gradient for 0-60 min followed by an increase to 80% ACN for 10 min and finally held at 80% ACN for 10 min. The mass spectrometer was operated in data-dependent acquisition mode with a maximum duty cycle of 3 seconds. Raw data were directly analyzed against the UniProt database restricted to Drosophila melanogaster using Proteome Discoverer 2.5 (Thermo Fisher Scientific) with the Sequest HT search engine. The search parameters were as follows: (a) Lys-C as an enzyme with up to 2 missed cleavages; (b) precursor mass tolerance of 10 ppm; (c) fragment mass tolerance of 0.6 Da; and (d) acetylation of protein N-terminus, oxidation of methionine, carbamidomethylation or N-ethylmaleimidation of cysteine, and di-glycine modification of lysine as variable modifications. Peptides were filtered at a false discovery rate of 1% using the Percolator node. Label-free quantification was performed based on intensities of precursor ions using the Precursor Ions Quantifier node. Normalization was performed such that the total sum of abundance values for each sample over all peptides was the same.

将磁珠上经抗Flag免疫沉淀（immunoprecipitated）得到的3×Flag-SUMO-T86K蛋白洗涤一次，重悬于50 mM碳酸氢铵（ammonium bicarbonate）缓冲液中，随后加入1 µg Lys-C酶，于37℃条件下孵育过夜。酶解产物经还原、烷基化处理后，用HBS（50 mM羟乙基哌嗪乙硫磺酸-氢氧化钠（HEPES-NaOH），pH 7.5，150 mM氯化钠（NaCl））稀释10倍，与PTMScan HS K-ε-GG残基磁免疫亲和磁珠（Cell Signaling Technology）于4℃孵育2小时。经HBS洗涤磁珠三次后，用200 µL 0.1%三氟乙酸（trifluoroacetic acid, TFA）与60%乙腈（acetonitrile, ACN）洗脱结合的肽段。洗脱液经真空蒸发浓缩后，重悬于0.1% TFA中，再通过GL-Tip SDB进行脱盐纯化。蒸发干燥后的肽段重悬于0.1% TFA与3% ACN混合液中，采用连接于轨道阱融合质谱仪（Orbitrap Fusion）的EASY-nLC 1200超高效液相色谱（UHPLC）进行分析。肽段在内径75 µm×150 mm的C18反相色谱柱上分离，采用线性乙腈梯度洗脱：0~60 min内乙腈浓度从4%升至32%，随后10 min内升至80%并维持10 min。质谱仪以数据依赖性采集模式运行，最大占空比为3秒。原始质谱数据直接以限定为黑腹果蝇（Drosophila melanogaster）的通用蛋白质资源数据库（UniProt）为参考库，使用Proteome Discoverer 2.5（赛默飞世尔科技，Thermo Fisher Scientific）搭载Sequest HT搜索引擎进行搜库分析。搜库参数设置如下：(a) 酶解酶为Lys-C，允许最多2个漏切位点；(b) 前体离子质量容忍度为10 ppm；(c) 碎片离子质量容忍度为0.6 Da；(d) 可变修饰包括蛋白质N端乙酰化、甲硫氨酸氧化、半胱氨酸的氨基甲酰甲基化或N-乙基马来酰亚胺修饰，以及赖氨酸的双甘氨酸修饰。肽段鉴定结果通过Percolator节点进行过滤，设定错误发现率（false discovery rate, FDR）为1%。基于前体离子强度，通过Precursor Ions Quantifier节点进行无标记定量分析，归一化方式为所有样本的全部肽段丰度总和保持一致。